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A Capillary Electrophoretic Assay for Acetyl CoA Carboxylase
Bryant, Sherrisse K.; Waldrop, Grover L.; Gilman, S. Douglass
A simple off-column capillary electrophoretic (CE) assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition was developed. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneous monitored in this assay. Acetyl coenzyme A (CoA), malonyl-CoA, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were separated by CE, and the depletion of ATP and acetyl-CoA as well as the production of ADP and malonyl-CoA were monitored. Inhibition of holo-ACC by the biotin carboxylase inhibitor, 2-amino-N,N-dibenzyloxazole-5-carboxamide, and the carboxyltransferase inhibitor, andrimid, was confirmed using this assay. A previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for the BC component of ACC also was developed. PMID:23435309
Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.
Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul; Wallace, John C; Jitrapakdee, Sarawut; Attwood, Paul V
We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity. PMID:26149215
Dilute acid hydrolysis of paper birch: kinetics studies of xylan and acetyl-group hydrolysis.
Maloney, M T; Chapman, T W; Baker, A J
Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18M and temperatures of between 100 and 170 degrees C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based on the existence of reactive and resistant xylan portions. The resulting rate equation predicts the experimental xylan concentrations in the residue to within 10%. Hydrolysis of xylan-associated acetyl groups was found to occur at the same rate as that of xylan, except at 100 degrees C, where acetyl is released preferentially. No effect of acid concentration on the rate of acetyl removal relative to that of xylan was evident. PMID:18553680
Acetyl CoA Carboxylase Shares Control of Fatty Acid Synthesis with Fatty Acid Synthase in Bovine Mammary Homogenate
T. C. Wright; J. P. Cant; J. T. Brenna; B. W. McBride
The objectives of this research were to determine the flux control coefficients for acetyl CoA carboxylase and fatty acid synthase using an in vitro preparation of bovine mammary homogenate. For an enzyme to be considered rate limiting with the use of metabolic control analysis, its control coefficient would be equal to unity. The hypothesis for this experiment was that the
Correlation of ATP citrate lyase and acetyl CoA levels with trichothecene production in Fusarium graminearum.
Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei
The correlation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride. PMID:24284828
Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes.
Perry, Rachel J; Camporez, Joăo-Paulo G; Kursawe, Romy; Titchenell, Paul M; Zhang, Dongyan; Perry, Curtis J; Jurczak, Michael J; Abudukadier, Abulizi; Han, Myoung Sook; Zhang, Xian-Man; Ruan, Hai-Bin; Yang, Xiaoyong; Caprio, Sonia; Kaech, Susan M; Sul, Hei Sook; Birnbaum, Morris J; Davis, Roger J; Cline, Gary W; Petersen, Kitt Falk; Shulman, Gerald I
Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulin's ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D. PMID:25662011
Correlation of ATP Citrate Lyase and Acetyl CoA Levels with Trichothecene Production in Fusarium graminearum
Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei
Thecorrelation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride. PMID:24284828
The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes
Michael J. Selig; William S. Adney; Michael E. Himmel; Stephen R. Decker
Analysis of variously pretreated corn stover samples showed neutral to mildly acidic pretreatments were more effective at\\u000a removing xylan from corn stover and more likely to maintain the acetyl to xylopyranosyl ratios present in untreated material\\u000a than were alkaline treatments. Retention of acetyl groups in the residual solids resulted in greater resistance to hydrolysis\\u000a by endoxylanase alone, although the synergistic
Hepatic enzymatic synthesis and hydrolysis of CoA esters of solvent-derived oxa acids.
Panuganti, Sree D; Penn, Jill M; Moore, Kathleen H
Many ethylene glycol-derived solvents are oxidized to xenobiotic alkoxyacetic acids (3-oxa acids) by hepatic enzymes. The toxicity of these ubiquitous solvents has been associated with their oxa acid metabolites. For many xenobiotic carboxylic acids, the toxicity is associated with the CoA ester of the acid. In this study, related alkoxyacetic acids were evaluated as potential substrates for acyl-CoA synthetases found in mitochondrial, peroxisomal, and microsomal fractions isolated from rat liver. Likewise, chemically synthesized oxa acyl-CoAs were used as substrates for acyl-CoA hydrolases associated with the same rat liver fractions. Activities of the xenobiotic oxygen-substituted substrates were compared with analogous physiologic aliphatic substrates by UV-vis spectrophotometric methods. All of the solvent-derived oxa acids were reasonable substrates for the acyl-CoA synthetases, although their activity was usually less than the corresponding physiologic acid. Acyl-CoA hydrolase activities were decreased compared with acyl-CoA synthetase activities for all substrates, especially for the oxa acyl-CoAs. These studies suggest that these xenobiotic carboxylic acids may be converted to reactive acyl-CoA moieties which will persist in areas of the cell proximal to lipid synthesis, beta-oxidation, protein acylation, and amino acid conjugation. The interaction of these xenobiotic acyl-CoAs with those processes may be important to their toxicity and/or detoxification. PMID:12717739
[3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue
NASA Technical Reports Server (NTRS)
Hall, P. J.; Bandurski, R. S.
[3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.
The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein
Neuberger, A.; Ratcliffe, Wendy A.
Rabbit Tamm–Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm–Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm–Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues. PMID:4349114
Hydrolysis of wheat arabinoxylan by two acetyl xylan esterases from Chaetomium thermophilum.
Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard; Busk, Peter Kamp
The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 126.96.36.199) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has been sequenced, its carbohydrate esterases are not annotated yet. We applied peptide pattern recognition (PPR) tool for sequence analysis of the C. thermophilum genome, and 11 carbohydrate esterase genes were discovered. Furthermore, we cloned and heterologously expressed two putative acetyl xylan esterase genes, CtAxeA and CtAxeB, in Pichia pastoris. The recombinant proteins, rCtAxeA and rCtAxeB, released acetic acids from p-nitrophenyl acetate and water-insoluble wheat arabinoxylan. These results indicate that CtAxeA and CtAxeB are true acetyl xylan esterases. For both recombinant esterases, over 93 % of the initial activity was retained after 24 h of incubation at temperatures up to 60 °C, and over 90 % of the initial activity was retained after 24 h of incubation in different buffers from pH 4.0 to 9.0 at 4 and 50 °C. The overall xylose yield from wheat arabinoxylan hydrolysis was 8 % with xylanase treatment and increased to 34 % when xylanase was combined with rCtAxeA and rCtAxeB. In sum, the present study first report the biochemical characterization of two acetyl xylan esterases from C. thermophilum, which are efficient in hydrolyzing hemicellulose with potential application in biomass bioconversion to high value chemicals or biofuels. PMID:25369895
Probing the allosteric activation of pyruvate carboxylase using 2?,3?-O-(2,4,6-trinitrophenyl) adenosine 5?-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
Adina-Zada, Abdussalam; Hazra, Rasmani; Sereeruk, Chutima; Jitrapakdee, Sarawut; Zeczycki, Tonya N.; Maurice, Martin St.; Cleland, W.Wallace; Wallace, John C.; Attwood, Paul V.
2?,3?-O-(2,4,6-Trinitrophenyl) adenosine 5?-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5 fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme. PMID:21426897
Synthesis and Kinetics of Hydrolysis of 3,5-Dimethyl-N-acetyl-p-benzoquinone Imine: An Undergraduate Laboratory
NASA Astrophysics Data System (ADS)
Buccigross, Jeanne M.; Metz, Christa; Elliot, Lori; Becker, Pamela; Earley, Angela S.; Hayes, Jerry W.; Novak, Michael; Underwood, Gayl A.
The synthesis of the title compound by a three-step procedure is described. The hydrolysis kinetics, which involve two consecutive psuedo-first-order processes, are also described. The synthesis and kinetics experiments described here are proposed for incorporation into undergraduate laboratory courses under a variety of formats. The compound described here is related to a toxic metabolite of the common analgesics acetaminophen and phenacetin.
Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation
The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA
Protein acetylation and acetyl coenzyme a metabolism in budding yeast.
Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron; Vancura, Ales
Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. PMID:25326522
Acetylation of woody lignocellulose: significance and regulation
Pawar, Prashant Mohan-Anupama; Koutaniemi, Sanna; Tenkanen, Maija; Mellerowicz, Ewa J.
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 toward 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. PMID:23734153
Identification of a novel CoA synthase isoform, which is primarily expressed in Brain
Nemazanyy, Ivan [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine)]. E-mail: firstname.lastname@example.com; Panasyuk, Ganna [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine); Breus, Oksana [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine); Zhyvoloup, Alexander [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine); Filonenko, Valeriy [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine); Gout, Ivan T. [Department of Structure and Function of Nucleic Acids, Institute of Molecular Biology and Genetics, 150 Zabolotnogo St, Kyiv 03680 (Ukraine) and Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, Gower Street, London WC1E 6BT (United Kingdom)]. E-mail: email@example.com
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 {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} 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 {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} 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.
Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase
Seravalli, Javier; Ragsdale, Stephen W.
Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60–70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA. PMID:18203715
Captan (N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide) undergoes hydrolysis readily in water with a maximum half-life of 710 min. Over the pH range 2-6, the reaction is pH independent and the pseudo-first-order rate constant is (1.8 + or - 0.1) x 10 to the -5th power/s....
Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.
Dupont, C; Clarke, A J
O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover. PMID:2066331
In vivo trapping of polyketide intermediates from an assembly line synthase using malonyl carba(dethia)-N-acetyl cysteamines.
Tosin, Manuela; Demydchuk, Yuliya; Parascandolo, James S; Per, Covadonga Blasco; Leeper, Finian J; Leadlay, Peter F
Early-stage intermediates in the biosynthesis of erythromycin A by Saccharopolyspora erythraea were intercepted by malonyl carba(dethia)-N-acetyl cysteamines, generated in vivo from the hydrolysis of the corresponding methyl esters. PMID:21301753
Autotrophic growth: the methyl binding site of CO dehydrogenase in the synthesis of acetyl-CoA
Pezacka, E.; Wood, H.G.
A pathway in which CO or CO/sub 2/ and H/sub 2/ is used as a source of energy and carbon to synthesize acetyl-CoA is used for autotrophic growth of acetogenes, methanogens and some sulfate-reducing bacteria. All enzymes involved in this pathway have been purified from C. thermoaceticum. Five of them: CO dehydrogenase (CODH), corrinoid protein, methyltransferase, CODH disulfide reductase (SSRd) and ferredoxin catalyzed synthesis of acetyl-CoA from methyltetrahydrofolate, CO and CoA. CODH is a central enzyme catalyzing the condensation of CH/sub 3/, CO and CoA and per se it catalyzes a reversible exchange of CO with acetyl-CoA. Thus, CODH must have binding sites for CH/sub 3/, CO and CoA. They have succeeded in methylating ..beta.. subunits of CODH using /sup 14/CH/sub 3/I or /sup 14/CH-corrinoid protein, a native donor of the CH/sub 3/ group in synthesis of acetyl-CoA. With resulting (/sup 14/CH/sub 3/)CODH, only SSRd is required for synthesis of (/sup 14/C)acetyl-CoA from CO and CoA. The kinetic studies show that CH/sub 3/I is a competitive inhibitor for exchange reaction between CO and acetyl-CoA. Acetaldehyde and acetyl-CoA but not acetic acid and CoA protected CODH against methylation by CH/sub 3/I. Methyl group bound to CODH is very slowly removed by CO and CoA and acetyl-CoA accelerated this process. These data confirm that CH/sub 3/ group from CH/sub 3/I and CH/sub 3/-corrinoid protein is bound to the methyl binding site of CODH.
Enzymatic synthesis of a novel acetylated neutral lipid (related to platelet activating factor, PAF) by acyl-CoA: 1-alkyl-2-acetyl-sn-glycerol acyltransferase
Kawasaki, Tomio; Synder, F. (Oak Ridge Associated Univ., TN (USA))
Recent data from our laboratory have described the complete enzymatic steps for the de novo synthesis of alkylacetylglycerols and their subsequent conversion to PAF. Experiments are reported here to show that the alkylacetylglycerols also are a substrate for an acyltransferase. Homogenates of HL-60 cells produced labeled 1-alkyl-2-acetyl-3-acyl-sn-glycerols from 1-({sup 3}H)hexadecyl-2-acetyl-sn-glycerol, 1-alkyl-2-({sup 3}H)acetyl-sn-glycerol, or (1-{sup 14}C)linoleic acid when incubated with CoA (0.1 mM), ATP (10 mM), and Mg{sup 2+} (95 mM). Formation of labeled alkylacetylacyl-glycerols by the acyltransferase required CoA, ATP, and Mg{sup 2+}. The labeled alkylacetylacylglycerol produced had an identical R{sub f} to that of an authenic standard; after treatment with pancreatic lipase, the sn-3 acyl moiety was hydrolyzed to regenerate the original 1-alkyl-2-acetyl-sn-glycerol precursor. (1-{sup 14}C)Linoleic acid was an excellent substrate for the acylation step, whereas (1-{sup 14}C)oleic acid was barely utilized. Kinetic properties and related characteristics of this enzyme that forms the acetyl analog of a triglyceride will be presented. Although the function of this new type of acetylated neutral lipid class is presently unknown, it could serve as a potential precursor reservoir in the de novo route of PAF synthesis.
Determination of the structure of lecithins via the formation of acetylated 1,2-diglycerides
O. S. Privett; L. J. Nutter
A detailed procedure for quantitative determinations of molecular species of lecithins is described and applied to several\\u000a lecithins isolated from natural sources. The method is based on the conversion of lecithin to acetylated 1,2-diglycerides\\u000a and analysis of these compounds by methodology used for the determination of triglyceride structure.\\u000a \\u000a The preparation of the acetylated 1,2-diglycerides was carried out via hydrolysis with
The diversity of acetylated proteins
Bogdan Polevoda; Fred Sherman
Acetylation of proteins, either on various amino-terminal residues or on the ?-amino group of lysine residues, is catalyzed by a wide range of acetyltransferases. Amino-terminal acetylation occurs on the bulk of eukaryotic proteins and on regulatory peptides, whereas lysine acetylation occurs at different positions on a variety of proteins, including histones, transcription factors, nuclear import factors, and ?-tubulin.
NASA Technical Reports Server (NTRS)
Hoagland, P. D.; Fox, S. W.
Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.
A type III polyketide synthase from Rhizobium etli condenses malonyl CoAs to a heptaketide pyrone with unusually high catalytic efficiency.
Jeya, Marimuthu; Kim, Tae-Su; Kumar Tiwari, Manish; Li, Jinglin; Zhao, Huimin; Lee, Jung-Kul
A novel type III polyketide synthase (RePKS) from Rhizobium etli produced a heptaketide pyrone using acetyl-CoA and six molecules of malonyl-CoA. Its catalytic efficiency (k(cat)/K(m) = 5230 mM(-1) min(-1)) for malonyl CoA was found to be the highest ever reported. Molecular dynamics studies revealed the unique features of RePKS. PMID:23059854
Inhibition of neutral lipase from castor bean lipid bodies by coenzyme A (CoA) and Oleoyl-CoA
Not Available
The neutral lipase (EC 18.104.22.168) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of ({sup 14}C)trioleoylglycerol, releasing ({sup 14}C)oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of ({sup 14}C)oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts ({sup 14}C)oleic acid produced from the lipase reaction to ({sup 14}C)oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important in vivo in coordinating lipase activity with fatty acid oxidation.
Acetyl-coenzyme A synthetase 2 is a nuclear protein required for replicative longevity in Saccharomyces cerevisiae
Aris, John P.
, including fungi, plants, and animals [1]. All catalyze the reaction: acetate ? CoA ? ATP ? acetyl-CoA ? AMP is an important aspect of the metabolic strategy yeast employ to protect sugar resources in their environment [email@example.com Present Address: A. A. Falco´n Department of Nutritional Sciences and Toxicology, University of California
Progressing batch hydrolysis process
Wright, J.D.
A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.
Autocatalytic activation of acetyl-CoA synthase.
Maynard, Ernest L; Tan, Xiangshi; Lindahl, Paul A
Acetyl-CoA synthase (ACS identical with ACS/CODH identical with CODH/ACS) from Moorella thermoacetica catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group of a corrinoid-iron-sulfur protein (CoFeSP). A time lag prior to the onset of acetyl-CoA production, varying from 4 to 20 min, was observed in assay solutions lacking the low-potential electron-transfer agent methyl viologen (MV). No lag was observed when MV was included in the assay. The length of the lag depended on the concentrations of CO and ACS, with shorter lags found for higher [ACS] and sub-saturating [CO]. Lag length also depended on CoFeSP. Rate profiles of acetyl-CoA synthesis, including the lag phase, were numerically simulated assuming an autocatalytic mechanism. A similar reaction profile was monitored by UV-vis spectrophotometry, allowing the redox status of the CoFeSP to be evaluated during this process. At early stages in the lag phase, Co(2+)FeSP reduced to Co(+)FeSP, and this was rapidly methylated to afford CH(3)-Co(3+)FeSP. During steady-state synthesis of acetyl-CoA, CoFeSP was predominately in the CH(3)-Co(3+)FeSP state. As the synthesis rate declined and eventually ceased, the Co(+)FeSP state predominated. Three activation reductive reactions may be involved, including reduction of the A- and C-clusters within ACS and the reduction of the cobamide of CoFeSP. The B-, C-, and D-clusters in the beta subunit appear to be electronically isolated from the A-cluster in the connected alpha subunit, consistent with the ~70 A distance separating these clusters, suggesting the need for an in vivo reductant that activates ACS and/or CoFeSP. PMID:15015040
AMP-forming acetylCoA synthetase from the extremely halophilic archaeon Haloarcula marismortui : purification, identification and expression of the encoding gene, and phylogenetic affiliation
Christopher Bräsen; Peter Schönheit
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA ? Acetyl-CoA + AMP + PPi). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas
Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria.
Ekstrom, Eileen B; Morel, François M M; Benoit, Janina M
Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains. PMID:12957930
Progressing batch hydrolysis process
Wright, John D. (Denver, CO)
A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.
Hepatic Acetylator Polymorphism in Breast Cancer Patients
José M. Ladero; María J. Fernandez; Ramiro Palmeiro; Juan J. Muńoz; Carlos Jara; Carmen Lazaro; Gumersindo Perez-Manga
Hepatic acetylator phenotype has been determined, using sulfamethazine, in 81 white Spanish women with histologically proven breast cancer and in 75 adequate female controls. No differences were detected in the distribution of acetylator phenotype between the two groups of slow acetylators, 49 patients (60.5%) and 45 controls (60%). The percentage of acetylated sulfamethazine in plasma for each phenotype was not
Particleboards from Acetylated Wood Flakes
Daniel Wagner; Clemens Schwarzinger; Manuela Leidl; Harald Schmidt; Andreas Endesfelder
Summary. Particleboards from acetylated wood flakes with various resins were pressed and compared with untreated control boards with\\u000a respect to their weathering resistance and mechanical strength. The industrially used melamine, urea, and phenol resins showed\\u000a a poor adhesion behavior to the acetylated flakes resulting in a high decrease of the mechanical strength of the particleboards.\\u000a A novel type of apolar resin
Molecular Structure of Acetyl Peroxide
NSDL National Science Digital Library
Acetyl peroxide is a colorless liquid with a pungent odor. It is generally stored as a 25% solution in dimethyl phthalate to prevent detonation. It may explode if heated, or in contact with combustible materials. As the pure material, acetyl peroxide is unstable and incompatible with organic materials. The compound is harmful by inhalation, ingestion and skin contact. It is used as an initiator and catalyst for resins, and it also promotes polymerization in the manufacture of certain plastics.
Global Hawk Pacific (GloPac) COA and Mission Coordination
NASA Technical Reports Server (NTRS)
Dillon, Mark; Hall, Philip
This slide presentation reviews the science objectives of the Global Hawk unmanned aircraft system (UAS) in the Pacific region, shows examp le flight tracks, the satellite under-flight requirement, the flight planning, and the agencies coordination of the airspace required for the Certificate of Authorization (COA).
Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.
Yu, Le; Zhao, Jingbo; Xu, Mengmeng; Dong, Jie; Varghese, Saju; Yu, Mingrui; Tang, I-Ching; Yang, Shang-Tian
The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA ? acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (?ack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (?ack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH 5.0 and 6.0. Compared to C. tyrobutyricum (?ack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22?-?0.26 g/g (vs. 0.10?-?0.13 g/g) and productivity to 0.35 g/l h (vs. 0.13 g/l h) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation. PMID:25851718
Acetyltransferase and human hemoglobin acetylation
Kasten-Jolly, J.; Qiu, C.C.; Abraham, E.C.
A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the ..gamma..-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-(/sup 14/C-acetyl)-CoA into both human Hb F/sub 0/ and HB A/sub 0/, but at a lower rate than for histones. Characterization of the labeled products indicated that the ..cap alpha..-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F/sub 0/ the amino-terminus of the ..gamma..-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F/sub 0/, ..gamma..-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction.
Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica
Jones, Cheryl P.
Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 126.96.36.199) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. PMID:25303954
Autotrophic growth: methylated carbon monoxide dehydrogenase as an intermediate of acetyl-CoA synthesis
Pezacka, E.; Wood, H.G.
A new pathway of autotrophic growth has been discovered in certain anaerobic bacteria in which acetyl-CoA is the product formed from CO/sub 2/ for initiation of anabolism rather than 3-phosphoglycerate as in the Calvin Cycle. CO/sub 2/ is reduced in combination with tetrahydrofolate to methyltetrahydrofolate (CH/sub 3/THF) and is the source of the CH/sub 3/ group. CO/sub 2/ or CO is the source of the carbonyl group. CO dehydrogenase (CODH), corrinoid enzyme, methyltransferase, ferredoxin and CODH disulfide reductase have been isolated from Clostridium thermoaceticum and shown to catalyze the synthesis of acetyl-CoA from CH/sub 3/THF, CO and CoA. The methyltransferase catalyzes transfer of the CH/sub 3/ group from CH/sub 3/THF to the corrinoid enzyme from which the methyl is transferred to CODH. CO is bound to the Ni of CODH forming a Ni-Fe-C center. When CO/sub 2/ is the source of carbon, H/sub 2/ and hydrogenase are required for reduction of the CO/sub 2/ by CODH. CODH disulfide reductase is required for the addition of CoA to the CODH (Pezacka, E. and Wood, H.G. J. Biol. Chem., in press). Then, CODH catalyzes the combination of the three groups forming acetyl-CoA. The authors have now succeeded in methylating CODH using /sup 14/CH/sub 3/I or /sup 14/CH/sub 3/-B/sub 12/. With the resulting /sup 14/CH/sub 3/-CODH, only CODH disulfide reductase is required for synthesis of (/sup 14/C)acetyl-CoA from CO and CoA. The amino acid sequence at the CH/sub 3/-site is being investigated.
N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation.
Moffett, John R; Arun, Peethambaran; Ariyannur, Prasanth S; Namboodiri, Aryan M A
N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury. PMID:24421768
Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.
Guranowski, A; Günther Sillero, M A; Sillero, A
Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 126.96.36.199) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605
The Evolution of Acetyl-CoA Synthase
NASA Astrophysics Data System (ADS)
Lindahl, Paul A.; Chang, Belinda
Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoA from CO_2 + 2e^-, CoA, and a methyl group, and contain 5 types of subunits (?, ?, ?, ?, and ?). Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition. Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO_2 and 2e^-, and are composed of 2 autonomous proteins, an ?_2?_2 tetramer and a ?? heterodimer. Class IV enzymes catabolize CO to CO_2 and are ?-subunit monomers. Phylogenetic analyses were performed on all five subunits. ACS ? sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residues that may function as ligands to the B- and C-clusters were identified. Other residues exclusively conserved in Class I/II sequences may be ligands to additional metal centers in Class I and II enzymes. ACS ? sequences also separated into two groups, but they were less divergent than the ?'s, and the separation was not as distinct. Class III-like ? sequences contained ~300 residues at their N-termini absent in Class I/II sequences. Conserved residues identified in ? sequences may function as ligands to active site residues used for acetyl-CoA synthesis. ACS ?-sequences separated into 3 groups (Classes I, II, and III), while ?-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of ? sequences. ACS ?-sequence topology showed greater divergence and less consistency vis-ŕ-vis the other subunits, possibly reflecting reduced evolutionary constraints due to the absence of metal centers. The ? subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms may have evolved are discussed.
Enzymatic hydrolysis of molasses.
Najafpour, Ghasem D; Shan, Cheong Poi
Kinetic studies of the enzymatic hydrolysis of molasses were conducted using glucoamylase. Central Sugar Refinery SDN BHD contains 13-20% glucose. The molasses was diluted and the kinetic experiments were conducted at 67 degrees C with 100-1000 mg/l of glucoamylase. The glucose contents of the molasses were enhanced after hydrolysis of molasses solution with 1000 mg/l glucoamylase. A Lineweaver-Burk plot was obtained based on enzyme kinetic data. The rate constant, Km and maximum reaction rate, Vmax for 500 mg/l of glucoamylase were 100 mmol/l (18 g/l) and 5 mmol/l min (0.9 g/l min), respectively. The maximum reaction rate, Vmax for 1000 mg/l of glucoamylase was doubled, to 100 mmol/l (18 g/l) and the rate constant, Km was the same for 500 mg/l of glucoamylase. The substrate inhibition model was noncompetitive based on the resulting Lineweaver-Burk plot for enzyme concentration of 500 and 1000 mg/l. PMID:12421015
Structural studies on 4-O-acetyl-?-N-acetylneuraminyl-(2?3)-lactose, the main oligosaccharide in echidna milk
J. F. G. Vliegenthart; J. P. Kamerling; L. Dorland; H. van Halbeek; M. Messer; R. Schauer
The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-?-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 hydrolysis) and trimethyl-silylated\\/esterified sialic acid, and by m.s. (after derivatisation) and 500-MHz, 1H-n.m.r. spectroscopy of the intact oligosaccharide. Information about the glycosidic bonds
Lysosomal cholesterol accumulation inhibits subsequent hydrolysis of lipoprotein cholesteryl ester.
Jerome, W Gray; Cox, Brian E; Griffin, Evelyn E; Ullery, Jody C
Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression. PMID:18312718
Lysosomal Cholesterol Accumulation Inhibits Subsequent Hydrolysis Of Lipoprotein Cholesteryl Ester
Jerome, W. Gray; Cox, Brian E.; Griffin, Evelyn E.; Ullery, Jody C.
Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long-lived and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression. PMID:18312718
Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation
Bryson, Bryan D.
Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine ...
Structure of an acetylCoA binding protein from Staphylococcus aureus representing a novel subfamily of GCN5-related N -acetyltransferase-like proteins
John R. Cort; Theresa A. Ramelot; Diana Murray; Thomas B. Acton; Li-Chung Ma; Rong Xiao; Gaetano T. Montelione; Michael A. Kennedy
We have determined the solution NMR structure of SACOL2532, a putative GCN5-like N-acetyltransferase (GNAT) from Staphylococcus aureus. SACOL2532 was shown to bind both CoA and acetyl-CoA, and structures with and without bound CoA were determined. Based on analysis\\u000a of the structure and sequence, a subfamily of small GCN5-related N-acetyltransferase (GNAT)-like proteins can be defined. Proteins from this subfamily, which is largely
Technology Transfer Automated Retrieval System (TEKTRAN)
Purified T. reesei RUT C-30 acetyl esterase catalyzes acetyl transfer to a variety of carbohydrates in water in the presence of vinyl acetate as the acetyl group donor. The degree of conversion and the number of formed acetates depended on the acceptor used. With some acceptors, such as methyl or ...
Hydrolysis reactor for hydrogen production
Davis, Thomas A.; Matthews, Michael A.
In accordance with certain embodiments of the present disclosure, a method for hydrolysis of a chemical hydride is provided. The method includes adding a chemical hydride to a reaction chamber and exposing the chemical hydride in the reaction chamber to a temperature of at least about 100.degree. C. in the presence of water and in the absence of an acid or a heterogeneous catalyst, wherein the chemical hydride undergoes hydrolysis to form hydrogen gas and a byproduct material.
Enzymatic fat hydrolysis and synthesis
Warner M. Linfield; Robert A. Barauskas; Lorraine Sivieri; Samuel Serota; Robert W. Stevenson
The hydrolysis of tallow, coconut oil and olive oil, by lipase fromCandida rugosa, was studied. The reaction approximates a firstorder kinetics model. Its rate is unaffected by temperature in the range of\\u000a 26–46 C. Olive oil is more rapidly hydrolyzed compared to tallow and coconut oil. Hydrolysis is adversely affected by hydrocarbon\\u000a solvents and a nonionic surfactant. Since amounts of
Determinants within the C-Terminal Domain of Streptomyces lividans Acetyl-CoA Synthetase that Block Acetylation of Its Active Site Lysine In Vitro by the Protein Acetyltransferase (Pat) Enzyme
Tucker, Alex C.; Escalante-Semerena, Jorge C.
Reversible lysine acetylation (RLA) is a widespread regulatory mechanism that modulates the function of proteins involved in diverse cellular processes. A strong case has been made for RLA control exerted by homologues of the Salmonella enterica protein acetyltransferase (SePat) enzyme on the broadly distributed AMP-forming CoA ligase (a.k.a. acyl-CoA synthetases) family of metabolic enzymes, with acetyl-CoA synthetase (Acs) being the paradigm in the field. Here we investigate why the Acs homologue in Streptomyces lividans (SlAcs) is poorly acetylated in vitro by the S. lividans protein acetyltransferase (SlPat) enzyme. Chimeras of S. enterica Acs (SeAcs) and S. lividans Acs (SlAcs) constructed during the course of this work were acetylated by SlPatA in vitro, retained most of their activity, and were under RLA control in a heterologous host. We identified SeAcs residues N- and C-terminal to the target lysine that when introduced into SlAcs, rendered the latter under RLA control. These results lend further support to the idea that Pat enzymes interact with extensive surfaces of their substrates. Finally, we suggest that acetylation of SlAcs depends on factors or conditions other than those present in our in vitro system. We also discuss possible explanations why SlAcs is not controlled by RLA as defined in other bacterial species. PMID:24918787
Cellular/Molecular Acetylation of Microtubules Influences Their Sensitivity
Baas, Peter W.
Cellular/Molecular Acetylation of Microtubules Influences Their Sensitivity to Severing by Katanin of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression
Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase.
Matsumoto, Ken'ichiro; Okei, Takehiro; Honma, Inori; Ooi, Toshihiko; Aoki, Hirobumi; Taguchi, Seiichi
(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by ?-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g?L(-1) (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g?L(-1), with a productivity of 0.22 g?L(-1) h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of (13)C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB. PMID:22592551
A Method to Determine Lysine Acetylation Stoichiometries
Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al
Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore »butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less
Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding.
Ahmad, P M; Feltman, D S; Ahmad, F
A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4'-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity. PMID:7103949
Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra.
Steghens, J P; Min, K L; Bernengo, J C
A laboratory-made spectroluminometer was used to analyse the light emitted by firefly (Photinus pyralis) luciferase reacting with several nucleotide derivatives. The analysis of the light emission in the presence of ATP or dATP provides some evidence that the enzyme has two nucleotide binding sites, each one leading to the formation of a complex emitting mainly at 575 nm (ATP) or 610 nm (dATP). AMP is able to displace dATP from the second site (610 nm) to the first one. Photoaffinity labelling of the second site by 8-azido-AMP gives similar results. The amplification effect of CoA and acetyl-CoA is also reconsidered according to this model. PMID:9806891
Efcient incorporation of CoA, NAD and FAD into RNA by in vitro transcription
Huang, Faqing
Ef®cient incorporation of CoA, NAD and FAD into RNA by in vitro transcription Faqing Huang has been developed to ef®ciently prepare RNA with coenzymes CoA, NAD and FAD covalently attached± and FAD±RNA, which may ®nd broad applications in generating coenzyme- utilizing ribozymes. In addition
Structural insights into rice straw pretreated by hot-compressed water in relation to enzymatic hydrolysis.
Yu, Guoce; Yano, Shinichi; Inoue, Hiroyuki; Inoue, Seiichi; Wang, Jianlong; Endo, Takashi
Pretreatment-induced structural alteration is critical in influencing the rate and extent of enzymatic saccharification of lignocellulosic biomass. The present work has investigated structural features of rice straw pretreated by hot-compressed water (HCW) from 140 to 240 °C for 10 or 30 min and enzymatic hydrolysis profiles of pretreated rice straw. Compositional profiles of pretreated rice straw were examined to offer the basis for structural changes. The wide-angle X-ray diffraction analysis revealed possible modification in crystalline microstructure of cellulose and the severity-dependent variation of crystallinity. The specific surface area (SSA) of pretreated samples was able to achieve more than 10-fold of that of the raw material and was in linear relationship with the removal of acetyl groups and xylan. The glucose yield by enzymatic hydrolysis of pretreated materials correlated linearly with the SSA increase and the dissolution of acetyl and xylan. A quantitatively intrinsic relationship was suggested to exist between enzymatic hydrolysis and the extraction of hemicellulose components in hydrothermally treated rice straw, and SSA was considered one important structural parameter signaling the efficiency of enzymatic digestibility in HCW-treated materials in which hemicellulose removal and lignin redistribution happened. PMID:25178420
The hydrolysis of pesticides that are sorbed to sterilized natural sediments has been investigated in aqueous systems at acid, neutral and alkaline pH's. The results show that the rate constants of pH independent ('neutral') hydrolyses are the same within experimental uncertainti...
Enzymatic hydrolysis of organic phosphorus
Technology Transfer Automated Retrieval System (TEKTRAN)
Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...
Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.
Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R; Hoppel, Charles
Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial ?-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins. PMID:25660059
Acetylation inactivates the transcriptional repressor BCL6
Oksana R. Bereshchenko; Wei Gu; Riccardo Dalla-Favera
The proto-oncogene BCL6 encodes a BTB\\/POZ-zinc finger transcriptional repressor that is necessary for germinal-center formation and has been implicated in the pathogenesis of B-cell lymphomas. Here we show that the co-activator p300 binds and acetylates BCL6 in vivo and inhibits its function. Acetylation disrupts the ability of BCL6 to recruit histone deacetylases (HDACs), thereby hindering its capacity to repress transcription
Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation
Bryson, Bryan D.; White, Forest M.
Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine acetylation profiles for particular biological states, but none to date have investigated the temporal dynamics regulating cellular response to perturbation. Reasoning that lysine acetylation may be altered in response to growth factors, we implemented quantitative mass spectrometry-based proteomics to investigate the temporal dynamics of lysine acetylation in response to growth factor stimulation in cultured carcinoma cell lines. We found that lysine acetylation changed rapidly in response to activation of several different receptor tyrosine kinases by their respective ligands. To uncover the effects of lysine acetylation dynamics on tyrosine phosphorylation signaling networks, cells were treated with an HDAC inhibitor. This short-term pharmacological inhibition of histone deacetylase activity modulated signaling networks involving phosphorylated tyrosine and thereby altered the response to receptor tyrosine kinase activation. This result highlights the interconnectivity of lysine acetylation and tyrosine phosphorylation signaling networks and suggests that HDAC inhibition may influence cellular responses by affecting both types of post-translational modifications. PMID:25978619
Cellulase hydrolysis of unsorted MSW.
Jensen, Jacob Wagner; Felby, Claus; Jřrgensen, Henning
A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition of surfactant. Concurrently, the enzyme performance was analysed on pure cellulose in a solution of MSW wastewater. Results showed no effect of surfactant addition to the hydrolysis media as measured by viscosity and particle size distribution. MSW treatment wastewater was found to contain a high amount of calcium, potassium, sodium, chloride and others that may affect cellulolytic enzymes. Cellulase performance showed no effect of adding the metal ion-chelating agent EDTA to the solution. The cellulases were stable, tolerated and functioned in the presence of several contaminants. PMID:21989799
An acetyl group deficit limits mitochondrial ATP production at the onset of exercise.
Greenhaff, Paul L; Campbell-O'Sullivan, S P; Constantin-Teodosiu, D; Poucher, S M; Roberts, P A; Timmons, J A
The oxygen deficit at the onset of submaximal exercise represents a period when the energy demand of contraction cannot be met solely by mitochondrial ATP generation, and as a consequence there is an acceleration of ATP re-synthesis from oxygen-independent routes (phosphocreatine hydrolysis and glycolysis). Historically, the origin of the oxygen deficit has been attributed to a lag in muscle blood flow and oxygen availability at the onset of exercise which limits mitochondrial respiration. However, more recent evidence suggests that considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that pharmacological activation of the pyruvate dehydrogenase complex (and consequent stockpiling of acetyl groups), using dichloroacetate or exercise interventions, can markedly reduce the degree of ATP re-synthesis from oxygen-independent routes during the rest-to-work transition period. This review will focus on these findings, and will offer the hypothesis that acetyl group delivery to the tricarboxylic acid cycle limits mitochondrial flux at the onset of exercise--the so-called acetyl group deficit. PMID:12023864
Hydrolysis of imidazole-2-ylidenes.
Hollóczki, Oldamur; Terleczky, Péter; Szieberth, Dénes; Mourgas, Georgios; Gudat, Dietrich; Nyulászi, László
The direct reaction of an imidazole-2-ylidene in a predominantly aqueous environment [about 0.1 M solution in a H(2)O (>60%)/THF solvent system] was investigated for the first time. The reaction yielded a stable solution of the corresponding imidazolium-hydroxide of pH 13, which is in agreement with results from an ab initio molecular dynamics simulation. In contrast, hydrolysis of the carbene in a mainly aprotic environment (>80% THF) gives a hydrogen-bridged carbene-water complex which could be detected by NMR and IR spectroscopies for the first time. This complex converts slowly to two isomeric ring opened products and is at higher water concentration in dynamic equilibrium with the imidazolium hydroxide. A computational mechanistic study of the carbene hydrolysis with a gradually increasing number of water molecules revealed that the imidazolium-hydroxide structure can only be optimized with three or more water molecules as reactants, and with the increasing number of water molecules its stability is increasing with respect to the carbene-water complex. In agreement with the experimental results, these findings point out that solvent stabilization and basicity of the hydroxide ion plays a crucial role in the reaction. With increasing number of water molecules the barriers connecting the reaction intermediates are getting smaller, and the ring opened hydrolysis products can be derived from imidazolium-hydroxide type intermediates. Computational studies on the hydrolysis of a nonaromatic imidazolidine-2-ylidene analogue clearly indicated the analogous ring-opened product to be by 10-12 kcal/mol more stable than the appropriate ion pair and the carbene-water complex, in agreement with the known aromatic stabilization of imidazol-2-ylidenes. Accordingly, these molecules hydrolyze with exclusive formation of the ring-opened product. PMID:21174475
A Jojoba P-Ketoacyl-COA Synthase cDNA Complements the Canola Fatty Acid Elongation Mutation in Transgenic Plants
Michael W. Lassner; Kathryn Lardizabal; James G. Metz
p-Ketoacyl-coenzyme A (COA) synthase (KCS) catalyzes the condensation of malonyl-COA with long-chain acyl-COA. This reaction is the initial step of the microsomal fatty acyl-COA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths >18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of
Mechanistic Insight with HBCH2CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases
Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 ?M, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [3H]-saturated trimer-CoA ([3H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [3H]-sTCoA ([3H]-sT-CH2-CoA), saturated dimer-([3H]-sD-CO2H), and trimer-acid ([3H]-sT-CO2H), distinct from the expected methylene analogue of [3H]-saturated tetramer-CoA ([3H]-sTet-CH2-CoA). Detection of [3H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation. PMID:24896226
Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula.
Zhou, Rui; Jackson, Lisa; Shadle, Gail; Nakashima, Jin; Temple, Stephen; Chen, Fang; Dixon, Richard A
Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase. PMID:20876124
Acetyl-L-carnitine in hepatic encephalopathy.
Malaguarnera, Michele
Hepatic encephalopathy is a common complication of hepatic cirrhosis. The clinical diagnosis is based on two concurrent types of symptoms: impaired mental status and impaired neuromotor function. Impaired mental status is characterized by deterioration in mental status with psychomotor dysfunction, impaired memory, and increased reaction time, sensory abnormalities, poor concentration, disorientation and coma. Impaired neuromotor function include hyperreflexia, rigidity, myoclonus and asterixis. The pathogenesis of hepatic encephalopathy has not been clearly defined. The general consensus is that elevated levels of ammonia and an inflammatory response work in synergy to cause astrocyte to swell and fluid to accumulate in the brain which is thought to explain the symptoms of hepatic encephalopathy. Acetyl-L-carnitine, the short-chain ester of carnitine is endogenously produced within mitochondria and peroxisomes and is involved in the transport of acetyl-moieties across the membranes of these organelles. Acetyl-L-carnitine administration has shown the recovery of neuropsychological activities related to attention/concentration, visual scanning and tracking, psychomotor speed and mental flexibility, language short-term memory, attention, and computing ability. In fact, Acetyl-L-carnitine induces ureagenesis leading to decreased blood and brain ammonia levels. Acetyl-L-carnitine treatment decreases the severity of mental and physical fatigue, depression cognitive impairment and improves health-related quality of life. The aim of this review was to provide an explanation on the possible toxic effects of ammonia in HE and evaluate the potential clinical benefits of ALC. PMID:23389620
Acetylation Is Indispensable for p53 Activation
Tang, Yi; Zhao, Wenhui; Chen, Yue; Zhao, Yingming; Gu, Wei
SUMMARY The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. We have now identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. Our study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response. PMID:18485870
The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification.
Laskar, Dhrubojyoti D; Jourdes, Michaël; Patten, Ann M; Helms, Gregory L; Davin, Laurence B; Lewis, Norman G
Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated. PMID:17092316
Inhibition of excitatory amino acid-induced phosphoinositide hydrolysis as a possible mechanism of nitroprusside neurotoxicity.
Yu, O; Chuang, D M
Inclusion of sodium nitroprusside (Na2[Fe(2+)-(CN)5NO]) into the culture medium is toxic to cultured rat cerebellar granule neurons. A possible underlying mechanism may be the inhibition of phosphoinositide (PI) response to excitatory amino acids (EAAs) because activation of glutamate receptors can be neuroprotective and neurotrophic in differentiating neurons. Sodium nitroprusside selectively inhibited the PI response to EAAs (NMDA > glutamate = quisqualate > kainate) without affecting that to carbachol or KCl. In contrast, S-nitroso-N-acetyl-penicillamine (SNAP), another nitric oxide (NO) donor, potentiated NMDA-induced PI hydrolysis. Hemoglobin reversed the effects of nitroprusside and SNAP. However, NO may not be involved because NO solution was without effect and N-acetylpenicillamine, a SNAP analogue that does not contain a NO moiety, also potentiated NMDA-induced PI hydrolysis in a hemoglobin-sensitive manner. Furthermore, the metabolites of NO (nitrate and nitrite), L-arginine, reduced glutathione, 8-bromo-cyclic guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), and atrial natriuretic peptide, which accelerates the production of cGMP independent of NO, were ineffective as modulators. However, potassium ferrocyanide (K4[Fe2+(CN)6]), but not potassium ferricyanide (K3[Fe3+(CN)6]), inhibited NMDA-induced PI hydrolysis as effectively as nitroprusside, but this inhibition was not reversed by hemoglobin. Cyanide, a product from the disintegration of nitroprusside, potentiated rather than inhibited NMDA-induced PI hydrolysis. Taken together, these results suggest that the parent molecule itself, nitroprusside, contributes primarily in inhibiting EAA-induced PI hydrolysis. Inhibition of EAA-induced PI hydrolysis may in part mediate the mechanisms of nitroprusside toxicity in primary cultures of differentiating cerebellar granule neurons. PMID:8522973
Acetylation of bleached Kraft pulp: effect of xylan content on properties of acetylated compounds.
Peredo, Karol; Reyes, Herna; Escobar, Danilo; Vega-Lara, Johana; Berg, Alex; Pereira, Miguel
Bleached Kraft pulp (BKP) from Eucalyptus globulus and cotton xylan blends (CXB) was acetylated. The effects of xylan content on cellulose acetylation and the properties of the acetylated material were studied. An increase in xylan content caused a slight decrease in the degree of substitution (2.98 to 2.68 for CXB; 2.93 to 2.84 for BKP). Thermal analysis showed that the melting temperature also decreases from 268.0 to 188.8 °C for CXB and from 221.4 to 212.8 °C for BKP. Moreover, the solubility decreased due to the partial dissolution of acetylated xylans. The presence of xylans during Kraft pulp acetylation does not have a significant negative effect on the physical properties of the acetylated material, but the decrease in melting temperature was beneficial for the application of acetylated polymer as a natural internal plasticizer. This is considered to be an important argument for BKP utilization in the cellulose acetate manufacturing process. PMID:25498729
3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients
Wang, S.P.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Quebec (Canada)] [and others] [Hopital Sainte-Justine, Quebec (Canada); and others
3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 126.96.36.199) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.
Hydrolytic processes provide the baseline loss rate for any chemical in an aqueous envi- ronment. Although various hydrolytic pathways account for significant degradation of certain classes of organic chemicals, other organic structures are completely inert. Strictly speaking, hy...
Discovery and characterization of Ku acetylation in Mycobacterium smegmatis.
Zhou, Ying; Chen, Tao; Zhou, Lin; Fleming, Joy; Deng, Jiaoyu; Wang, Xude; Wang, Liwei; Wang, Yingying; Zhang, Xiaoli; Wei, Wenjing; Bi, Lijun
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. PMID:25767122
Temporal analysis of protein lysine acetylation during adipocyte differentiation
Xu, Zuyuan; Ande, Sudharsana Rao; Mishra, Suresh
The post-translational modification of protein by acetylation has been emerging as a prevalent modification in enzymes that catalyze intermediary metabolism. However, the dynamics of protein acetylation during adipocyte differentiation that involves a major shift in cellular metabolism is not known. In this study, we investigated the temporal changes in acetylation during adipocyte differentiation. Almost all acetylated proteins identified showed a sequential change in acetylation during the differentiation process. While the majority of the acetylated proteins showed a sequential upregulation during adipocyte differentiation, in a few proteins a sequential downregulation of protein acetylation was also observed. Our findings suggest that a wide-ranging temporal change in protein acetylation occurs during adipocyte differentiation including differentially expressed proteins signifying an important role in adipocyte differentiation. PMID:23700550
Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.
Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj
Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well. PMID:20512516
Gene encoding acetyl-coenzyme A carboxylase
Roessler, P.G.; Ohlrogge, J.B.
A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.
Isolation, purification and structural characterization of an acetylated heteroglycan from the unripe fruits of Manilkara zapota L.
Mondal, Subhas; Das, Debsankar; Roy, Sadhan K; Islam, Syed S
A water soluble polysaccharide isolated from the hot water extract of the unripe fruits of Manilkara zapota L. was found to consist of 3-O-acyl-L-rhamnose, L-arabinose, 3-O-acetyl-D-methyl galacturonate in a molar proportion of nearly 1:1:1. Structural investigation of the polysaccharide was carried out using total hydrolysis, methylation analysis; periodate oxidation followed by GLC-MS, and NMR experiments. On the basis of the above experiments it is concluded that the following repeating unit is present in the polysaccharide. PMID:22560630
Technology Transfer Automated Retrieval System (TEKTRAN)
The classic wheat evolutionary history is one of adaptive radiation of the diploid Triticum/Aegilops species (A, S, D) , genome convergence and divergence of the tetraploid (T. turgidum AABB, and T. timopheevii AAAGG) and hexaploid (T. aestivum, AABBDD) species. The objective of this study was to a...
Retarded hydrolysis-condensing reactivity of tetrabutyl titanate by acetylacetone and the application in dye-sensitized solar cells
Zhou, Conghua, E-mail: firstname.lastname@example.com; Ouyang, Jun; Yang, Bingchu
Graphical abstract: - Highlights: • Effect of acetone acetyl on coarsening rate of TiO{sub 2} nanocrystallites was studied. • Hydrolysis reactivity of alkoxide was retarded with addition of acetone acetyl. • Coarsening rate of TiO{sub 2} nanocrystallites is retarded with addition of acetone acetyl. • The synthesized TiO{sub 2} sols were utilized in dye sensitized solar cells. • Small particles formed by Ti-complexes were beneficial for device performance. - Abstract: TiO{sub 2} nanocrystallites have been synthesized by hydrothermal reaction using tetrabutyl titanate as source material. Acetylacetone was utilized to modify hydrolysis-condensation behavior of the alkoxide and thus coarsening dynamics of TiO{sub 2} nanocrystallites in the reaction. With assistance of Fourier transformation infrared spectrum, transmission electron microscopy, selected area electron diffraction and X-ray diffraction, interaction between acetylacetone and tetrabutyltitanate was explored, crystallographic and morphological properties of TiO{sub 2} nanocrystallites were monitored. Less hydrolysable complex was formed by "method of chelating" as tetrabutyltitanate was mixed with acetylacetone, leading to retarded coarsening rate of nanocrystallites. The obtained TiO{sub 2} nanocrystallites were applied to fabricate nanoporous photoanode of dye sensitized solar cells. Improvement of 18% has been achieved for photo-to-electric energy conversion efficiency of the devices due to both upgraded open circuit voltage and photocurrent density.
Determination of the Barrier Height for Acetyl Radical Dissociation from Acetyl Chloride Photodissociation at 235 nm Using Velocity Map Imaging
Butler, Laurie J.
Determination of the Barrier Height for Acetyl Radical Dissociation from Acetyl Chloride velocity map imaging to determine the barrier height for acetyl radical, CH3CO, dissociation to CH3 + CO at a translational energy of 25.0 ( 0.4 kcal/mol. From this onset we can calculate the barrier height for CH3CO f CH3
Ethanol from biomass by enzymatic hydrolysis
Enzymes are biological catalysts that generally are designed to do one job well, but to do one job only. Therefore, the enzymes that catalyze the hydrolysis of cellulose to sugar do not break down the sugars. Thus, enzymatic processes are capable of yields approaching 100%. Enzymatic hydrolysis processes have been under development for only 10 years. Although improvements have been
Dilute acid hydrolysis of lignocellulosic biomass
P. Lenihan; A. Orozco; E. O'Neill; M. N. M. Ahmad; D. W. Rooney; G. M. Walker
The overall aim of this work was to establish the optimum conditions for acid hydrolysis of hemicellulosic biomass in the form of potato peel. The hydrolysis reaction was undertaken in a 1l high pressure pilot batch reactor using dilute phosphoric acid. Analysis of the decomposition rate of hemicellulosic biomass (namely Cellulose, Hemicellulose and lignin) was undertaken using HPLC of the
Alkaline hydrolysis rate constants were measured for dimethyl, diethyl, di-n-butyl, di-iso-butyl, and di-(2-ethylhexyl) phthalate esters in water. A linear free energy relationship (LFER) was established for estimating alkaline hydrolysis rate constants for other phthalate esters...
Technology Transfer Automated Retrieval System (TEKTRAN)
Many lipases display some degree of sn-1, sn-3 selectivity toward acyl groups linked to the glycerol backbone. We have found conditions which provide selective hydrolysis of acyl chains from the triacylglycerol. These conditions depend on the lipase itself, the solvent in which hydrolysis or alcohol...
Bromodomains shake the hegemony of pan-acetyl antibodies
Champleboux, Morgane; Govin, Jérôme
Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan-acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch-to-batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470–1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes. PMID:25825234
Acetylation and characterization of banana (Musa paradisiaca) starch.
Bello-Pérez, L A; Contreras-Ramos, S M; Jěmenez-Aparicio, A; Paredes-López, O
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. PMID:11265448
Factors affecting hydrolysis of condensed phosphates in soils
Stewart, William M.
under the various treatments LIST OF FIGURES FIGURE Page I Effect of soil temperature on hydrolysis of pyrophosphate 2 Effect of soil pH on hydrolysis of pyrophosphate 30 3 Pyrophosphate hydrolysis plotted accot. ding to first order kinetics 34... 4 Effect of soil temperature on hydrolysis of tripolyphosphate . . . . . . . ~ . ~ ~ 37 5 Effect of soil pH on hydrolysis of tripolyphosphate 38 6 Tripolyphosphate hydrolysis plotted according to first order kinetics 4l INTRODUCTION Phosphorous...
COA's Barney Bright Sculpture In 1964, Louisville sculptor Jeptha Barnard "Barney" Bright was
Hayes, Jane E.
COA's Barney Bright Sculpture In 1964, Louisville sculptor Jeptha Barnard "Barney" Bright was commissioned to create a sculpture for the University of Kentucky's College of Agriculture. Presumably, Bright of Barney Bright's first commissioned sculptures. The untitled piece features three intertwining abstract
Research Publishing and Outreach Contact: email@example.com, 541-737-3295, Burt 128
Kurapov, Alexander
Research Publishing and Outreach Contact: firstname.lastname@example.com, 541-737-3295, Burt 128 a proposal budget is approved by Accounting, Publishing can help copyedit, proofread project descriptions. If you have a news story, contact Publishing or write directly to Mark Floyd (Mark
Technology Transfer Automated Retrieval System (TEKTRAN)
Several transcription factors are involved in regulating lipid metabolism in various animal tissues. Peroxisome proliferator activated receptor (PPAR) gamma and PPAR alpha regulate both lipogenesis and fatty acid oxidation. Gene fragments for PPAR gamma, PPAR alpha, and acyl CoA oxidase (ACO) have b...
A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans
Svetlitchnyi, Vitali; Dobbek, Holger; Meyer-Klaucke, Wolfram; Meins, Thomas; Thiele, Bärbel; Römer, Piero; Huber, Robert; Meyer, Ortwin
In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACSMt/CODHMt from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACSMt, and a zink-nickel site in the closed form. The ACSCh of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIIICh. Homogenous ACSCh and ACSCh/CODHIIICh catalyzed the exchange between [1-14C]acetyl-CoA and 12CO with specific activities of 2.4 or 5.9 ?mol of CO per min per mg, respectively, at 70°C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 ?mol of acetyl-CoA formed per min per mg, respectively, at 70°C and pH 7.3. The functional cluster A of ACSCh contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACSCh, a 2.2-Ĺ crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy. PMID:14699043
A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans.
Svetlitchnyi, Vitali; Dobbek, Holger; Meyer-Klaucke, Wolfram; Meins, Thomas; Thiele, Bärbel; Römer, Piero; Huber, Robert; Meyer, Ortwin
In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS(Mt)/CODH(Mt) from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS(Mt), and a zink-nickel site in the closed form. The ACS(Ch) of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII(Ch). Homogeneous ACS(Ch) and ACS(Ch)/CODHIII(Ch) catalyzed the exchange between [1-(14)C]acetyl-CoA and (12)CO with specific activities of 2.4 or 5.9 micromol of CO per min per mg, respectively, at 70 degrees C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 micromol of acetyl-CoA formed per min per mg, respectively, at 70 degrees C and pH 7.3. The functional cluster A of ACS(Ch) contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS(Ch), a 2.2-A crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy. PMID:14699043
Restricted mitochondrial protein acetylation initiates mitochondrial autophagy
Webster, Bradley R.; Scott, Iain; Han, Kim; Li, Jian H.; Lu, Zhongping; Stevens, Mark V.; Malide, Daniela; Chen, Yong; Samsel, Leigh; Connelly, Patricia S.; Daniels, Mathew P.; McCoy, J. Philip; Combs, Christian A.; Gucek, Marjan; Sack, Michael N.
Summary Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced 'stress-resilience'. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects. PMID:24006259
Pds5 promotes and protects cohesin acetylation
Chan, Kok-Lung; Gligoris, Thomas; Upcher, William; Kato, Yuki; Shirahige, Katsuhiko; Nasmyth, Kim; Beckouët, Frédéric
Cohesin's Smc1 and Smc3 subunits form V-shaped heterodimers, the nucleotide binding domains (NBDs) of which bind the C- and N-terminal domains, respectively, of the ?-kleisin subunit, forming a large tripartite ring within in which sister DNAs are entrapped, and thereby held together (sister chromatid cohesion). During replication, establishment of stable cohesion is dependent on Eco1-mediated acetylation of Smc3's NBD, which is thought to prevent dissociation of ?-kleisin from Smc3, thereby locking shut a "DNA exit gate." How Scc3 and Pds5, regulatory subunits bound to ?-kleisin, regulate cohesion establishment and maintenance is poorly understood. We show here that by binding to ?-kleisin adjacent to its Smc3 nucleotide binding N-terminal domain, Pds5 not only promotes cohesin's release from chromatin but also mediates de novo acetylation of Smc3 by Eco1 during S phase and subsequently prevents de-acetylation by the deacetylase Hos1/HDAC8. By first promoting cohesin's release from chromosomes and subsequently creating and guarding the chemical modification responsible for blocking release, Pds5 enables chromosomal cohesin to switch during S phase from a state of high turnover to one capable of tenaciously holding sister chromatids together for extended periods of time, a duality that has hitherto complicated analysis of this versatile cohesin subunit. PMID:23878248
N-acetyl-l-histidine, a Prominent Biomolecule in Brain and Eye of Poikilothermic Vertebrates
Baslow, Morris H.; Guilfoyle, David N.
N-acetyl-l-histidine (NAH) is a prominent biomolecule in brain, retina and lens of poikilothermic vertebrates. In fish lens, NAH exhibits an unusual compartmentalized metabolism. It is synthesized from l-histidine (His) and acetyl Co-enzyme A. However, NAH cannot be catabolized by lens cells. For its hydrolysis, NAH is exported to ocular fluid where a specific acylase cleaves His which is then actively taken up by lens and re-synthesized into NAH. This energy-dependent cycling suggested a pump mechanism operating at the lens/ocular fluid interface. Additional studies led to the hypothesis that NAH functioned as a molecular water pump (MWP) to maintain a highly dehydrated lens and avoid cataract formation. In this process, each NAH molecule released to ocular fluid down its gradient carries with it 33 molecules of bound water, effectively transporting the water against a water gradient. In ocular fluid the bound water is released for removal from the eye by the action of NAH acylase. In this paper, we demonstrate for the first time the identification of NAH in fish brain using proton magnetic resonance spectroscopy (MRS) and describe recent evidence supporting the NAH MWP hypothesis. Using MRS, we also document a phylogenetic transition in brain metabolism between poikilothermic and homeothermic vertebrates. PMID:25919898
Expression of an acetyl-CoA synthase and a CoA-transferase in Escherichia coli to produce modified taxanes in vivo.
Loncaric, Catherine; Ward, Amanda F; Walker, Kevin D
Previous in vitro studies revealed that the 10-deacetylbaccatin III 10beta-O-acetyltransferase (DBAT) from Taxus can catalyze the transfer of acetyl, propionyl or n-butyryl from CoA to the C10-hydroxyl of 10-deacetylbaccatin III. Accordingly, Escherichia coli JM109 were transformed to recombinantly express dbat, and this enzyme function was coupled to that of acetyl-CoA synthase (acs, EC 188.8.131.52) expressed from and regulated by genes encoded on the bacterial chromosome. Incubation of the bacteria with 10-deacetylbaccatin III and increasing concentrations of acetic acid revealed an in vivo conversion ( approximately 10%) of substrate to natural product baccatin III (C10-acetylated), which was remarkably similar to the relative conversion without acid supplementation. Incubation of the modified E. coli with 5 mM propionic acid, revealed a fivefold increase in the conversion ( approximately 13%) of 10-deacetylbaccatin III to 10-deacetyl-10-propionylbaccatin III, compared to approximately 2% conversion in the absence of exogenous propionate. To produce the butyrylbaccatin III analog in vivo, bacteria were engineered to co-express the dbat and atoAD (EC 126.96.36.199) genes; the latter encodes an acetoacetate: acetyl-CoA CoA-transferase that activates butyrate to butyryl CoA. The bacteria were incubated with 10-deacetylbaccatin III and 25-100 mM butyrate, and a maximum of approximately 2.6% conversion to 10-butyrylbaccatin III was observed compared to approximately 0.6% conversion when no exogenous butyrate was supplied. PMID:17183509
Continuous steam hydrolysis of tulip poplar
Fieber, C.A.; Roberts, R.S.; Faass, G.S.; Muzzy, J.D.; Colcord, A.R.; Bery, M.K.
The continuous hydrolysis of poplar chips by steam at 300-350 psi resulted in the separation of hemicellulose (I) cellulose and lignin components. The I fraction was readily depolymerised by steam to acetic acid, furfural, methanol, and xylose.
Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency.
Ghosh, Alok; Trivedi, Prachi P; Timbalia, Shrishiv A; Griffin, Aaron T; Rahn, Jennifer J; Chan, Sherine S L; Gohil, Vishal M
Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx?CxnCx??C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6? cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx?CxnCx??C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6? cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. PMID:24549041
Enzymatic hydrolysis of low substituted carboxymethyl cellulose
Chanona Dominquez, Guadalupe
ENZYMATIC HYDROLYSIS OF LOW SUBSTITUTED CARBOXYMETHYL CELLULOSE A Thesis by GUADALUPE CHANONA DOMINGUEZ Submitted to the Graduate College of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE... December 1984 Major Subject: Agricultural Engineering ENZYMATIC HYDROLYSIS OF LOW SUBSTITUTED CARBOXYMETHYL CELLULOSE A Thesis by GUADALUPE CHANONA DOMINGUEZ Approved as to style and content by: Cady R. Engler airman) Ed. J. Soltes (Member...
Lactose Utilization and Hydrolysis in Saccharomyces fragilis
SUMMARY Sodium azide, 2,4-dinitrophenol and iodoacetate did not inhibit hydrolysis of lactose by cell-free preparations of Sacchuromyces fragilis ,8-galactosidase, but with intact organisms fermentation and hydrolysis were inhibited to a similar extent. This suggests that these inhibitors may interfere with the transport of lactose into the cell. Galactose fermentation was inhibited by sodium azide and dinitrophenol to a much greater
Hydrolysis of steam-pretreated lignocellulose
Johan Karlsson; József Medve; Folke Tjerneld
The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose.\\u000a Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures.\\u000a Adsorption isotherms were determined at 4
Selective Acetylation of per-O-TMS-Protected Monosaccharides
Witschi, Mark A.
Selective acetylation of various per-O-TMS-protected carbohydrates has been accomplished. Using a protecting group exchange strategy and microwave assistance, monosaccharides (glucose, galactose and mannose) can be selectively acetylated producing either the 6-O-monoacetate or 1,6-O-diacetylated species. This new class of molecules can be deprotected without migration of the acetyl groups providing useful synthetic intermediates. To demonstrate the scope of the reaction, the methodology was successfully extended to TMS-protected ceramide. PMID:20799705
Physical properties of chemically acetylated rat liver chromatin.
Wallace, R B; Sargent, T D; Murphy, R F; Bonner, J
The physical properties of rat liver chromatin and nucleosomes acetylated with acetic anhydride were examined in order to clarify the mechanism by which chemical acetylation of histones increases template activity in vitro [Marushige, K. (1976) Proc. Natl. Acad. Sci. USA 73, 3937-3941]. Acetylation was found to have dramatic effects on the magnesium solubility, nuclease sensitivity, thermal denaturation, and sedimentation of chromatin and nucleosomes. The significance of the results to models of gene activation and chromatin replication is considered. Images PMID:269387
Differential patterns of histone acetylation in inflammatory bowel diseases
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. PMID:21272292
Structure, morphology and functionality of acetylated and oxidised barley starches.
El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreńo, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa
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. PMID:25172707
Role of transcription factor acetylation in diabetic kidney disease.
Liu, Ruijie; Zhong, Yifei; Li, Xuezhu; Chen, Haibing; Jim, Belinda; Zhou, Ming-Ming; Chuang, Peter Y; He, John Cijiang
Nuclear factor (NF)-?B and signal transducer and activator of transcription 3 (STAT3) play a critical role in diabetic nephropathy (DN). Sirtuin-1 (SIRT1) regulates transcriptional activation of target genes through protein deacetylation. Here, we determined the roles of Sirt1 and the effect of NF-?B (p65) and STAT3 acetylation in DN. We found that acetylation of p65 and STAT3 was increased in both mouse and human diabetic kidneys. In human podocytes, advanced glycation end products (AGEs) induced p65 and STAT3 acetylation and overexpression of acetylation-incompetent mutants of p65 and STAT3 abrogated AGE-induced expression of NF-?B and STAT3 target genes. Inhibition of AGE formation in db/db mice by pyridoxamine treatment attenuated proteinuria and podocyte injury, restored SIRT1 expression, and reduced p65 and STAT3 acetylation. Diabetic db/db mice with conditional deletion of SIRT1 in podocytes developed more proteinuria, kidney injury, and acetylation of p65 and STAT3 compared with db/db mice without SIRT1 deletion. Treatment of db/db mice with a bromodomain and extraterminal (BET)-specific bromodomain inhibitor (MS417) which blocks acetylation-mediated association of p65 and STAT3 with BET proteins, attenuated proteinuria, and kidney injury. Our findings strongly support a critical role for p65 and STAT3 acetylation in DN. Targeting protein acetylation could be a potential new therapy for DN. PMID:24608443
Gelation behavior of native and acetylated konjac glucomannan.
Huang, Long; Takahashi, Rheo; Kobayashi, Shinsaku; Kawase, Tokuzo; Nishinari, Katsuyoshi
Gelation kinetics of native and acetylated konjac glucomannan (KGM) samples in the presence of alkali (sodium carbonate) was studied by dynamic viscoelastic measurements. Molecular weight and other molecular parameters of KGM were determined by static light scattering and viscosity measurements. It was found that KGM molecules were degraded during acetylation treatment, but the molecular weights of acetylated samples were almost independent of the degree of acetylation (DA) and were about a half of that of a native sample. At a fixed alkaline concentration, increasing concentration of KGM or temperature shortened the gelation time, but increasing DA delayed it. The deacetylation reaction and subsequent aggregation process of acetylated samples needed longer time than that of native sample, and acetylated samples formed finally more elastic gels. It implied that the presence of acetyl groups exerts a strong influence on gelation behavior of KGM. It was suggested that the gelation rate of acetylated KGM and native KGM, which depends on the alkaline concentration and temperature, is an important factor that determines the elastic modulus of gels. This was supported by the experimental finding that the saturated elastic modulus tends to the same value when the ratio of alkali concentration to acetylated groups was kept constant. In slower gelation processes, junction zones are more homogeneously distributed and more numerous, leading to the more elastic gels. PMID:12425668
Upregulation of Endothelial Nitric Oxide Synthase by HMG CoA Reductase Inhibitors
Ulrich Laufs; Vito La Fata; Jorge Plutzky; James K. Liao
Background—Oxidized low-density lipoprotein (ox-LDL) causes endothelial dysfunction in part by decreasing the availability of endothelial nitric oxide (NO). Although HMG CoA reductase inhibitors restore endothelial function by reducing serum cholesterol levels, it is not known whether they can also directly upregulate endothelial NO synthase (ecNOS) activity. Methods and Results—Human saphenous vein endothelial cells were treated with ox-LDL (50 mg\\/mL thiobarbituric
Regulation of schistosome egg production by HMG CoA reductase
VandeWaa, E.A.; Bennett, J.L.
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 /sup 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.
Down-regulation of autoreactive T-cells by HMG CoA reductase inhibitors
Teodor-D. Brumeanu; Robert Goldstein; Sofia Casares
The inhibitors of HMG CoA reductase (statins) are widely used as cholesterol-lowering drugs with excellent safety records in hypercholesterolemic patients. Statins exert pleiotropic effects on a variety of cells, and they were recently described as a new class of immune modulators. Depending on their structure, dose, and route of administration, statins regulate the function of both the antigen-presenting cells and
Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation
Burrier, R.E.; Manson, C.R.; Brecher, P.
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.
New Targets for Acetylation in Autophagy
NSDL National Science Digital Library
Ahmed Hamai (Paris; INSERM REV)
Macroautophagy is an evolutionarily conserved homeostatic process that mediates the degradation of long-lived cytoplasmic components in eukaryotes, which allows cells to survive stresses such as inflammation, hypoxia, and deprivation of nutrients or growth factors. At least 30 members of the Atg (autophagy-related) protein family orchestrate this degradative process. Additional complexity resides in the signaling networks controlling the autophagic process, which include various posttranslational modifications of key components. Evidence is accumulating that protein acetylation represents an evolutionarily conserved mechanism tightly regulating macroautophagy.
Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases
Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.
Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.
Formation of the thioester, N-acetyl, S-lactoylcysteine, by reaction of N-acetylcysteine with pyruvaldehyde in aqueous solution. [in prebiotic evolution
NASA Technical Reports Server (NTRS)
Weber, A. L.
N-acetylcysteine reacts efficiently with pyruvaldehyde (methylglyoxal) in aqueous solution (pH 7.0) in the presence of a weak base, like imidazole or phosphate, to give the thioester, N-acetyl, S-lactoylcysteine. Reactions of 100 mM N-acetylcysteine with 14 mM, 24 mM and 41 mM pyruvaldehyde yield, respectively, 86%, 76% and 59% N-acetyl, S-lactoylcysteine based on pyruvaldehyde. The decrease in the percent yield at higher pyruvaldehyde concentrations suggests that during its formation the thioester is not only consumed by hydrolysis, but also by reaction with some substance in the pyruvaldehyde preparation. Indeed, purified N-acetyl, S-lactoylcysteine disappears much more rapidly in the presence of pyruvaldehyde than in its absence. Presumably, N-acetyl, S-lactoylcysteine synthesis occurs by rearrangement of the hemithioacetal of N-acetylcysteine and pyruvaldehyde. The significance of this pathway of thioester formation to molecular evolution is discussed.
Study on Dendrobium officinale O-acetyl-glucomannan (Dendronan®): part II. Fine structures of O-acetylated residues.
Xing, Xiaohui; Cui, Steve W; Nie, Shaoping; Phillips, Glyn O; Goff, H Douglas; Wang, Qi
Main objective of this study was to investigate the detailed structural information about O-acetylated sugar residues in Dendronan(®). A water solution (2%, w/w) of Dendronan(®) was treated with endo-?-mannanase to produce oligosaccharides rich in O-acetylated sugar residues. The oligosaccharides were partly recovered by ethanol precipitation (70%, w/w). The recovered sample (designated Hydrolyzed Dendrobium officinale Polysaccharide, HDOP) had a yield of 24.7% based on the dry weight of Dendronan(®) and was highly O-acetylated. A D2O solution of HDOP (6%, w/w) generated strong signals in (1)H, (13)C, 2D (1)H-(1)H COSY, 2D (1)H-(1)H TOCSY, 2D (1)H-(1)H NOESY, 2D (1)H-(13)C HMQC, and 2D (1)H-(13)C HMBC NMR spectra. Results of NMR analyses showed that the majority of O-acetylated mannoses were mono-substituted with acetyl groups at O-2 or O-3 position. There were small amounts of mannose residues with di-O-acetyl substitution at both O-2 and O-3 positions. Minor levels of mannoses with 6-O-acetyl, 2,6-di-O-acetyl, and 3,6-di-O-acetyl substitutions were also identified. Much information about sugar residue sequence was extracted from 2D (1)H-(13)C HMBC and 2D (1)H-(1)H NOESY spectra. (1)J(C-H) coupling constants of major sugar residues were obtained. Evidences for the existence of branches or O-acetylated glucoses in HDOP were not found. The major structure of Dendronan(®) is shown as follows: [Formula: see text] M: ?-D-mannopyranose; G: ?-D-glucopyranose; a: O-acetyl group. PMID:25498655
A Biotin Analog Inhibits Acetyl-CoA Carboxylase
Stephens, Jacqueline
A Biotin Analog Inhibits Acetyl-CoA Carboxylase Activity and Adipogenesis* Received for publication the induction of PPAR , STAT1, and STAT5A expression that normally occurs with adipogenesis. Moreover, addition the course of adipogenesis the activities of several lipogenic enzymes such as acetyl-CoA car- boxylase
Diet, acetylator phenotype, and risk of colorectal neoplasia
I. C. Roberts-Thomson; K. K. Khoo; W. J. Hart; R. N. Butler; P. Ryan; A. J. McMichael
SummaryBackground Inherited or acquired differences in metabolic pathways that activate or inactivate dietary carcinogens may influence the risk of developing cancer. A polymorphism in N-acetyltransferase classifies people into fast and slow acetylators. This enzyme catalyses the formation of mutagenic products from foodstuffs, especially cooked meat and fish. Some data suggest that fast acetylators are at higher risk of colorectal cancer.
Ethanol from biomass by enzymatic hydrolysis
Wright, J.D.
Enzymes are biological catalysts that generally are designed to do one job well, but to do one job only. Therefore, the enzymes that catalyze the hydrolysis of cellulose to sugar do not break down the sugars. Thus, enzymatic processes are capable of yields approaching 100%. Enzymatic hydrolysis processes have been under development for only 10 years. Although improvements have been made in enzymatic technology, more are both possible and necessary. The important research issues include understanding the processes necessary to render the crystalline cellulose easily digestible, understanding and improving the basic mechanisms in the hydrolysis step, and developing better and less expensive enzymes. The hemicellulose fraction (25%) is primarily composed of xylan, which is simple to convert to the simple sugar xylose, but the xylose is difficult to ferment to ethanol. There were no practical systems for xylose fermentation 10 years ago. Today, methods have been identified using new yeasts, fungi, bacteria, and processes combining enzymes and yeasts. Although none of the fermentations is yet ready for commercial use, considerable progress has been made. The following sections describe current research efforts in each of the major areas (cellulose hydrolysis, xylose fermentation, and lignin conversion), with an emphasis on enzymatic hydrolysis using fungal enzymes.
Semi-synthetic preparation of 1-O-(1'-/sup 14/C)hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures
Weber, N.; Mangold, H.K.
Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-(1'-/sup 14/C)hexadecyl-sn-glycerol or rac-1-O-(1'-/sup 14/C)hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-(1'-/sup 14/C)hexadecyl-sn-glycero-3-phosphocholine. 1-O-(1'-14C)Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.
Self-scrolling ability of differentially acetylated chitosan film.
Saito, Yukie; Luchnikov, Valeriy; Inaba, Ayano; Tamura, Katsuhito
Chitosan film cast on a glass slide was exposed to acetic anhydride vapor, resulting in an acetylation gradient in the film, with preferential acetylation of the exposed surface. The difference in degree of acetylation between the two surfaces of the peeled film was confirmed by attenuated total reflection infrared spectroscopy. Upon immersion of the film in water, differential swelling occurred because the more highly acetylated surface absorbed less water, and the resulting bending moment caused self-scrolling with the more highly acetylated surface inside. Simultaneous peeling and scrolling of films with a thickness of micrometer order, using dilute aqueous hydrofluoric acid, afforded tightly rolled chitosan microtubes. This simple self-scrolling mechanism is potentially applicable for micro-scale design with various naturally occurring polymers. PMID:24815399
A Novel Functional Site in the PB2 Subunit of Influenza A Virus Essential for Acetyl-CoA Interaction, RNA Polymerase Activity, and Viral Replication*
Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m7GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. PMID:25063805
Reversible acetylation regulates acetate and propionate metabolism in Mycobacterium smegmatis
Hayden, Jennifer D.; Brown, Lanisha R.; Gunawardena, Harsha P.; Perkowski, Ellen F.; Chen, Xian
Carbon metabolic pathways are important to the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, extremely little is known about metabolic regulation in mycobacteria. There is growing evidence for lysine acetylation being a mechanism of regulating bacterial metabolism. Lysine acetylation is a post-translational modification in which an acetyl group is covalently attached to the side chain of a lysine residue. This modification is mediated by acetyltransferases, which add acetyl groups, and deacetylases, which remove the acetyl groups. Here we set out to test whether lysine acetylation and deacetylation impact acetate metabolism in the model mycobacteria Mycobacterium smegmatis, which possesses 25 candidate acetyltransferases and 3 putative lysine deacetylases. Using mutants lacking predicted acetyltransferases and deacetylases we showed that acetate metabolism in M. smegmatis is regulated by reversible acetylation of acetyl-CoA synthetase (Ms-Acs) through the action of a single pair of enzymes: the acetyltransferase Ms-PatA and the sirtuin deacetylase Ms-SrtN. We also confirmed that the role of Ms-PatA in regulating Ms-Acs regulation depends on cAMP binding. We additionally demonstrated a role for Ms-Acs, Ms-PatA and Ms-SrtN in regulating the metabolism of propionate in M. smegmatis. Finally, along with Ms-Acs, we identified a candidate propionyl-CoA synthetase, Ms5404, as acetylated in whole-cell lysates. This work lays the foundation for studying the regulatory circuit of acetylation and deacetylation in the cellular context of mycobacteria. PMID:23813678
Acylglucuronide in alkaline conditions: migration vs. hydrolysis.
Di Meo, Florent; Steel, Michele; Nicolas, Picard; Marquet, Pierre; Duroux, Jean-Luc; Trouillas, Patrick
This work rationalizes the glucuronidation process (one of the reactions of the phase II metabolism) for drugs having a carboxylic acid moiety. At this stage, acylglucuronides (AG) metabolites are produced, that have largely been reported in the literature for various drugs (e.g., mycophenolic acid (MPA), diclofenac, ibuprofen, phenylacetic acids). The competition between migration and hydrolysis is rationalized by adequate quantum calculations, combing MP2 and density functional theory (DFT) methods. At the molecular scale, the former process is a real rotation of the drug around the glucuconic acid. This chemical-engine provides four different metabolites with various toxicities. Migration definitely appears feasible under alkaline conditions, making proton release from the OH groups. The latter reaction (hydrolysis) releases the free drug, so the competition is of crucial importance to tackle drug action and elimination. From the theoretical data, both migration and hydrolysis appear kinetically and thermodynamically favored, respectively. PMID:23420401
A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids.
Lindenkamp, Nicole; Schürmann, Marc; Steinbüchel, Alexander
In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (?4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16?pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating other CoA-transferase(s) or CoA-synthetase(s), thereby compensating for the lacking Pct. The ability of R. eutropha H16 to substitute absent enzymes by isoenzymes has been already shown in different other studies in the past. PMID:23250223
Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase.
Hurtado-Guerrrero, Ramón; Peńa-Díaz, Javier; Montalvetti, Andrea; Ruiz-Pérez, Luis M; González-Pacanowska, Dolores
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 proportion compared to the anabolic reaction. We report that the catalytically active species of HMGR in solution is the tetrameric form. Fluvastatin inhibited competitively the enzyme while cerivastatin binds by a mechanism which is more accurately described by a biphasic process characteristic of a class of 'slow, tight-binding' inhibitors. PMID:11801242
A Bacterial Indole3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean ( Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery
Kevin Liu; Han-Jung Lee; Sio San Leong; Chen-Lun Liu; Jyh-Ching Chou
Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin\\u000a metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes\\u000a in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver\\u000a a bacterial IAA-Asp hydrolase into cells of mung bean
Hydrolysis of iodine: equilibria at high temperatures
Palmer, D.A.; Ramette, R.W.; Mesmer, R.E.
The hydrolysis (or disproportionation) of molecular iodine to form iodate and iodide ions has been studied by emf measurements over the temperature range, 3.8/sup 0/ to 209.0/sup 0/C. The interpretation of these results required a knowledge of the formation constant for triiodide ion and the acid dissociation constant of iodic acid, both of which were measured as a function of temperature. The resulting thermodynamic data have been incorporated into a general computer model describing the hydrolysis equilibria of iodine as a function of initial concentration, pH and temperature.
The antibiotic CJ-15,801 is an antimetabolite that hijacks and then inhibits CoA biosynthesis.
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
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
The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis
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
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
Novel domain arrangement in the crystal structure of a truncated acetyl-CoA synthase from Moorella thermoacetica.
Volbeda, Anne; Darnault, Claudine; Tan, Xiangshi; Lindahl, Paul A; Fontecilla-Camps, Juan C
Ni-dependent acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) constitute the central enzyme complex of the Wood-Ljungdahl pathway of acetyl-CoA formation. The crystal structure of a recombinant bacterial ACS lacking the N-terminal domain that interacts with CODH shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape, and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that, according to anomalous scattering data, is most likely Cu or Zn. Incorporation of a functional Ni(2)Fe(4)S(4) A-cluster would require only minor structural rearrangements. Using available structures, a plausible model of the interaction between CODH and the smaller ACS in archaeal multienzyme complexes is presented, along with a discussion of evolutionary relationships of the archaeal and bacterial enzymes. PMID:19650626
Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata.
Avidan, Omri; Brandis, Alexander; Rogachev, Ilana; Pick, Uri
Triglycerides (TAGs) from microalgae can be utilized as food supplements and for biodiesel production, but little is known about the regulation of their biosynthesis. This work aimed to test the relationship between acetyl-CoA (Ac-CoA) levels and TAG biosynthesis in green algae under nitrogen deprivation. A novel, highly sensitive liquid chromatography mass spectrometry (LC-MS/MS) technique enabled us to determine the levels of Ac-CoA, malonyl-CoA, and unacetylated (free) CoA in green microalgae. A comparative study of three algal species that differ in TAG accumulation levels shows that during N starvation, Ac-CoA levels rapidly rise, preceding TAG accumulation in all tested species. The levels of Ac-CoA in the high TAG accumulator Chlorella desiccata exceed the levels in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Similarly, malonyl-CoA and free CoA levels also increase, but to lower extents. Calculated cellular concentrations of Ac-CoA are far lower than reported K mAc-CoA values of plastidic Ac-CoA carboxylase (ptACCase) in plants. Transcript level analysis of plastidic pyruvate dehydrogenase (ptPDH), the major chloroplastic Ac-CoA producer, revealed rapid induction in parallel with Ac-CoA accumulation in C. desiccata, but not in D. tertiolecta or C. reinhardtii. It is proposed that the capacity to accumulate high TAG levels in green algae critically depends on their ability to divert carbon flow towards Ac-CoA. This requires elevation of the chloroplastic CoA pool level and enhancement of Ac-CoA biosynthesis. These conclusions may have important implications for future genetic manipulation to enhance TAG biosynthesis in green algae. PMID:25922486
Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata
Avidan, Omri; Brandis, Alexander; Rogachev, Ilana; Pick, Uri
Triglycerides (TAGs) from microalgae can be utilized as food supplements and for biodiesel production, but little is known about the regulation of their biosynthesis. This work aimed to test the relationship between acetyl-CoA (Ac-CoA) levels and TAG biosynthesis in green algae under nitrogen deprivation. A novel, highly sensitive liquid chromatography mass spectrometry (LC-MS/MS) technique enabled us to determine the levels of Ac-CoA, malonyl-CoA, and unacetylated (free) CoA in green microalgae. A comparative study of three algal species that differ in TAG accumulation levels shows that during N starvation, Ac-CoA levels rapidly rise, preceding TAG accumulation in all tested species. The levels of Ac-CoA in the high TAG accumulator Chlorella desiccata exceed the levels in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Similarly, malonyl-CoA and free CoA levels also increase, but to lower extents. Calculated cellular concentrations of Ac-CoA are far lower than reported K mAc-CoA values of plastidic Ac-CoA carboxylase (ptACCase) in plants. Transcript level analysis of plastidic pyruvate dehydrogenase (ptPDH), the major chloroplastic Ac-CoA producer, revealed rapid induction in parallel with Ac-CoA accumulation in C. desiccata, but not in D. tertiolecta or C. reinhardtii. It is proposed that the capacity to accumulate high TAG levels in green algae critically depends on their ability to divert carbon flow towards Ac-CoA. This requires elevation of the chloroplastic CoA pool level and enhancement of Ac-CoA biosynthesis. These conclusions may have important implications for future genetic manipulation to enhance TAG biosynthesis in green algae. PMID:25922486
Acyl CoA Binding Protein (ACBP) Gene Ablation Induces Pre-Implantation Embryonic Lethality in Mice
Landrock, Danilo
Palmitoyltransferase 1 Dbi Diazepam Binding Inhibitor DNA Deoxyribonucleic acid dpc Days post coitum EDTA Ethylenediaminetetraacetic acid ES cells Embryonic Stemm Cells FABP1 Fatty Acid Binding Protein 1 (liver fatty acid binding protein, L-FABP) FABP2 Fatty... INTRODUCTION AND LITERATURE REVIEW* Known Physiological Role of Acyl CoA Bindin g Protein Acyl CoA binding protein (ACBP), also known as diazepam binding inhibitor (DBI), is a soluble 10 kDa lipid binding protein ubiquitously expressed in all tissues...
Cooperation between COA6 and SCO2 in COX2 Maturation during Cytochrome c Oxidase Assembly Links Two Mitochondrial Cardiomyopathies.
Pacheu-Grau, David; Bareth, Bettina; Dudek, Jan; Juris, Lisa; Vögtle, F-Nora; Wissel, Mirjam; Leary, Scot C; Dennerlein, Sven; Rehling, Peter; Deckers, Markus
Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency. PMID:25959673
Regulation of Autophagy and Mitophagy by Nutrient Availability and Acetylation
Webster, Bradley R.; Scott, Iain; Traba, Javier; Han, Kim; Sack, Michael N.
Normal cellular function is dependent on a number of highly regulated homeostatic mechanisms, which act in concert to maintain conditions suitable for life. During periods of nutritional deficit, cells initiate a number of recycling programs which break down complex intracellular structures, thus allowing them to utilize the energy stored within. These recycling systems, broadly named "autophagy", enable the cell to maintain the flow of nutritional substrates until they can be replenished from external sources. Recent research has shown that a number of regulatory components of the autophagy program are controlled by lysine acetylation. Lysine acetylation is a reversible post-translational modification that can alter the activity of enzymes in a number of cellular compartments. Strikingly, the main substrate for this modification is a product of cellular energy metabolism: acetyl-CoA. This suggests a direct and intricate link between fuel metabolites and the systems which regulate nutritional homeostasis. In this review, we examine how acetylation regulates the systems that control cellular autophagy, and how global protein acetylation status may act as a trigger for recycling of cellular components in a nutrient-dependent fashion. In particular, we focus on how acetylation may control the degradation and turnover of mitochondria, the major source of fuel-derived acetyl-CoA. PMID:24525425
Identification of Coa2 as an assembly factor for cytochrome c oxidase biogenesis1 Fabien Pierrel, Oleh Khalimonchuk, Paul A. Cobine and Dennis R. Winge*3
Paris-Sud XI, Université de
mitochondria is dependent on a new4 assembly factor designated Coa2. Coa2 was identified from its ability characterization of stalled intermediate complexes in human cells of patients with mutations16 in assembly factors1 Identification of Coa2 as an assembly factor for cytochrome c oxidase biogenesis1 2 Fabien
Lipase-mediated hydrolysis of blackcurrant oil
Miroslav Vacek; Marie Zarevúcka; Zden?k Wimmerb; Karel Stránský; Bohum??r Koutek; Martina Macková; Kate?ina Demnerová
Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40°C, but the latter series of enzymic processes were
Enhanced enzymatic hydrolysis of cellulose in microgels.
Chang, Aiping; Wu, Qingshi; Xu, Wenting; Xie, Jianda; Wu, Weitai
A cellulose-based microgel, where an individual microgel contains approximately one cellulose chain on average, is synthesized via free radical polymerization of a difunctional small-molecule N,N'-methylenebisacrylamide in cellulose solution. This microgelation leads to a low-ordered cellulose, favoring enzymatic hydrolysis of cellulose to generate glucose. PMID:26035077
Fermentable sugars by chemical hydrolysis of biomass
Raines, Ronald T.
Fermentable sugars by chemical hydrolysis of biomass Joseph B. Binder and Ronald T. Raines1 of carbon for a scalable biorefinery. biofuel carbohydrate ethanol fermentation ionic liquid, diluted with water, and heated was trans- formed into a fermentable sugar (17, 18). The concentrated acid
The program RATE was developed to aid in the extrapolation and interpretation of hydrolysis rate data to a format that is useful for environmental risk assessment. ydrolysis data typically are reported in the literature as pseudo-first-order rate constants at the temperature and ...
Rate constants for hydrolysis in water of ten classes of organic compounds are examined with the objective of establishing new, or expanding existing, property reactivity correlations. These relationships then can be used to predict the environmental hydrolysis of chemicals that ...
Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development
Jin, Huanan; Song, Zhihong; Nikolau, Basil J.
Acetoacetyl CoA thiolase (AACT, EC 18.104.22.168) 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.
Beating the acetyl coenzyme A-pathway to the origin of life.
Nitschke, Wolfgang; Russell, Michael J
Attempts to draft plausible scenarios for the origin of life have in the past mainly built upon palaeogeochemical boundary conditions while, as detailed in a companion article in this issue, frequently neglecting to comply with fundamental thermodynamic laws. Even if demands from both palaeogeochemistry and thermodynamics are respected, then a plethora of strongly differing models are still conceivable. Although we have no guarantee that life at its origin necessarily resembled biology in extant organisms, we consider that the only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfil the thermodynamic requirements for life to come into being. Wood-Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism. Based on a review of our present understanding of the biochemistry and biophysics of acetogenic, methanogenic and methanotrophic pathways and on a phylogenetic analysis of involved enzymes, we propose that a variant of modern methanotrophy is more likely than traditional WL systems to date back to the origin of life. The proposed model furthermore better fits basic thermodynamic demands and palaeogeochemical conditions suggested by recent results from extant alkaline hydrothermal seeps. PMID:23754811
Beating the acetyl coenzyme A-pathway to the origin of life
Nitschke, Wolfgang; Russell, Michael J.
Attempts to draft plausible scenarios for the origin of life have in the past mainly built upon palaeogeochemical boundary conditions while, as detailed in a companion article in this issue, frequently neglecting to comply with fundamental thermodynamic laws. Even if demands from both palaeogeochemistry and thermodynamics are respected, then a plethora of strongly differing models are still conceivable. Although we have no guarantee that life at its origin necessarily resembled biology in extant organisms, we consider that the only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfil the thermodynamic requirements for life to come into being. Wood–Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism. Based on a review of our present understanding of the biochemistry and biophysics of acetogenic, methanogenic and methanotrophic pathways and on a phylogenetic analysis of involved enzymes, we propose that a variant of modern methanotrophy is more likely than traditional WL systems to date back to the origin of life. The proposed model furthermore better fits basic thermodynamic demands and palaeogeochemical conditions suggested by recent results from extant alkaline hydrothermal seeps. PMID:23754811
9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.
Baumann, Anna-Maria T; Bakkers, Mark J G; Buettner, Falk F R; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A; de Groot, Raoul J; Mühlenhoff, Martina
Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1-a previously identified human candidate gene-is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044
Ultrafast photodissociation studies of acetyl cyanide and acetic acid and unimolecular decomposition rates of the acetyl radical products
NASA Astrophysics Data System (ADS)
Owrutsky, J. C.; Baronavski, A. P.
Unimolecular decomposition rates for acetyl radical following the photodissociation of acetyl cyanide and acetic acid near 193 nm have been studied using ultrafast mass-resolved photoionization spectroscopy. In both cases, the parent decays with an instrumentally limited lifetime, while the acetyl radical behaves in a manner consistent with an RRKM mechanism, in contrast to our previous results on acetone. It is necessary to convolute the population distribution with the microcanonical RRKM rates in order to achieve this agreement. We have also undertaken an ab initio study of the excited states of acetyl cyanide to clarify the assignments of these states. The state excited at 193 nm arises from a ???* transition with a calculated transition velocity dipole moment oriented at an angle of 57° with respect to the C-C?N bond, resulting in an anisotropy parameter of -0.22. This is in reasonable agreement with the previous data of North et al. [J. Phys. Chem. A 101, 9224 (1997)]. The apparent RRKM behavior of the acetyl radical formed by the photodissociation of acetic acid and acetyl cyanide indicates that acetyl radical produced by the photodissociation of acetone at 193 nm may exhibit "extrinsic non-RRKM" effects, i.e., dynamic bottlenecks or mode specific effects.
Technical bases for precipitate hydrolysis process operating parameters
Bannochie, C.J.
This report provides the experimental data and rationale in support of the operating parameters for precipitate hydrolysis specified in WSRC-RP-92737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF).
Enzymatic hydrolysis of rawhide using papain and neutrase
Siriporn Damrongsakkul; Kongpob Ratanathammapan; Kittinan Komolpis; Wiwut Tanthapanichakoon
Rawhide split was hydrolysed separately by two proteolytic enzymes, papain and neutrase. The effects of enzymatic conditions of the hydrolysis reaction were investigated. During the first 10min of the enzymatic hydrolysis, the yield of the hydrolysed protein increased sharply, then it slowly increased or became essentially constant due to the limited availability of the substrate. The optimum hydrolysis conditions of
Enhancing water resistance of welded dowel wood joints by acetylated lignin
A. Pizzi; X. Zhou; P. Navarrete; C. Segovia; H. R. Mansouri; M. I. Placentia Pena; F. Pichelin
Low molecular mass acetylated organosolv lignin from wheat straw and from depolymerised low sulphur organosolv wood lignin have been shown to markedly improve both the water resistance and the mechanical performance of welded dowel wood joints. The acetylated oligomers distribution and extent of acetylation of the two lignins were determined by Matrix assisted laser desorption ionization-time-of-flight mass spectrometry. Extensive acetylation
Acetylation and hydroxylation of sulphatroxazole in the snail Cepaea hortensis and the slug Arion rufus.
Vree, T B; Vree, M L; Nouws, J F
The snail Cepaea hortensis can acetylate and hydroxylate sulphatroxazole via a pathway similar to that in man. The principal metabolic pathways, in order of decreasing rate, are hydroxylation, glucuronidation and acetylation. The slug Arion rufus also can acetylate and hydroxylate sulphatroxazole, although, in contrast to the snail, is rate of acetylation is higher than that of hydroxylation. PMID:3564322
Acetylation and hydroxylation of sulphatroxazole in the snail Cepaea hortensis and the slug Arion rufus
T. B. Vree; M. L. Vree; J. F. M. Nouws
The snail Cepaea hortensis can acetylate and hydroxylate sulphatroxazole via a pathway similar to that in man. The principal metabolic pathways, in order of decreasing rate, are hydroxylation, glucuronidation and acetylation. The slug Anion rufus also can acetylate and hydroxylate sulphatroxazole, although, in contrast to the snail, is rate of acetylation is higher than that of hydroxylation.
An improved chemo-enzymatic synthesis of 1-beta-O-acyl glucuronides: highly chemoselective enzymatic removal of protecting groups from corresponding methyl acetyl derivatives.
Baba, Akiko; Yoshioka, Tadao
An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-beta-O-acyl glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the nature of their 1-beta-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-beta-O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6. PMID:17985922
Can changes in histone acetylation contribute to memory formation?
Lopez-Atalaya, Jose P; Barco, Angel
Neuronal histone acetylation has been postulated to be a mnemonic substrate and a target for memory enhancers and neuropsychiatric drugs. Here we critically evaluate this view and examine the apparent conflict between the proposed instructive role for histone acetylation in memory-related transcription and the insights derived from genomic and genetic studies in other systems. We next discuss the suitability of activity-dependent neuronal histone acetylation as a mnemonic substrate and debate alternative interpretations of current evidence. We believe that further progress in our understanding of the role of histone acetylation and other epigenetic modifications in neuronal plasticity, memory, and neuropsychiatric disorders requires a clear discrimination between cause and effect so that novel epigenetics-related processes can be distinguished from classical transcriptional mechanisms. PMID:25269450
Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands
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 ...
The role of acetylation in rDNA transcription
Iwona Hirschler-Laszkiewicz; Alice Cavanaugh; Qiyue Hu; Jason Catania; Maria Laura Avantaggiati; Lawrence I. Rothblum
Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl- histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chro- matin. TSA did not reverse inhibition of transcription
Mechanisms of Ischemic Neuroprotection by Acetyl-L-carnitine
Acetyl-L-carnitine is a naturally occurring substance that, when administered at supraphysiologic concentrations, is neuroprotective in several animal models of global and focal cerebral ischemia. Three primary mechanisms of action are supported by neurochemical outcome measures performed with these models and with in vitro models of acute neuronal cell death. The metabolic hypothesis is based on the oxidative metabolism of the acetyl component of acetyl-L-carnitine and is a simple explanation for the reduction in postischemic brain lactate levels and elevation of ATP seen with drug administration. The antioxidant mechanism is supported by reduction of oxidative stress markers, for example, protein oxidation, in both brain tissue and cerebrospinal fluid. The relatively uncharacterized mechanism of inhibiting excitotoxicity could be extremely important in both acute brain injury and chronic neurodegenerative disorders. New experiments performed with primary cultures of rat cortical neurons indicate that the presence of acetyl-L-carnitine significantly inhibits both acute and delayed cell death following exposure to NMDA, an excitotoxic glutamate antagonist. Finally, several other mechanisms of action are possible, including a neurotrophic effect of acetyl-L-carnitine and inhibition of mitochondrial permeability transition. While the multiple potential mechanisms of neuroprotection by acetyl-L-carnitine limit an accurate designation of the most important mode of action, they are compatible with the concept that several brain injury pathways must be inhibited to optimize therapeutic efficacy. PMID:16179519
Reduced microtubule acetylation in cystic fibrosis epithelial cells
Rymut, Sharon M.; Harker, Alyssa; Corey, Deborah A.; Burgess, James D.; Sun, Hongtao; Clancy, John P.
Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-?-tubulin (Ac-tub) content is reduced by ?40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr?/? mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-?B activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110? is also identified as a regulator of MT acetylation. PMID:23873844
Reduced microtubule acetylation in cystic fibrosis epithelial cells.
Rymut, Sharon M; Harker, Alyssa; Corey, Deborah A; Burgess, James D; Sun, Hongtao; Clancy, John P; Kelley, Thomas J
Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-?-tubulin (Ac-tub) content is reduced by ?40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr-/- mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-?B activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110? is also identified as a regulator of MT acetylation. PMID:23873844
An acetylation switch controls TDP-43 function and aggregation propensity
Cohen, Todd J.; Hwang, Andrew W.; Restrepo, Clark R.; Yuan, Chao-Xing; Trojanowski, John Q.; Lee, Virginia M.Y.
TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA-binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signaling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies. PMID:25556531
Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apolipoproteins in mouse peritoneal macrophages
Claus Langer; Yadong Huang; Paul Cullen; Bernd Wiesenhütter; Robert W. Mahley; Gerd Assmann; Arnold von Eckardstein
We investigated the effect of endogenous apolipoprotein (apo) E synthesis in mouse peritoneal macrophages on cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 (HDL3) and lipid-free apolipoproteins (apo). After loading with acetylated LDL (acLDL) peritoneal macrophages from wild-type (apoE+\\/+) and apoE-deficient (apoE-\\/-) mice were incubated with medium alone or with liposomes, HDL3, lipid-free apoA-I, or lipid-free apoE3.
Continuous steam hydrolysis of tulip poplar
Fieber, C.; Colcord, A.R.; Faass, S.; Muzzy, J.D.; Roberts, R.S.
To produce ethanol from hardwood it is desirable to fractionate the hardwood in order to produce a relatively pure cellulosic pulp for dilute acid hydrolysis. An experimental investigation of continuous steam hydrolysis of tulip poplar wood chips indicates that over 90% of the lignin present can be extracted by 0.1N sodium hydroxide, resulting in a cellulose pulp containing over 90% hexosan. The study was performed using a Stake Technology, Ltd., continuous digester rated at one oven dry ton per hour of wood chips. The yields of hexosans, hexoses, xylan, xylose, lignin, furfural, acetic acid and methanol were determined as a function of residence time and steam pressure in the digester. The information provides a basis for establishing a material and energy balance for a hardwood to ethanol plant.
Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase
Berger, Stefanie; Welte, Cornelia; Deppenmeier, Uwe
The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM = 0.27 ± 0.05?mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell. PMID:22927778
N-Acetyl-L-Aspartate is a Major Source of Acetyl Groups for Lipid Synthesis during Rat Brain Development
R. Burri; C. Steffen; N. Herschkowitz
The function of N-acetyl-L-aspartate (NAA), a predominant substance in the CNS, has not yet been determined. To investigate the possible function of NAA as a lipid precursor [14C]-N-acetyl-L-aspartate (NAA) or [14C]-acetate (AcA) was injected intracerebrally into 8, 15- and 22-day-old rats. These time points were selected because NAA concentration and the activity of the NAA synthetizing enzyme L-aspartate-N-acetyltransferase (ANAT) were
?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis
Jin, Xin; Zhang, Zhen-hai; Sun, E.; Jia, Xiao-Bin
Background: The content of icaritin and genistein in herba is very low, preparation with relatively large quantities is an important issue for extensive pharmacological studies. Objective: This study focuses on preparing and enzymic hydrolysis of flavonoid glycosides /?-cyclodextrin inclusion complex to increase the hydrolysis rate. Materials and Methods: The physical property of newly prepared inclusion complex was tested by differential scanning calorimetry (DSC). The conditions of enzymatic hydrolysis were optimized for the bioconversion of flavonoid glycosides /?-cyclodextrin inclusion complex by mono-factor experimental design. The experiments are using the icariin and genistein as the model drugs. Results: The solubility of icariin and genistein were increased almost 17 times from 29.2 ?g/ml to 513.5 ?g/ml at 60°C and 28 times from 7.78 ?g/ml to 221.46 ?g/ml at 50°C, respectively, demonstrating that the inclusion complex could significantly increase the solubility of flavonoid glycosides. Under the optimal conditions, the reaction time of icariin and genistin decreased by 68% and 145%, when compared with that without ?-CD inclusion. By using this enzymatic condition, 473 mg icaritin (with the purity of 99.34%) and 567 mg genistein(with the purity of 99.46%), which was finally determined by melt point, ESI-MS, UV, IR, 1H NMR and 13C NMR, was obtained eventually by transforming the inclusion complex(contains 1.0 g substrates). Conclusion: This study can clearly indicate a new attempt to improve the speed of enzyme-hydrolysis of poorly water-soluble flavonoid glycosides and find a more superior condition which is used to prepare icaritin and genistein. PMID:24143039
Kinetics of ptaquiloside hydrolysis in aqueous solution.
Ayala-Luis, Karina B; Hansen, Pernille B; Rasmussen, Lars H; Hansen, Hans Christian B
Ptaquiloside (PTA) is a well-known toxin produced by the bracken fern (Pteridium aquilinum (L.) Kuhn). It is proposed that PTA from bracken stands can leach through soil and sediments into drinking-water reservoirs, thus representing a concern for human health. To predict the persistence of the toxin, a full understanding of the PTA degradation in aqueous environments is important. The kinetics of PTA hydrolysis was examined at 22 degrees C in aqueous buffered solutions (pH 2.88-8.93). The reaction was found to follow first-order kinetics with respect to PTA at all pH and temperature conditions. At pH lower than 4.43 (+/- 0.32), the reaction is acid-mediated, whereas the reaction is base-mediated at pH higher than 6.39 (+/- 0.28). The rate constants for the acid-catalyzed, base-catalyzed, and neutral hydrolysis are 25.70 (+/- 0.96), 4.83 (+/- 0.03) X 10(4), and 9.49 (+/- 6.02) x 10(-4) h(-1), respectively. The PTA hydrolysis at pH 4.46 is strongly dependent on temperature, with an activation energy of 74.4 (+/- 2.6) kJ mol(-1). Stoichiometric calculations, reaction kinetics, and ultraviolet-visible spectrophotometry strongly indicates the formation of an intermediary compound at pH 5.07 and 6.07 via a mechanism comprising two first-order consecutive reactions. Ptaquiloside has the lowest rate of hydrolysis at slightly acidic pH and low temperatures. Therefore, because PTA is not sorbed in soil, slightly acidic sandy soils in cold climates are most prone to PTA leaching to deeper soil layers and aquifers. PMID:17022402
Enzymatic hydrolysis of PTT polymers and oligomers
A. Eberl; S. Heumann; R. Kotek; F. Kaufmann; S. Mitsche; A. Cavaco-Paulo; G. M. Gübitz
Oligomers and polymers (film, fabrics) of the linear aromatic polyester poly(trimethylene terephthalate) (PTT) were treated with polyesterases from Thermomyces lanuginosus, Penicillium citrinum, Thermobifida fusca and Fusarium solani pisi. The cutinase from T. fusca was found to release the highest amounts of hydrolysis products from PTT materials and was able to open and hydrolyse a cyclic PTT dimer according to RP-HPLC–UV
Fungal secretomes enhance sugar beet pulp hydrolysis.
Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland
The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771
Sugarcane bagasse hydrolysis using yeast cellulolytic enzymes.
Souza, Angelica Cristina de; Carvalho, Fernanda Paula; Silva e Batista, Cristina Ferreira; Schwan, Rosane Freitas; Dias, Disney Ribeiro
Ethanol fuel production from lignocellulosic biomass is emerging as one of the most important technologies for sustainable development. To use this biomass, it is necessary to circumvent the physical and chemical barriers presented by the cohesive combination of the main biomass components, which hinders the hydrolysis of cellulose and hemicellulose into fermentable sugars. This study evaluated the hydrolytic capacity of enzymes produced by yeasts, isolated from the soils of the Brazilian Cerrado biome (savannah) and the Amazon region, on sugarcane bagasse pre-treated with H2SO4. Among the 103 and 214 yeast isolates from the Minas Gerais Cerrado and the Amazon regions, 18 (17.47%) and 11 (5.14%) isolates, respectively, were cellulase-producing. Cryptococcus laurentii was prevalent and produced significant ?- glucosidase levels, which were higher than the endo- and exoglucanase activities. In natura sugarcane bagasse was pre-treated with 2% H2SO4 for 30 min at 150oC. Subsequently, the obtained fibrous residue was subjected to hydrolysis using the Cryptococcus laurentii yeast enzyme extract for 72 h. This enzyme extract promoted the conversion of approximately 32% of the cellulose, of which 2.4% was glucose, after the enzymatic hydrolysis reaction, suggesting that C. laurentii is a good ?-glucosidase producer. The results presented in this study highlight the importance of isolating microbial strains that produce enzymes of biotechnological interest, given their extensive application in biofuel production. PMID:23851270
Palm date fibers: analysis and enzymatic hydrolysis.
Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J
Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438
Acetylation of Acetyl-CoA Synthetase from Mycobacterium tuberculosis Leads to Specific Inactivation of the Adenylation Reaction
Noy, Tahel; Xu, Hua; Blanchard, John S.
Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A, allowing the organism to grow on acetate as the sole carbon source. ACS was the first enzyme in Mycobacterium tuberculosis shown to be regulated by posttranslational acetylation by the cAMP-dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD+ and a mycobacterial sirtuin-like deacetylase. In this study we characterize the kinetic mechanism of MtACS, where the overall reaction can be divided into two half-reactions: the acetyl-adenylate forming reaction and the thiol-ligation reaction. We also provide evidence for the existence of the acetyl-adenylate intermediate via 31P-NMR spectroscopy. Furthermore, we dissect the regulatory role of K617 acetylation and show that acetylation inhibits only the first, adenylation half-reaction while leaving the second half reaction unchanged. Finally, we demonstrate that the chemical mechanism of the enzyme relies on a conformational change which is controlled by the protonation state of aspartate 525. Together with our earlier results, this suggests a degree of regulation of enzyme activity that is appropriate for the role of the enzyme in central carbon metabolism. PMID:24751484
4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.
Urashima, Tadasu; Inamori, Hiroaki; Fukuda, Kenji; Saito, Tadao; Messer, Michael; Oftedal, Olav T
Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(?2-3)Gal(?1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(?2-3)Gal(?1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(?2-3)Gal(?1-4)GlcNAc(?1-3)Gal(?1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(?2-3)Gal(?1-4)[Fuc(?1-3)]GlcNAc(?1-3)Gal(?1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (?20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces. PMID:25601457
Synthesis and antimelanoma activity of tertiary amide analogues of N-acetyl-4-S-cysteaminylphenol.
Pearson, Vikki C; Ferguson, Jillian; Rogers, Paul M; Kelland, Lloyd R; Robins, David J
The biosynthetic pathway to melanin is a realistic target for therapeutic intervention in the development of new drugs to combat malignant melanoma. N-Acetyl-4-S-cysteaminylphenol (1) is an analogue of a biosynthetic intermediate in the pathway to melanin. It probably acts as a prodrug and is oxidized selectively in melanocytes to an o-quinone, which can alkylate cellular nucleophiles resulting in interference with cell growth and proliferation. We previously synthesized a range of more lipophilic analogues of 1 by varying the acyl portion and introducing substitution alpha to the nitrogen. Most of the new compounds displayed greater cytotoxicity than the original lead compound 1. We have now prepared 12 new compounds with varying acyl portions and three different substituents on the nitrogen of the amide in order to increase lipophilicity and to reduce the possibility of hydrolysis of the amides. Most of the tertiary amides showed greater cytotoxicity towards five representative melanoma cell lines than the parent secondary amide. The highest cytotoxicity, comparable to cisplatin, was observed for the benzyl substituted compounds 4, 8, 12, and 16 and the cyclohexylacetamide derivatives 13-15 against these five cell lines. The moderate activity of 13-16 against SK-Mel-24 (non-tyrosinase containing) and an ovarian cell line suggests that interference with the melanin pathway may not be the only mode of action of these new compounds. PMID:12812364
Identification and preliminary characterization of acsF, a Putative Ni-insertase used in the biosynthesis of acetyl-CoA synthase from Clostridium thermoaceticum
Huay-Keng Loke; Paul A. Lindahl
OAK-B135 The acsABCDE genes in the Clostridium thermoaceticum genome are used for autotrophic acetyl-CoA synthesis using the Wood/Ljungdahl pathway. A 2.8 kb region between acsC and acsD was cloned and sequenced. Two open reading frames, orf7 ({approx} 1.9 kb) and acsF ({approx} 0.7 kb) were identified. orf7 appears to encode an Fe-S protein, in that it contains 5 conserved cysteine residues, 3 of which are present in a motif (CXXXXXCXXC) commonly used to coordinate Fe-S clusters. However, Orf7 is probably not involved in autotrophic acetyl-CoA synthesis, as homologous genes are present in organisms that do not utilize this pathway and are absent in many that do. In contrast, acsF is probably involved in this pathway. Sequence alignment of AcsF and 11 homologs reveals a number of conserved regions, including a P-loop that binds nucleoside triphosphates and catalyzes their hydrolysis. One homolog is CooC, an ATPase/GTPase that inserts Ni into a precursor form of the C-cluster of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified AcsF lacked Ni and Fe, and slowly catalyzed the hydrolysis of ATP. Such similarities to CooC suggest that AcsF may function to insert Ni into a Ni-deficient form of the bifunctional acetyl-CoA synthase/CODH from C. thermoaceticum (ACSCt). However, this could not be established, as expression of acsF did not effect activation of recombinant AcsAB expressed in E. coli. Also, E. coli cells defective in hypB retained the ability to synthesize active recombinant AcsAB. Rather, the concentration of extracellular Ni2+ ions was critical to activation.
Organosolv pretreatment for enzymatic hydrolysis of poplars: I. enzyme hydrolysis of cellulosic residues
Chum, H.L.; Johnson, D.K.; Black, S.; Baker, J.; Grohmann, K.; Sarkanen, K.V.; Wallace, K.; Schroeder, H.A.
Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H/sub 2/SO/sub 4/ and H/sub 3/PO/sub 4/) such that the cooking liquor pH less than or equal to 3 are easily digested by enzymes. The total yields of hydrolysis residues (pulps) are in the 40-60% range; the acid-catalyzed delignification followed by enzyme hydrolysis can generate 70-88% of the original six-carbon sugars contained in the wood. Glucomannan and arabinogalactan are dissolved in to the pulping liquor in the pH range of 2-4.5. Lower pH (less than or equal to 3) leads to additional solubilization of six-carbon sugars. These sugars may be fermented directly. From the insoluble hydrolysis residues, 36-41% conversions of wood into fermentable sugars were obtained after enzyme hydrolysis; the starting feedstocks contain 50.8 and 46.6% hexosans, respectively, for aspen and black cottonwood. The kinetics of enzymatic hydrolysis of cellulose can be formally treated as two simultaneous pseudo-first-order reactions in which fast and slow hydrolysis of cellulose occur. Correlations between the glucan digestibility and the effect of the pretreatment have been made. The higher residual xylan content reduces the amount of the rapidly hydrolyzable glucan fraction and lowers the glucan digestibility. The proposed simple kinetic treatment is very helpful in assessing the effect of the pretreatment on pulp enzyme hydrolyzability.
Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.
Kassovska-Bratinova, S; Fukao, T; Song, X Q; Duncan, A M; Chen, H S; Robert, M F; Pérez-Cerdá, C; Ugarte, M; Chartrand, C; Vobecky, S; Kondo, N; Mitchell, G A
Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.18.104.22.168) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes > fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. PMID:8751852
Targeted proteomics for quantification of histone acetylation in Alzheimer's disease.
Zhang, Kangling; Schrag, Matthew; Crofton, Andrew; Trivedi, Rishi; Vinters, Harry; Kirsch, Wolff
The epigenetic remodeling of chromatin histone proteins by acetylation has been the subject of recent investigations searching for biomarkers indicative of late onset cognitive loss. Histone acetylations affect the regulation of gene transcription, and the loss of learning induced deacetylation at specific histone sites may represent biomarkers for memory loss and Alzheimer's disease (AD). Selected-reaction-monitoring (SRM) has recently been advanced to quantitate peptides and proteins in complex biological systems. In this paper, we provide evidence that SRM-based targeted proteomics can reliably quantify specific histone acetylations in both AD and control brain by identifying the patterns of H3 K18/K23 acetylations Results of targeted proteomics assays have been validated by Western blot (WB) analysis. As compared with LC-MS/MS-TMT (tandem-mass-tagging) and WB methods, the targeted proteomics method has shown higher throughput, and therefore promised to be more suitable for clinical applications. With this methodology, we find that histone acetylation is significantly lower in AD temporal lobe than found in aged controls. Targeted proteomics warrants increased application for studying epigenetics of neurodegenerative diseases. PMID:22577027
Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.
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
Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ?-oxidative or nonoxidative pathways. The first step in the ?-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 ?-oxidative pathway. PMID:22649270
?-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice
Jeong, Moon Hee; Tran, Nguyen Khoi Song; Kwak, Tae Hwan; Park, Byung Keon; Lee, Chul Soon; Park, Tae-Sik; Lee, Young-Hoon; Park, Woo Jin; Yang, Dong Kwon
Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of ?-lapachone (?-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD+ level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of ?-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of ?-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, ?-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. ?-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, ?-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that ?-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. ?-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy. PMID:24614171
Multiple mass isotopomer tracing of acetyl-CoA metabolism in Langendorff-perfused rat hearts: channeling of acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase.
Li, Qingling; Deng, Shuang; Ibarra, Rafael A; Anderson, Vernon E; Brunengraber, Henri; Zhang, Guo-Fang
We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (?citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA. PMID:25645937
Xylan as limiting factor in enzymatic hydrolysis of nanocellulose.
Penttilä, Paavo A; Várnai, Anikó; Pere, Jaakko; Tammelin, Tekla; Salmén, Lennart; Siika-aho, Matti; Viikari, Liisa; Serimaa, Ritva
The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture. PMID:23238342
Influence of clay minerals on the hydrolysis of carbamate pesticides.
Wei, J; Furrer, G; Kaufmann, S; Schulin, R
Using batch experiments, we investigated the influence of clay minerals (montmorillonite, beidellite, illite, and vermiculite) on the hydrolysis of five carbamate pesticides: carbosulfan, carbofuran, aldicarb, pirimicarb, and chlorpropham. Compared to the other minerals, montmorillonite had the strongest influence on the hydrolysis of these carbamates. Montmorillonite enhanced the hydrolysis of carbosulfan and aldicarb. In contrast, the hydrolysis of chlorpropham was inhibited by montmorillonite, probably because of its strong adsorption on montmorillonite. The hydrolysis of pirimicarb was not affected by montmorillonite. The presence of organic substances, phosphate, and fluoride in suspensions decreased the catalytic activity of montmorillonite. Surface acidity of montmorillonite and/or formation of surface chelates are probably the key factors of surface catalysis in the case of the hydrolysis of carbosulfan. PMID:11414023
The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review
Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang
Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697
Ultrasound enhanced enzymatic hydrolysis of Parthenium hysterophorus: A mechanistic investigation.
Singh, Shuchi; Agarwal, Mayank; Bhatt, Aditya; Goyal, Arun; Moholkar, Vijayanand S
This study has attempted to establish the mechanism of the ultrasound-induced enhancement of enzymatic hydrolysis of pretreated and delignified biomass of Parthenium hysterophorus. A dual approach of statistical optimization of hydrolysis followed by application of sonication at optimum conditions has been adopted. The kinetics of hydrolysis shows a marked 6× increase with sonication, while net sugar yield shows marginal rise of ?20%. The statistical experimental design reveals the hydrolysis process to be enzyme limited. Profile of sugar yield in ultrasound-assisted enzymatic hydrolysis has been analyzed using HCH-1 model coupled with Genetic Algorithm optimization. The trends in the kinetic and physiological parameters of HCH-1 model reveal that sonication enhances enzyme/substrate affinity and reaction velocity of hydrolysis. The product inhibition of enzyme in all forms (free, adsorbed, complexed) also reduces with ultrasound. These effects are attributed to intense micro-convection induced by ultrasound and cavitation in the liquid medium. PMID:26094188
Hydrolysis of ferric chloride in solution
Lussiez, G.; Beckstead, L.
The Detox{trademark} process uses concentrated ferric chloride and small amounts of catalysts to oxidize organic compounds. It is under consideration for oxidizing transuranic organic wastes. Although the solution is reused extensively, at some point it will reach the acceptable limit of radioactivity or maximum solubility of the radioisotopes. This solution could be cemented, but the volume would be increased substantially because of the poor compatibility of chlorides and cement. A process has been developed that recovers the chloride ions as HCl and either minimizes the volume of radioactive waste or permits recycling of the radioactive chlorides. The process involves a two-step hydrolysis at atmospheric pressure, or preferably under a slight vacuum, and relatively low temperature, about 200{degrees}C. During the first step of the process, hydrolysis occurs according to the reaction below: FeCl{sub 3 liquid} + H{sub 2}O {r_arrow} FeOCl{sub solid} + 2 HCl{sub gas} During the second step, the hot, solid, iron oxychloride is sprayed with water or placed in contact with steam, and hydrolysis proceeds to the iron oxide according to the following reaction: 2 FeOCl{sub solid} + H{sub 2}O {r_arrow} Fe{sub 2}O{sub 3 solid} + 2 HCl{sub gas}. The iron oxide, which contains radioisotopes, can then be disposed of by cementation or encapsulation. Alternately, these chlorides can be washed off of the solids and can then either be recycled or disposed of in some other way.
Technology Transfer Automated Retrieval System (TEKTRAN)
An understanding of the lignification process is of vital importance, especially for the pulp and paper industry. Cinnamoyl-coa reductase (CCR) is an enzyme that plays a central role in the lignification process. Previous results have shown that down-regulation of CCR decreases the lignin content. B...
Mechanistic Insight with HBCH[subscript 2]CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases
Zhang, Wei
Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH[subscript 2]CoA, in which the S in HBCoA is replaced ...
New insights into structure-function relationships of oxalyl CoA decarboxylase from Escherichia coli.
Werther, Tobias; Zimmer, Agnes; Wille, Georg; Golbik, Ralph; Weiss, Manfred S; König, Stephan
The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5'-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5'-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. PMID:20553497
Cytoskeleton Dynamics: A Continuum Cooperative Hydrolysis Model
NASA Astrophysics Data System (ADS)
Xu, Jian-Wei; Cheng, Bo; Feng, Yu-Yu; Wang, Zi-Qing; Wang, Guo-Dong
Cytoskeleton is a network of filamentous proteins, such as actin filaments and microtubules. We propose a continuum cooperative hydrolysis model which possesses exactly analytical solution to describe the dynamics of filament. The results show that the cooperativity leads to non negative-exponential distribution of T (ATP or GTP) subunits. As an application, we investigate the treadmilling phenomenon using our model. It is shown that the cooperativity remarkably affects the length of filament. Supported by Chinese Universities Scientific Fund under Grant No. 2014YB029 and National Natural Science Foundation of China under Grant No. 11205123
Improved method for detection of starch hydrolysis
Ohawale, M.R.; Wilson, J.J.; Khachatourians, G.G.; Ingledew, W.M.
A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and alpha-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of alpha-amylase activities in culture filtrates or column effluents. (Refs. 18).
Hydrolysis of the chlorophosphazenes: cyclic trimer and linear polymer
Gabler, Douglas G
been previously proposed as a polymerization catalyst. Two of the species are oxo-bridged dimers of two trimer rings that may be useful as model compounds for hydrolytic cross-linking reactions in the polymer. Hydrolysis of poly... Experiments 1 4 8 9 II. TRIMER HYDROLYSIS. Background. NMR Experiments Sample Preparation. Results and Discussion. Dimer Formation Trends in NMR Spectroscopic Parameters. 11 14 19 21 31 37 III. POLYMER HYDROLYSIS. 38 Background. Sample...
An acetylation rheostat for the control of muscle energy homeostasis.
Menzies, Keir; Auwerx, Johan
In recent years, the role of acetylation has gained ground as an essential modulator of intermediary metabolism in skeletal muscle. Imbalance in energy homeostasis or chronic cellular stress, due to diet, aging, or disease, translate into alterations in the acetylation levels of key proteins which govern bioenergetics, cellular substrate use, and/or changes in mitochondrial content and function. For example, cellular stress induced by exercise or caloric restriction can alter the coordinated activity of acetyltransferases and deacetylases to increase mitochondrial biogenesis and function in order to adapt to low energetic levels. The natural duality of these enzymes, as metabolic sensors and effector proteins, has helped biologists to understand how the body can integrate seemingly distinct signaling pathways to control mitochondrial biogenesis, insulin sensitivity, glucose transport, reactive oxygen species handling, angiogenesis, and muscle satellite cell proliferation/differentiation. Our review will summarize the recent developments related to acetylation-dependent responses following metabolic stress in skeletal muscle. PMID:23999889
Synthetic biology for engineering acetyl coenzyme A metabolism in yeast.
Nielsen, Jens
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-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol. PMID:25370498
Histone Deacetylase Inhibitors Globally Enhance H3/H4 Tail Acetylation Without Affecting H3 Lysine 56 Acetylation
Drogaris, Paul; Villeneuve, Valérie; Pomičs, Christelle; Lee, Eun-Hye; Bourdeau, Véronique; Bonneil, Éric; Ferbeyre, Gerardo; Verreault, Alain; Thibault, Pierre
Histone deacetylase inhibitors (HDACi) represent a promising avenue for cancer therapy. We applied mass spectrometry (MS) to determine the impact of clinically relevant HDACi on global levels of histone acetylation. Intact histone profiling revealed that the HDACi SAHA and MS-275 globally increased histone H3 and H4 acetylation in both normal diploid fibroblasts and transformed human cells. Histone H3 lysine 56 acetylation (H3K56ac) recently elicited much interest and controversy due to its potential as a diagnostic and prognostic marker for a broad diversity of cancers. Using quantitative MS, we demonstrate that H3K56ac is much less abundant than previously reported in human cells. Unexpectedly, in contrast to H3/H4 N-terminal tail acetylation, H3K56ac did not increase in response to inhibitors of each class of HDACs. In addition, we demonstrate that antibodies raised against H3K56ac peptides cross-react against H3 N-terminal tail acetylation sites that carry sequence similarity to residues flanking H3K56. PMID:22355734
[Conformation aspects of peptide interaction with proteolytic enzymes. Alpha-chymotrypsin catalyzed hydrolysis of the cyclopeptides containing leucyltyrosyl fragments].
Tsetlin, M M; Ivanov, V T; Ovchinnikov, Y A; Klyosov, A A
The studies were made on the interaction of alpha-chymotrypsin with a series of cyclopeptides cyclo(-L-leucyl-L-tyrosyl-glycyln-), n=4, 6 and 8 (I, II and III respectively), and cyclo(-L-leucyl-L-tryosyl-beta-aminovalero-yl2-) (IV). Compounds I and IV are resistant to enzyme action whereas cyclopeptides II and III proved to be the substrates, their kinetic constants being Km=15.4 and 13.2 mM and kcat=0.54 and 9.53 sec-1 respectively. The binding capacity of cyclopeptides I-IV is evaluated by their competitive inhibition of alpha-chymotrypsin catalyzed hydrolysis of N-acetyl-L-tyrosine methyl ester. PMID:1203366
The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.
Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel
The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes. PMID:18184660
Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase
Reger,A.; Carney, J.; Gulick, A.
The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.
4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.
Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K
Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway. PMID:23677922
Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase\\/Etf Complex from Clostridium kluyveri
Fuli Li; Julia Hinderberger; Henning Seedorf; Jin Zhang; Wolfgang Buckel; Rudolf K. Thauer
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 ferre- doxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehy- drogenase\\/Etf complex
Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.
Ritter, G; Boosfeld, E; Markstein, E; Yu, R K; Ren, S L; Stallcup, W B; Oettgen, H F; Old, L J; Livingston, P O
Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse. PMID:2302705
Metabolic Regulation of Protein N-Alpha-Acetylation by Bcl-xL Promotes Cell Survival
Yi, Caroline H.
Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and ...
Regulation of Acetyl Coenzyme A Synthetase in Escherichia coli
Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl
MICROBIOLOGY: Bacteria Seize Control by Acetylating Host Proteins
NSDL National Science Digital Library
Carolyn A. Worby (University of California; Departments of Pharmacology, Cellular and Molecular Medicine, and Chemistry and Biochemistry)
Access to the article is free, however registration and sign-in are required. The plague-causing bacterium Yersinia pestis injects toxic proteins into its hosts' cells. One of these interferes with the host's secretion of a protective factor by adding acetyl groups to a signaling kinase, blocking its activation.
Acetylated starch-polylactic acid loose-fill packaging materials
Junjie Guan; Kent M. Eskridge; Milford A. Hanna
Different genetic and botanical sources of starches are available for use in hydrophobic starch-based packaging materials. The objectives of this study were to evaluate the effects of the type of acetylated starch and the presence of polylactic acid (PLA) and ethanol on the functional properties of extruded foams, and to compare the specific mechanical energy requirements for preparing these foams.
Acetylation of the human DNA glycosylase NEIL2 and inhibition of its activity
Kishor K. Bhakat; Tapas K. Hazra; Sankar Mitra
Post-translational modifications of proteins, includ- ing acetylation, modulate their cellular functions. Several human DNA replication and repair enzymes have recently been shown to be acetylated, leading to their inactivation in some cases. Here we show that the transcriptional coactivator p300 stably inter- acts with, and acetylates, the recently discovered human DNA glycosylase NEIL2, involved in the repair of oxidized bases
Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation.
Gamble, J; Lopaschuk, G D
Acetyl coenzyme A (CoA) carboxylase (ACC) is an important regulator of fatty acid oxidation in the heart, since it produces malonyl CoA, a potent inhibitor of mitochondrial fatty acid uptake. Under conditions of metabolic stress, 5'adenosine monophosphate-activated protein kinase (AMPK), which is highly expressed in cardiac muscle, can phosphorylate and decrease ACC activity. In this study, we determined if fatty acid oxidation in the heart could be regulated by insulin, due to alterations in AMPK regulation of ACC activity. Isolated working rat hearts were perfused with Krebs-Henseleit solution containing 11 mmol/L glucose, 0.4 mmol/L [9,10(-3)H]palmitate, and either 100 microU/mL insulin or 1,000 microU/mL insulin. Increasing insulin concentration resulted in a decrease in fatty acid oxidation rates (P < .05), a decrease in AMPK activity (P < .05), and an increase in ACC activity (P < .05) compared with the low-insulin group. A negative correlation was observed between AMPK and ACC activity (r = -.76). We conclude that insulin, acting through inhibition of AMPK and stimulation of ACC, is capable of inhibiting myocardial fatty acid oxidation. PMID:9361684
Dilek Kiliç Apar; Meltem Turhan; Belma Özbek
In the present study, the ultrasonication method was used to investigate the effect of ultrasonic energy on the hydrolysis of corn, rice, and wheat starch by using the alpha-amylase enzymes produced by Bacillus species and Bacillus licheniformis. The effects of sonifier operation variables such as duty cycle and acoustic power rate on the stability of alpha-amylase enzymes and hydrolysis degrees
Enhanced functional properties of tannic acid after thermal hydrolysis
Technology Transfer Automated Retrieval System (TEKTRAN)
Thermal hydrolysis processing of fresh tannic acid was carried out in a closed reactor at four different temperatures (65, 100, 150 and 200°C). Pressures reached in the system were 1.3 and 4.8 MPa at 150 and 200°C, respectively. Hydrolysis products (gallic acid and pyrogallol) were separated and qua...
Acid hydrolysis of pretreated lignocellulose from corn residue
P. R. Bienkowski; M. R. Ladisch; M. Voloch; G. T. Tsao
The lignocellulose derived from the hemicellulose hydrolysis of corn residue was steeped in 15 to 25% sulfuric acid at 40 to 103 degrees C, filtered to recover solids, and then dried in a fluidized bed dryer to concentrate the acid. Acid concentration, steeping temperature, drying time, and temperature effects are described by the current work. Hydrolysis of the pretreated lignocelloses
Acid hydrolysis and pretreatment of lignocellulosic substrates: Final subcontract report
H. Grethlein; A. Converse; J. VickRoy; J. Holland; W. Grous; J. Ward
This report presents findings on acid hydrolysis and pretreatment of lignocellulosic substrates. Acetone was incorporated as a major fluid component to promote the hydrolysis of wood in the presence of dilute sulfuric acid and water. The use of a novel cyclone reactor design was explored through computer simulation and prototype construction. An investigation of using sulfur dioxide near the critical
Hydrolysis of dietary fat by pancreatic lipase stimulates cholecystokinin release
Pius Hildebrand; Christophe Petrig; Beat Burckhardt; Silvia Ketterer; Hans Lengsfeld; André Fleury; Paul Hadváry; Christoph Beglinger
Background & Aims: The hypothesis that cholecystokinin release requires adequate dietary fat digestion in the small intestine was investigated in 10 healthy volunteers, and the consequences of reduced fat hydrolysis on pancreaticobiliary secretions were assessed. Methods: Fat hydrolysis was inhibited by intraduodenal perfusion of tetrahydrolipstatin, an irreversible lipase inhibitor. An oil emulsion containing 0, 30, 60, or 120 mg tetrahydrolipstatin
Selective hydrolysis of wastewater sludge Part 1, December 2008
Report Selective hydrolysis of wastewater sludge Part 1, December 2008 Revised Model calculations and cost benefit analysis for Esbjerg Vest wastewater treatment plant, Denmark PSO-F&U project nr. 2006 This project "Selective hydrolysis of wastewater sludge" is supported by EnergiNet .DK under the PSO
Selective hydrolysis of wastewater sludge Part 1, September 2007
Report Selective hydrolysis of wastewater sludge Part 1, September 2007 Model calculations and cost "Selective hydrolysis of wastewater sludge" is supported by EnergiNet.DK under the PSO-F&U projects having National Laboratory, Rambřll, the Estate of Overgaard and SamRas. The wastewater treatment plant Esbjerg
Avicel hydrolysis by cellulase enzyme in supercritical CO2
Yizhou Zheng; George T. Tsao
The enzymatic hydrolysis reaction in supercritical CO2 to produce glucose from cellulosic material Avicel was investigated. In comparison with the result from the enzymatic hydrolysis reaction of Avicel without CO2 introduced as a reaction medium, the reaction rate and glucose concentration are increased.
Supercritical carbon dioxide explosion as a pretreatment for cellulose hydrolysis
Yizhou Zheng; Ho-Mu Lin; Jingquan Wen; Ningjun Cao; Xuezhi Yu; George T. Tsao
Cellulosic material Avicel was treated with supercritical carbon dioxide to increase the reactivity of cellulose, thereby to enhance the rate and the extent of cellulose hydrolysis. Upon an explosive release of the carbon dioxide pressure, the disruption of the cellulosic structure increases the accessible surface area of the cellulosic substrate to enzymatic hydrolysis. This explosion pretreatment enhances the rate of
A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.
Schneiter, R; Hitomi, M; Ivessa, A S; Fasch, E V; Kohlwein, S D; Tartakoff, A M
The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope. PMID:8943372
Modeling of autocatalytic hydrolysis of adefovir dipivoxil in solid formulations.
Dong, Ying; Zhang, Yan; Xiang, Bingren; Deng, Haishan; Wu, Jingfang
The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs. PMID:21467805
DNA-Catalyzed Hydrolysis of Esters and Aromatic Amides
Brandsen, Benjamin M.; Hesser, Anthony R.; Castner, Marissa A.; Chandra, Madhavaiah
We previously reported that DNA catalysts (deoxyribozymes) can hydrolyze DNA phosphodiester linkages, but DNA-catalyzed amide bond hydrolysis has been elusive. Here we used in vitro selection to identify DNA catalysts that hydrolyze ester linkages as well as DNA catalysts that hydrolyze aromatic amides, for which the leaving group is an aniline moiety. The aromatic amide-hydrolyzing deoxyribozymes were examined using linear free energy relationship analysis. The hydrolysis reaction is unaffected by substituents on the aromatic ring (? ? 0), suggesting general acid-catalyzed elimination as the likely rate-determining step of the addition-elimination hydrolysis mechanism. These findings establish that DNA has the catalytic ability to achieve hydrolysis of esters and aromatic amides as carbonyl-based substrates, and they suggest a mechanism-based approach to achieve DNA-catalyzed aliphatic amide hydrolysis. PMID:24127695
Fermentable sugars by chemical hydrolysis of biomass
Binder, Joseph B.; Raines, Ronald T.
Abundant plant biomass has the potential to become a sustainable source of fuels and chemicals. Realizing this potential requires the economical conversion of recalcitrant lignocellulose into useful intermediates, such as sugars. We report a high-yielding chemical process for the hydrolysis of biomass into monosaccharides. Adding water gradually to a chloride ionic liquid-containing catalytic acid leads to a nearly 90% yield of glucose from cellulose and 70–80% yield of sugars from untreated corn stover. Ion-exclusion chromatography allows recovery of the ionic liquid and delivers sugar feedstocks that support the vigorous growth of ethanologenic microbes. This simple chemical process, which requires neither an edible plant nor a cellulase, could enable crude biomass to be the sole source of carbon for a scalable biorefinery. PMID:20194793
Storage oil hydrolysis during early seedling growth.
Quettier, Anne-Laure; Eastmond, Peter J
Storage oil breakdown plays an important role in the life cycle of many plants by providing the carbon skeletons that support seedling growth immediately following germination. This metabolic process is initiated by lipases (EC: 126.96.36.199), which catalyze the hydrolysis of triacylglycerols (TAGs) to release free fatty acids and glycerol. A number of lipases have been purified to near homogeneity from seed tissues and analysed for their in vitro activities. Furthermore, several genes encoding lipases have been cloned and characterised from plants. However, only recently has data been presented to establish the molecular identity of a lipase that has been shown to be required for TAG breakdown in seeds. In this review we briefly outline the processes of TAG synthesis and breakdown. We then discuss some of the biochemical literature on seed lipases and describe the cloning and characterisation of a lipase called SUGAR-DEPENDENT1, which is required for TAG breakdown in Arabidopsis thaliana seeds. PMID:19136267
Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation
Wubben, T.; Mesecar, A.D. (Purdue); (UIC)
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.
Trihalomethane hydrolysis in drinking water at elevated temperatures.
Zhang, Xiao-Lu; Yang, Hong-Wei; Wang, Xiao-Mao; Karanfil, Tanju; Xie, Yuefeng F
Hydrolysis could contribute to the loss of trihalomethanes (THMs) in the drinking water at elevated temperatures. This study was aimed at investigating THM hydrolysis pertaining to the storage of hot boiled water in enclosed containers. The water pH value was in the range of 6.1-8.2 and the water temperature was varied from 65 to 95 °C. The effects of halide ions, natural organic matter, and drinking water matrix were investigated. Results showed that the hydrolysis rates declined in the order following CHBrCl2 > CHBr2Cl > CHBr3 > CHCl3. THM hydrolysis was primarily through the alkaline pathway, except for CHCl3 in water at relatively low pH value. The activation energies for the alkaline hydrolysis of CHCl3, CHBrCl2, CHBr2Cl and CHBr3 were 109, 113, 115 and 116 kJ/mol, respectively. No hydrolysis intermediates could accumulate in the water. The natural organic matter, and probably other constituents, in drinking water could substantially decrease THM hydrolysis rates by more than 50%. When a drinking water was at 90 °C or above, the first order rate constants for THM hydrolysis were in the magnitude of 10(-2)?10(-1) 1/h. When the boiled real tap water was stored in an enclosed container, THMs continued increasing during the first few hours and then kept decreasing later on due to the competition between hydrolysis and further formation. The removal of THMs, especially brominated THMs, by hydrolysis would greatly reduce one's exposure to disinfection by-products by consuming the boiled water stored in enclosed containers. PMID:25898249
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
You, Di; Yao, Li-li; Huang, Dan; Escalante-Semerena, Jorge C.
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 Lys237, Lys380, Lys611, and Lys628 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. PMID:24957627
Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties.
Escamilla-Trevińo, Luis L; Shen, Hui; Uppalapati, Srinivasa Rao; Ray, Tui; Tang, Yuhong; Hernandez, Timothy; Yin, Yanbin; Xu, Ying; Dixon, Richard A
The down-regulation of enzymes of the monolignol pathway results in reduced recalcitrance of biomass for lignocellulosic ethanol production. Cinnamoyl CoA reductase (CCR) catalyzes the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. However, plants contain multiple CCR-like genes, complicating the selection of lignin-specific targets. This study was undertaken to understand the complexity of the CCR gene family in tetraploid switchgrass (Panicum virgatum) and to determine the biochemical properties of the encoded proteins. Four switchgrass cDNAs (most with multiple variants) encoding putative CCRs were identified by phylogenetic analysis, heterologously expressed in Escherichia coli, and the corresponding enzymes were characterized biochemically. Two cDNAs, PvCCR1 and PvCCR2, encoded enzymes with CCR activity. They are phylogenetically distinct, differentially expressed, and the corresponding enzymes exhibited different biochemical properties with regard to substrate preference. PvCCR1 has higher specific activity and prefers feruloyl CoA as substrate, whereas PvCCR2 prefers caffeoyl and 4-coumaroyl CoAs. Allelic variants of each cDNA were detected, but the two most diverse variants of PvCCR1 encoded enzymes with similar catalytic activity. Based on its properties and expression pattern, PvCCR1 is probably associated with lignin biosynthesis during plant development (and is therefore a target for the engineering of improved biomass), whereas PvCCR2 may function in defense. PMID:19761442
Hydrolysis of glucosinolates to isothiocyanates after ingestion of raw or microwaved cabbage by human volunteers.
Rouzaud, Gabrielle; Young, Sheila A; Duncan, Alan J
Cabbage contains the glucosinolate sinigrin, which is hydrolyzed by myrosinase to allyl isothiocyanate. Isothiocyanates are thought to inhibit the development of cancer cells by a number of mechanisms. The effect of cooking cabbage on isothiocyanate production from glucosinolates during and after their ingestion was examined in human subjects. Each of 12 healthy human volunteers consumed three meals, at 48-h intervals, containing either raw cabbage, cooked cabbage, or mustard according to a cross-over design. At each meal, watercress juice, which is rich in phenethyl isothiocyanate, was also consumed to allow individual and temporal variation in postabsorptive isothiocyanate recovery to be measured. Volunteers recorded the time and volume of each urination for 24 h after each meal. Samples of each urination were analyzed for N-acetyl cysteine conjugates of isothiocyanates as a measure of entry of isothiocyanates into the peripheral circulation. Excretion of isothiocyanates was rapid and substantial after ingestion of mustard, a source of preformed allyl isothiocyanate. After raw cabbage consumption, allyl isothiocyanate was again rapidly excreted, although to a lesser extent than when mustard was consumed. On the cooked cabbage treatment, excretion of allyl isothiocyanate was considerably less than for raw cabbage, and the excretion was delayed. The results indicate that isothiocyanate production is more extensive after consumption of raw vegetables but that isothiocyanates still arise, albeit to a lesser degree, when cooked vegetables are consumed. The lag in excretion on the cooked cabbage treatment suggests that the colon microflora catalyze glucosinolate hydrolysis in this case. PMID:14744743
Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity
Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J.; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee
Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level. PMID:22693256
Metabolic Regulation of Protein N-alpha-acetylation by Bcl-xL Promotes Cell Survival
Yi, Caroline H.; Pan, Heling; Seebacher, Jan; Jang, Il-Ho; Hyberts, Sven G.; Heffron, Gregory J.; Vander Heiden, Matthew G.; Yang, Renliang; Li, Fupeng; Locasale, Jason W.; Sharfi, Hadar; Zhai, Bo; Rodriguez-Mias, Ricard; Luithardt, Harry; Cantley, Lewis C.; Daley, George Q.; Asara, John M.; Gygi, Steven P.; Wagner, Gerhard; Liu, Chuan-Fa; Yuan, Junying
Summary Previous experiments suggest a connection between the N-alpha-acetylation of proteins and the sensitivity of cells to apoptotic signals. Here, we describe a novel biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the anti-apoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We conclude that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation. PMID:21854985
Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics
Abshiru, Nebiyu; Ippersiel, Kevin; Tang, Yong; Yuan, Hua; Marmorstein, Ronen; Verreault, Alain; Thibault, Pierre
Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme to substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. PMID:23036725
N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex
Scott, Daniel C.; Monda, Julie K.; Bennett, Eric J.; Harper, J. Wade; Schulman, Brenda A. (Harvard-Med); (SJCH)
Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.
Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review.
Kasaai, Mohammad R
Chitin, chitosan, and their derivatives have been identified as versatile biopolymers for a broad range of agriculture and food applications. Up to now, several methods have been developed to determine degree of N-acetylation, DA, for chitin and chitosan. In this article, an effort has been made to review the available literature information on the DA determination. These methods are classified into three categories: (1) spectroscopy (IR, (1)H NMR, (13)C NMR, (15)N NMR, and UV); (2) conventional (various types of titration, conductometry, potentiometry, ninhydrin assay, adsorption of free amino groups of chitosan by pictric acid); (3) destructive (elemental analysis, acid or enzymatic hydrolysis of chitin/chitosan and followed by the DA measurement by colorimetry or high performance liquid chromatography, pyrolysis-gas chromatography, and thermal analysis using differential scanning calorimetry) methods. These methods have been compared for their performances and limitations as well as their advantages and disadvantages. The use of IR and NMR spectroscopy methods provides a number of advantages. They do not need long-term procedures to prepare samples, and they provide information on the chemical structure. (1)H NMR and UV techniques are more sensitive than IR, (13)C NMR, and (15)N NMR spectroscopy. The IR technique is mostly used for a qualitative evaluation and comparison studies. Conventional methods are not applicable for highly acetylated chitin. The results of the latter methods are affected by ionic strength of the solvent, pH, and temperature of solution. In destructive methods, longer times are needed for the measurements compared to spectroscopy and conventional methods, but they are applicable for the entire range of the DA. PMID:19187020
Facile microencapsulation of curcumin in acetylated starch microparticles.
Nata, Iryanti Fatyasari; Chen, Kuan-Jung; Lee, Cheng-Kang
Highly acetylated starch was prepared by reacting corn starch with acetic anhydride. Acetylated starch (AS) is soluble in acetone and can be precipitated out from AS-acetone solution when deionised (DI) water is added as a non-solvent. The curcumin dissolved in AS-acetone solution could be effectively encapsulated in AS as spherical microparticles (1-3??m) when mixed with the DI water. The encapsulation yield of curcumin could reach 96.3%. AS encapsulation not only resulted in five-fold lower UV degradation rate of curcumin, the free radical scavenging activity of curcumin still could be well maintained. Approximately, 40% of the 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) was consumed by curcumin encapsulated in AS microparticles within 2?min. PMID:24697176
Elevated cholesterol reduces acetylsalicylic acid-mediated platelet acetylation
Magdalena Boncler; Peter Gresner; Marek Nocun; Joanna Rywaniak; Martin Dolnik; Jacek Rysz; Radoslaw Wilk; Malgorzata Czyz; Leszek Markuszewski; Maciej Banach; Cezary Watala
We describe the role of plasma and platelet cholesterol content in the ability of acetylsalicylic acid (ASA) to acetylate platelet proteins and inhibit platelet function. Platelet susceptibility to ASA was monitored in subjects differing in plasma total cholesterol and in suspensions of cholesterol-enriched or cholesterol-depleted platelets. Platelets from subjects with higher plasma cholesterol (>6 mmol\\/l) showed reduced platelet sensitivity to ASA
Acetylcholinesterase inhibition activity of acetylated depsidones from Lobaria pulmonaria
Boris Pejin; Giuseppina Tommonaro; Carmine Iodice; Vele Tesevic; Vlatka Vajs
As part of our ongoing project of new acetylcholinesterase inhibitors from lower marine and terrestrial species, a phytochemical investigation was conducted on a foliose lichen, Lobaria pulmonaria (L.) Hoffm. (Lobariaceae), from Bosnia and Herzegovina. The study led to the isolation of a mixture of acetylated depsidones which showed a moderate activity (0.5?µg) in the acetylcholinesterase inhibition test on Thin-layer chromatography
Acetylcholinesterase inhibition activity of acetylated depsidones from Lobaria pulmonaria
Boris Pejin; Giuseppina Tommonaro; Carmine Iodice; Vele Tesevic; Vlatka Vajs
As part of our ongoing project of new acetylcholinesterase inhibitors from lower marine and terrestrial species, a phytochemical investigation was conducted on a foliose lichen, Lobaria pulmonaria (L.) Hoffm. (Lobariaceae), from Bosnia and Herzegovina. The study led to the isolation of a mixture of acetylated depsidones which showed a moderate activity (0.5?µg) in the acetylcholinesterase inhibition test on Thin-layer chromatography
Histone acetylations mark origins of polycistronic transcription in Leishmania major
Thomas, Sean; Green, Amanda; Sturm, Nancy R; Campbell, David A; Myler, Peter J
Background Many components of the RNA polymerase II transcription machinery have been identified in kinetoplastid protozoa, but they diverge substantially from other eukaryotes. Furthermore, protein-coding genes in these organisms lack individual transcriptional regulation, since they are transcribed as long polycistronic units. The transcription initiation sites are assumed to lie within the 'divergent strand-switch' regions at the junction between opposing polycistronic gene clusters. However, the mechanism by which Kinetoplastidae initiate transcription is unclear, and promoter sequences are undefined. Results The chromosomal location of TATA-binding protein (TBP or TRF4), Small Nuclear Activating Protein complex (SNAP50), and H3 histones were assessed in Leishmania major using microarrays hybridized with DNA obtained through chromatin immunoprecipitation (ChIP-chip). The TBP and SNAP50 binding patterns were almost identical and high intensity peaks were associated with tRNAs and snRNAs. Only 184 peaks of acetylated H3 histone were found in the entire genome, with substantially higher intensity in rapidly-dividing cells than stationary-phase. The majority of the acetylated H3 peaks were found at divergent strand-switch regions, but some occurred at chromosome ends and within polycistronic gene clusters. Almost all these peaks were associated with lower intensity peaks of TBP/SNAP50 binding a few kilobases upstream, evidence that they represent transcription initiation sites. Conclusion The first genome-wide maps of DNA-binding protein occupancy in a kinetoplastid organism suggest that H3 histones at the origins of polycistronic transcription of protein-coding genes are acetylated. Global regulation of transcription initiation may be achieved by modifying the acetylation state of these origins. PMID:19356248
Photodecomposition of acetyl chloride on the excited singlet state surface
R. Sumathi; A. K. Chandra
We investigate the plausible mechanism of fast decomposition of acetyl chloride upon 1n?* (CO) excitation through the selective C–Cl bond-fission on the lowest excited singlet state surface using abinitio quantum chemical methods. Effects of zero point energy corrections and of electron correlation have been considered. The pathway involves dissociation, via a ?Cl?*CO and a ?Cl?*C–Cl configurations where ?'s stand for
Human platelet stimulation by acetyl glyceryl ether phosphorylcholine.
McManus, L M; Hanahan, D J; Pinckard, R N
Acetyl glyceryl ether phosphorylcholine (AGEPC) induced dose-dependent platelet aggregation and release of [3H]serotonin and platelet factor 4 in citrated human platelet-rich plasma. ADP scavengers or indomethacin prevented irreversible platelet aggregation responses induced by 0.2 microM AGEPC but had no effect upon platelet secretion; prostacyclin inhibited AGEPC-induced aggregation and secretion. EDTA or EGTA inhibited AGEPC-induced aggregation but had no effect on platelet secretion. PMID:7204562
Histone acetylation modifiers in the pathogenesis of malignant disease.
Mahlknecht, U.; Hoelzer, D.
Chromatin structure is gaining increasing attention as a potential target in the treatment of cancer. Relaxation of the chromatin fiber facilitates transcription and is regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which modify the acetylation state of histone proteins and other promoter-bound transcription factors. While HATs, which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, HDACs tend to associate with multisubunit core-pressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. HATs and HDACs are known to be involved both in the pathogenesis as well as in the suppression of cancer. Some of the genes encoding these enzymes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products that may interfere with regulatory cascades controlling cell growth and differentiation. On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation. We review herein the recent achievements, which further help to elucidate the biological role of histone acetylation modifying enzymes and their potential impact on our current understanding of the molecular changes involved in the development of solid tumors and leukemias. PMID:11055583
Hydrolysis of hydrogen cyanide in ammonia liquors
Kamennykh, B.M.; Nazarov, V.G.; Rus'yanova, N.D.; Lebedeva, G.N.
A thermodynamic analysis was performed on the conversion of hydrocyanic acid to anhydrous ammonia in concentrated ammonia liquors obtained from overall purification schemes used for coke oven gas. The temperature range of treatment (90 to 140/sup 0/C) was chosen in view of the standard low pressure (to 588 kPa) available for the process, and with consideration for the industrial liquor regeneration regime. Samples were collected while retaining the liquor in the reactor without agitation at a constant temperature. Changes in the HCN concentration as a function of retention time are described by logarithmic functions. An effective rate constant was calculated. Results show that in comprehensive schemes for gas treatment by cyclic methods with production of anhydrous ammonia and with ammonia, sulfur and cyanide removal, the principal part of the HCN extracted from the gas in the regneration of the wash liquors and treatment of the NH/sub 3/ liquors will be hydrolyzed at high temperature, and the hydrolysis products will be removed. 4 figures, 1 table.
Elevated cholesterol reduces acetylsalicylic acid-mediated platelet acetylation.
Boncler, Magdalena; Gresner, Peter; Nocun, Marek; Rywaniak, Joanna; Dolnik, Martin; Rysz, Jacek; Wilk, Radoslaw; Czyz, Malgorzata; Markuszewski, Leszek; Banach, Maciej; Watala, Cezary
We describe the role of plasma and platelet cholesterol content in the ability of acetylsalicylic acid (ASA) to acetylate platelet proteins and inhibit platelet function. Platelet susceptibility to ASA was monitored in subjects differing in plasma total cholesterol and in suspensions of cholesterol-enriched or cholesterol-depleted platelets. Platelets from subjects with higher plasma cholesterol (>6 mmol/l) showed reduced platelet sensitivity to ASA (inhibition of platelet aggregation and thromboxane generation by 60% and 68% in 'lower-' vs. 32% and 56% in 'higher-cholesterol' donors; n=13 in each group; p=0.056 and p<0.04, respectively). [Acetyl-1-(14)C] incorporation to platelet proteins in subjects with higher plasma cholesterol was significantly reduced (11.0 vs. 14.6 nmol/g protein, p<0.0001) and correlated significantly with blood total cholesterolemia (R(K)=-0.430, p<0.003) and LDL-cholesterol (R(K)=-0.349, p<0.012), but not with platelet cholesterol content. In conclusion, elevated plasma cholesterol is an important determinant of ASA-induced acetylation of platelets and platelet diminished sensitivity to ASA. The molecular basis of such an association remains obscure, notwithstanding it may constitute a link between sub-optimal platelet response to aspirin and lipid metabolic disorders. PMID:17950536
Carbon isotope fractionation and the acetyl-CoA pathway
NASA Astrophysics Data System (ADS)
Blaser, Martin; Conrad, Ralf
Homoacetogenic bacteria can catalyze the reductive synthesis of acetate from CO2 via the acetyl-CoA pathway. Besides this unifying property homoacetogenic bacteria constitute a metabolically and phylogenetically diverse bacteriological group. Therefore their environmental role is difficult to address. It has been recognized that in methanogenic environments homoacetogenic bacteria contribute to the degradation of organic matter. The natural abundance of 13C may be used to understand the functional impact of homoacetogenic bacteria in the soil environment. To distinguish the acetyl-CoA pathway from other dominant processes, the isotopic composition of acetate and CO2 can be determined and the fractionation factors of the individual processes may be used to discriminate between the dominant pathways. To characterize the fractionation factor associated with the acetyl-CoA pathway the phylogenetic and metabolic diversity needs to be considered. Therefore the fractionation factor of substrate utilization and product formation of different homoacetogens (Acetobacterium woodii, Sporomusa ovata, Thermoanaerobacter kivui, Morella thermoautotrophica) has been studied under pure culture conditions in two defined minimal medium with H2/CO2 as sole source of carbon and energy. It became obvious that the cultivation conditions have a major impact on the obtained fractionation factors.
Histone acetylation regulates osteodifferentiation of hDPSCs via DSPP.
Gu, Shensheng; Liang, Jingping; Wang, Jia; Liu, Bin
Dental pulp stem cells (DPSCs) are a unique population of precursor cells isolated from postnatal human dental pulp, with the ability to regenerate a reparative dentin-like complex. We examined the regulation of odontoblast-like differentiation of DPSCs by histone acetylation. Western blot analysis showed that histone H3 acetylation was strongly induced in osteodifferentiation medium. Inhibition of histone acetyltransferase by garcinol reversed osteodifferentiation and mineral formation. Real-time polymerase chain reaction assay indicated that the dentin sialophosphoprotein (DSPP) gene, which is mainly expressed in odontoblasts and preameloblasts in teeth and plays an important role in tooth function, was also down-regulated in garcinol-treated cells. Moreover, lentivirus-mediated knockdown of DSPP in human DPSCs was associated with significant inhibition of mineral formation, but not osteoblast differentiation. In conclusion, the results of this study suggest that DSPP positively affects mineral formation, and that odontoblast-like differentiation and maturation of DPSCs can be regulated by histone acetylation of the DSPP gene. PMID:23747867
Demonstration and cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver.
Pierce, R J; Spik, G; Montreuil, J
An endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type glycoasparagine substrate was demonstrated in rat liver. This activity was optimal at pH 7.0 and was predominantly present in the soluble (cytosolic) fraction. PMID:7378051
Actin Polymerization Overshoots and Hydrolysis as Assayed by Pyrene Fluorescence
Carlsson, Anders
undergo a two-step hydrolysis (12). First, the ATP con- taining subunits (F-ATP) hydrolyze to a state- ing an independent measurement of the amount of polymerized actin that had completely hydrolyzed (F
Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates
Neilson, M.J.; Kelsey, R.G.; Shafizadeh, F.
It has been shown that simultaneous attrition of cellulose in an attritor containing stainless-steel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.
Optimization of enzymatic hydrolysis of cassava to obtain fermentable sugars.
Collares, Renata M; Miklasevicius, Luiza V S; Bassaco, Mariana M; Salau, Nina P G; Mazutti, Marcio A; Bisognin, Dilson A; Terra, Lisiane M
This work evaluates the enzymatic hydrolysis of starch from cassava using pectinase, ?-amylase, and amyloglucosidase. A central composite rotational design (CCRD) was carried out to evaluate the effects of amyloglucosidase, pectinase, reaction time, and solid to liquid ratio. All the experiments were carried out in a bioreactor with working volume of 2 L. Approximately 98% efficiency hydrolysis was obtained, resulting in a concentration of total reducing sugar released of 160 g/L. It was concluded that pectinase improved the hydrolysis of starch from cassava. Reaction time was found to be significant until 7 h of reaction. A solid to liquid ratio of 1.0 was considered suitable for hydrolysis of starch from cassava. Amyloglucosidase was a significant variable in the process: after its addition to the reaction media, a 30%-50% increase in the amount of total reducing sugar released was observed. At optimal conditions the maximum productivity obtained was 22.9 g/(L·h). PMID:22761249
Energetic approach of biomass hydrolysis in supercritical water.
Cantero, Danilo A; Vaquerizo, Luis; Mato, Fidel; Bermejo, M Dolores; Cocero, M José
Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams. PMID:25536511
SPARC chemical reactivity models were extended to calculate hydrolysis rate constants for carboxylic acid esters from molecular structure. The energy differences between the initial state and the transition state for a molecule of interest are factored into internal and external...
Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.
He, Hongzhen; Ding, Yi; Bartlam, Mark; Sun, Fei; Le, Yi; Qin, Xincheng; Tang, Hong; Zhang, Rongguang; Joachimiak, Andrzej; Liu, Jinyuan; Zhao, Nanming; Rao, Zihe
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. PMID:12527305
Software interface for high-speed readout of particle detectors based on the CoaXPress communication standard
NASA Astrophysics Data System (ADS)
Hejtmánek, M.; Neue, G.; Voleš, P.
This article is devoted to the software design and development of a high-speed readout application used for interfacing particle detectors via the CoaXPress communication standard. The CoaXPress provides an asymmetric high-speed serial connection over a single coaxial cable. It uses a widely available 75 ? BNC standard and can operate in various modes with a data throughput ranging from 1.25 Gbps up to 25 Gbps. Moreover, it supports a low speed uplink with a fixed bit rate of 20.833 Mbps, which can be used to control and upload configuration data to the particle detector. The CoaXPress interface is an upcoming standard in medical imaging, therefore its usage promises long-term compatibility and versatility. This work presents an example of how to develop DAQ system for a pixel detector. For this purpose, a flexible DAQ card was developed using the XILINX Spartan 6 FPGA. The DAQ card is connected to the framegrabber FireBird CXP6 Quad, which is plugged in the PCI Express bus of the standard PC. The data transmission was performed between the FPGA and framegrabber card via the standard coaxial cable in communication mode with a bit rate of 3.125 Gbps. Using the Medipix2 Quad pixel detector, the framerate of 100 fps was achieved. The front-end application makes use of the FireBird framegrabber software development kit and is suitable for data acquisition as well as control of the detector through the registers implemented in the FPGA.
Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase
Kaschabek, Stefan R.; Kuhn, Bernd; Müller, Dagmar; Schmidt, Eberhard; Reineke, Walter
The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 ± 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-Oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 ± 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found. PMID:11741862
Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase.
Shim, H; Hong, S B; Raushel, F M
The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters. PMID:9651332
Hydrolysis of the chlorophosphazenes: cyclic trimer and linear polymer
Gabler, Douglas G
. Haw The hydrolysis chemistry of the chlorophosphazene cyclic trimer (phosphonitrilic chloride) has long been of interest, because of its possible relevance to polymerization catalysis, as well as the undesirable role of water in the cross-linking... been previously proposed as a polymerization catalyst. Two of the species are oxo-bridged dimers of two trimer rings that may be useful as model compounds for hydrolytic cross-linking reactions in the polymer. Hydrolysis of poly...
Dilute-acid hydrolysis of sugarcane bagasse at varying conditions
Markus Neureiter; Herbert Danner; Christiane Thomasser; Bamusi Saidi; Rudolf Braun
Sugarcane bagasse, a byproduct of the cane sugar industry, is an abundant source of hemicellulose that could be hydrolyzed\\u000a to yield a fermentation feedstock for the production of fuel ethanol and chemicals. The effects of sulfuric acid concentration,\\u000a temperature, time, and dry matter concentration on hemicellulose hydrolysis were studied with a 20-L batch hydrolysis reactor\\u000a using a statistical experimental design.
Hydrolysis of esters in subcritical and supercritical water
Petra Krammer; Herbert Vogel
The task of this work was to gain a better understanding of the hydrolysis of esters in sub- and supercritical water (SCW). Therefore, the kinetics of the hydrolysis of ethyl acetate as a model reaction was studied in the pressure and temperature range of 23–30MPa and 250–400°C, respectively. The corresponding reaction mechanisms in sub- and supercritical water were discussed.
Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF)
Steven P. Harvey; Jan E. Kolakowski; Tu-Chen Cheng; Vipin K. Rastogi; Louis P. Reiff; Joseph J. DeFrank; Frank M. Raushel; Craig Hill
Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O-cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3mM GF but was evident at 0.5mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp.
Crystal Structure of Tabtoxin Resistance Protein Complexed with Acetyl Coenzyme A Reveals the Mechanism for ?-Lactam Acetylation
Hongzhen He; Yi Ding; Mark Bartlam; Fei Sun; Yi Le; Xincheng Qin; Hong Tang; Rongguang Zhang; Andrzej Joachimiak; Jinyuan Liu; Nanming Zhao; Zihe Rao
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 1.55Ĺ resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related
The effect of microwave irradiation on enzymatic hydrolysis of rice straw
Shengdong Zhu; Yuanxin Wu; Ziniu Yu; Xia Zhang; Hui Li; Ming Gao
A series of experiments involving microwave irradiation were carried out to evaluate the effect of microwave irradiation on enzymatic hydrolysis of rice straw. Compared with microwave irradiation free hydrolysis, rice straw pretreated by combining microwave irradiation with alkali could increase the initial hydrolysis rate but the hydrolysis yield remained unchanged. When the enzyme solution was treated by microwave irradiation, the
Toxicology 212 (2005) 107115 Carbofuran and malathion inhibit nucleotide hydrolysis in
Eizirik, Eduardo
Toxicology 212 (2005) 107115 Carbofuran and malathion inhibit nucleotide hydrolysis in zebrafish and ADP hydrolysis in an uncompetitive manner, but no effect was observed on AMP hydrolysis. Malathion decreased ATP and ADP hydrolysis in competitive and an uncompetitive manner, respectively, but not altered
Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.
Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K; Dean, Dennis R; Hoffman, Brian M; Antony, Edwin; Seefeldt, Lance C
The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein. PMID:24062462
Intramolecular general base catalyzed ester hydrolysis. The hydrolysis of 2-aminobenzoate esters.
Fife, Thomas H; Singh, Randhir; Bembi, Ramesh
Rate constants have been obtained for the hydrolysis of the trifluoroethyl, phenyl, and p-nitrophenyl esters of 2-aminobenzoic acid at 50 degrees C in H(2)O. The pseudo-first-order rate constants, k(obsd), are pH independent from pH 8 to pH 4 (the pK(a) of the amine group conjugate acid). The 2-aminobenzoate esters hydrolyze with similar rate constants in the pH-independent reactions, and these water reactions are approximately 2-fold slower in D(2)O than in H(2)O. The most likely mechanism involves intramolecular general base catalysis by the neighboring amine group. The rate enhancements in the pH-independent reaction in comparison with the pH-independent hydrolysis of the corresponding para substituted esters or the benzoate esters are 50-100-fold. In comparison with the hydroxide ion catalyzed reaction, the enhancement in k(obsd) at pH 4 with the phenyl ester is 10(5)-fold. Intramolecular general base catalyzed reactions are assessed in respect to their relative advantages and disadvantages in enzyme catalysis. A general base catalyzed reaction can be more rapid at low pH than a nucleophilic reaction that has a marked dependence on pH and the leaving group. PMID:12003523
Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum
Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl
In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.
New short and general synthesis of three key Maillard flavour compounds: 2-Acetyl-1-pyrroline, 6-acetyl-1,2,3,4-tetrahydropyridine and 5-acetyl-2,3-dihydro-4H-1,4-thiazine.
Deblander, Jurgen; Van Aeken, Sam; Adams, An; De Kimpe, Norbert; Abbaspour Tehrani, Kourosch
A new general synthetic route towards three key Maillard flavour compounds, namely 2-acetyl-1-pyrroline, 6-acetyl-1,2,3,4-tetrahydropyridine and 5-acetyl-2,3-dihydro-4H-1,4-thiazine, was developed. The key step in the process is the methylenation reaction of azaheterocyclic carboxylic esters by means of dimethyltitanocene, giving rise to intermediate vinyl ethers which can be considered as excellent and stable precursors for the title compounds, as a simple acidic treatment of these precursors suffices to release the characteristic Maillard flavours. PMID:25172717
Production of N-acetyl- d-neuraminic acid by coupling bacteria expressing N-acetyl- d-glucosamine 2-epimerase and N-acetyl- d-neuraminic acid synthetase
Kazuhiko Tabata; Satoshi Koizumi; Tetsuo Endo; Akio Ozaki
N-acetyl-d-glucosamine (GlcNAc) 2-epimerase catalyzes the interconversion between GlcNAc and N-acetyl-d-mannosamine (ManNAc) that is a precursor of N-acetyl-d-neuraminic acid (NeuAc). Homology search using the sequence of the porcine GlcNAc 2-epimerase as a query revealed that a gene product (Slr1975) of Synechocystis sp. PCC6803 showed significant homology. When the gene of slr1975 was cloned by PCR and expressed in Escherichia coli, the
Acetylation of MyoD by p300 requires more than its histone acetyltransferase domain.
Polesskaya, A; Harel-Bellan, A
MyoD, an essential transcription factor involved in muscle cell terminal differentiation, is regulated by acetylation, as are a number of other transcription factors, but the histone acetyltransferase enzyme responsible for this acetylation is a matter of controversy. In particular, contradictory findings have been reported concerning the ability of CBP/p300 to acetylate MyoD in vitro. Here we provide an explanation for this discrepancy: although full-length p300 does indeed acetylate MyoD, a fragment of p300 corresponding to its histone acetyltransferase domain does not. In addition to clearly demonstrating that p300 acetylates MyoD in vitro, these results underscore the necessity of using full-length histone acetyltransferase enzymes to draw valid conclusions from acetylation experiments. PMID:11577095
FANCJ\\/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response
Jenny Xie; Min Peng; Shawna Guillemette; Steven Quan; Stephanie Maniatis; Yuliang Wu; Aditya Venkatesh; Scott A. Shaffer; Robert M. Brosh; Sharon B. Cantor
BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1\\/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive
Autoimmune regulator is acetylated by transcription coactivator CBP/p300
Saare, Mario, E-mail: email@example.com [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)] [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) [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)] [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)] [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)
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 target genes.
Identification of Carboxylesterase-Dependent Dabigatran Etexilate Hydrolysis
Parker, Robert B.; Herring, Vanessa L.; Hu, Zhe-Yi
Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted to the active thrombin inhibitor, dabigatran (DAB), by serine esterases. The aims of the present study were to investigate the in vitro kinetics and pathway of DABE hydrolysis by human carboxylesterase enzymes, and the effect of alcohol on these transformations. The kinetics of DABE hydrolysis in two human recombinant carboxylesterase enzymes (CES1 and CES2) and in human intestinal microsomes and human liver S9 fractions were determined. The effects of alcohol (a known CES1 inhibitor) on the formation of DABE metabolites in carboxylesterase enzymes and human liver S9 fractions were also examined. The inhibitory effect of bis(4-nitrophenyl) phosphate on the carboxylesterase-mediated metabolism of DABE and the effect of alcohol on the hydrolysis of a classic carboxylesterase substrate (cocaine) were studied to validate the in vitro model. The ethyl ester of DABE was hydrolyzed exclusively by CES1 to M1 (Km 24.9 ± 2.9 ?M, Vmax 676 ± 26 pmol/min per milligram protein) and the carbamate ester of DABE was exclusively hydrolyzed by CES2 to M2 (Km 5.5 ± 0.8 ?M; Vmax 71.1 ± 2.4 pmol/min per milligram protein). Sequential hydrolysis of DABE in human intestinal microsomes followed by hydrolysis in human liver S9 fractions resulted in complete conversion to DAB. These results suggest that after oral administration of DABE to humans, DABE is hydrolyzed by intestinal CES2 to the intermediate M2 metabolite followed by hydrolysis of M2 to DAB in the liver by CES1. Carboxylesterase-mediated hydrolysis of DABE was not inhibited by alcohol. PMID:24212379
Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells
Barjaktarovic, Zarko; Kempf, Stefan J.; Sriharshan, Arundhathi; Merl-Pham, Juliane; Atkinson, Michael J.; Tapio, Soile
Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium. PMID:25840449
Glucose and SIRT2 reciprocally mediate the regulation of keratin 8 by lysine acetylation.
Snider, Natasha T; Leonard, Jessica M; Kwan, Raymond; Griggs, Nicholas W; Rui, Liangyou; Omary, M Bishr
Lysine acetylation is an important posttranslational modification that regulates microtubules and microfilaments, but its effects on intermediate filament proteins (IFs) are unknown. We investigated the regulation of keratin 8 (K8), a type II simple epithelial IF, by lysine acetylation. K8 was basally acetylated and the highly conserved Lys-207 was a major acetylation site. K8 acetylation regulated filament organization and decreased keratin solubility. Acetylation of K8 was rapidly responsive to changes in glucose levels and was up-regulated in response to nicotinamide adenine dinucleotide (NAD) depletion and in diabetic mouse and human livers. The NAD-dependent deacetylase sirtuin 2 (SIRT2) associated with and deacetylated K8. Pharmacologic or genetic inhibition of SIRT2 decreased K8 solubility and affected filament organization. Inhibition of K8 Lys-207 acetylation resulted in site-specific phosphorylation changes of K8. Therefore, K8 acetylation at Lys-207, a highly conserved residue among type II keratins and other IFs, is up-regulated upon hyperglycemia and down-regulated by SIRT2. Keratin acetylation provides a new mechanism to regulate keratin filaments, possibly via modulating keratin phosphorylation. PMID:23358244
Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury
Yildirim, Ferah
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 ...
Enzymatic 2'-N-acetylation of arbekacin and antibiotic activity of its product.
Hotta, K; Zhu, C B; Ogata, T; Sunada, A; Ishikawa, J; Mizuno, S; Ikeda, Y; Kondo, S
Aminoglycoside antibiotics (AGs) with a free 2'-amino group were subjected to enzymatic N-acetylation using a cell free extract that contained an aminoglycoside 2'-N-acetyltransferase, AAC (2'), derived from a kasugamycin-producing strain of Streptomyces kasugaensis. TLC and antibiotic assay of the incubated reaction mixtures revealed that a modified compound retaining substantial antibiotic activity was formed from arbekacin (ABK), while modification of the other AGs resulted in the marked decrease in antibiotic activity. Structure determination following isolation from a large scale reaction mixture showed the modified ABK to be 2'-N-acetyl ABK. In addition, 2',6'-di-N-acetyl ABK was formed as a minor product. The same conversion also occurred with dibekacin (DKB) resulting in the formation of 2'-N-acetyl DKB and 2',6'-di-N-acetyl DKB. MIC determination showed antibacterial activity (1.56 approximately 3.13 micrograms/ml) of 2'-N-acetyl ABK against a variety of organisms. By contrast, 2'-N-acetyl DKB showed no substantial antibiotic activity. We believe 2'-N-acetyl ABK has the highest and broadest antibacterial activity, compared with known N-acetylated AGs. PMID:8682723
Somnogenic activity of O-acetylated and dimeric muramyl peptides.
Johannsen, L; Rosenthal, R S; Martin, S A; Cady, A B; Obal, F; Guinand, M; Krueger, J M
Slow-wave sleep-promoting factors in brain and urine were identified as muramyl peptides (MPs), the building blocks of bacterial cell wall peptidoglycan. In this study, structural variations of MPs that occur naturally in bacterial peptidoglycan were investigated for somnogenic activity. Monomeric and dimeric MPs were isolated and purified from Neisseria gonorrhoeae and Actinomadura sp. strain R39. The structures of these MPs were verified by fast atom bombardment mass spectroscopy and tandem mass spectroscopy. After intracerebroventricular administration of MPs, electroencephalograms and brain temperatures of rabbits were recorded for 6 h and were analyzed to determine durations of slow-wave sleep, rapid-eye-movement sleep, and wakefulness. The 6-O acetylation of muramic acid enhanced the somnogenic effects of certain monomeric MPs relative to their non-O-acetylated (but otherwise identical) counterparts. Two monomeric MPs containing an unsubstituted amide (i.e., Iso-Gln) were inactive, thus confirming previous results showing that amidation of a variety of MPs can block somnogenic activity. Two peptide-cross-linked MP dimers tested had no effect on slow-wave sleep, although a third peptide-cross-linked MP containing a 1,6-anhydro muramyl end on one of its monomeric subunits, a structure that enhances somnogenic potency of un-cross-linked monomers, was somnogenic. Two dimers connected by glycosidic bonds and containing an Iso-Gln moiety were inactive. Two other glycosidically linked dimers that also contained an Iso-Gln moiety, but were of lower molecular weight, were somnogenic. In summary, 6-O acetylation of muramic acid in somnogenic MPs enhances activity, and as a class, peptide-linked dimeric MPs tend to be less active than their constituent monomers. PMID:2759708
Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties.
Orfila, Caroline; Dal Degan, Florence; Jřrgensen, Bodil; Scheller, Henrik Vibe; Ray, Peter M; Ulvskov, Peter
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. PMID:22293853
Hydrolysis and fractionation of lignocellulosic biomass
Torget, R.W.; Padukone, N.; Hatzis, C.; Wyman, C.E.
A multi-function process is described for the hydrolysis and fractionation of lignocellulosic biomass to separate hemicellulosic sugars from other biomass components such as extractives and proteins; a portion of the solubilized lignin; cellulose; glucose derived from cellulose; and insoluble lignin from said biomass comprising one or more of the following: optionally, as function 1, introducing a dilute acid of pH 1.0--5.0 into a continual shrinking bed reactor containing a lignocellulosic biomass material at a temperature of about 94 to about 160 C for a period of about 10 to about 120 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of extractives, lignin, and protein by keeping the solid to liquid ratio constant throughout the solubilization process; as function 2, introducing a dilute acid of pH 1.0--5.0, either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing either fresh biomass or the partially fractionated lignocellulosic biomass material from function 1 at a temperature of about 94--220 C for a period of about 10 to about 60 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of hemicellulosic sugars, semisoluble sugars and other compounds, and amorphous glucans by keeping the solid to liquid ratio constant throughout the solubilization process; as function 3, optionally, introducing a dilute acid of pH 1.0--5.0 either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing the partially fractionated lignocellulosic biomass material from function 2 at a temperature of about 180--280 C for a period of about 10 to about 60 minutes at a volumetric flow rate of 1 to about 5 reactor volumes to effect solubilization of cellulosic sugars by keeping the solid to liquid ratio constant throughout the solubilization process; and as function 4, optionally, introducing a dilute acid of pH 1.0--5.0 either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing the partially fractionated lignocellulosic biomass material from function 3 at a temperature of about 180--280 C for a period of about 10 to about 60 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of cellulosic sugars by keeping the solid to liquid ratio constant throughout the solubilization process.
Acetylcholinesterase inhibition activity of acetylated depsidones from Lobaria pulmonaria.
Pejin, Boris; Tommonaro, Giuseppina; Iodice, Carmine; Tesevic, Vele; Vajs, Vlatka
As part of our ongoing project of new acetylcholinesterase inhibitors from lower marine and terrestrial species, a phytochemical investigation was conducted on a foliose lichen, Lobaria pulmonaria (L.) Hoffm. (Lobariaceae), from Bosnia and Herzegovina. The study led to the isolation of a mixture of acetylated depsidones which showed a moderate activity (0.5?µg) in the acetylcholinesterase inhibition test on Thin-layer chromatography plate. Our results indicate for the first time the significance of depsidones, highly specific metabolites from lichen species, in searching for these inhibitors which still represent the best drugs currently available for the management of Alzheimer's disease. PMID:21985528
Piperazine oxadiazole inhibitors of acetyl-CoA carboxylase.
Bourbeau, Matthew P; Siegmund, Aaron; Allen, John G; Shu, Hong; Fotsch, Christopher; Bartberger, Michael D; Kim, Ki-Won; Komorowski, Renee; Graham, Melissa; Busby, James; Wang, Minghan; Meyer, James; Xu, Yang; Salyers, Kevin; Fielden, Mark; Véniant, Murielle M; Gu, Wei
Acetyl-CoA carboxylase (ACC) is a target of interest for the treatment of metabolic syndrome. Starting from a biphenyloxadiazole screening hit, a series of piperazine oxadiazole ACC inhibitors was developed. Initial pharmacokinetic liabilities of the piperazine oxadiazoles were overcome by blocking predicted sites of metabolism, resulting in compounds with suitable properties for further in vivo studies. Compound 26 was shown to inhibit malonyl-CoA production in an in vivo pharmacodynamic assay and was advanced to a long-term efficacy study. Prolonged dosing with compound 26 resulted in impaired glucose tolerance in diet-induced obese (DIO) C57BL6 mice, an unexpected finding. PMID:24294923
Theoretical structure investigations of N-acetyl-l-proline amide
NASA Astrophysics Data System (ADS)
Ramek, Michael; Kelterer, Anne-Marie; Teppen, Brian J.; Schäfer, Lothar
The potential energy surface of N-acetyl-l-proline amide has been investigated via RHF, AM1, and PM3 calculations. The results show significant differences between these methods: seven local minima can be found with RHF, three with AM1, 17 with PM3. The conformation of the RHF/6-31G? global minimum corresponds to the ?-turn structure of polypeptides. In contrast to this, the proline conformer that participates in the formation of ten-membered ?-turns in peptide chains has a relatively high energy in the dipeptide.
Sulfation of deoxynivalenol, its acetylated derivatives, and T2-toxin?
Fruhmann, Philipp; Skrinjar, Philipp; Weber, Julia; Mikula, Hannes; Warth, Benedikt; Sulyok, Michael; Krska, Rudolf; Adam, Gerhard; Rosenberg, Erwin; Hametner, Christian; Fröhlich, Johannes
The synthesis of several sulfates of trichothecene mycotoxins is presented. Deoxynivalenol (DON) and its acetylated derivatives were synthesized from 3-acetyldeoxynivalenol (3ADON) and used as substrate for sulfation in order to reach a series of five different DON-based sulfates as well as T2-toxin-3-sulfate. These substances are suspected to be formed during phase-II metabolism in plants and humans. The sulfation was performed using a sulfuryl imidazolium salt, which was synthesized prior to use. All protected intermediates and final products were characterized via NMR and will serve as reference materials for further investigations in the fields of toxicology and bioanalytics of mycotoxins. PMID:25170180
Sulfation of deoxynivalenol, its acetylated derivatives, and T2-toxin.
Fruhmann, Philipp; Skrinjar, Philipp; Weber, Julia; Mikula, Hannes; Warth, Benedikt; Sulyok, Michael; Krska, Rudolf; Adam, Gerhard; Rosenberg, Erwin; Hametner, Christian; Fröhlich, Johannes
The synthesis of several sulfates of trichothecene mycotoxins is presented. Deoxynivalenol (DON) and its acetylated derivatives were synthesized from 3-acetyldeoxynivalenol (3ADON) and used as substrate for sulfation in order to reach a series of five different DON-based sulfates as well as T2-toxin-3-sulfate. These substances are suspected to be formed during phase-II metabolism in plants and humans. The sulfation was performed using a sulfuryl imidazolium salt, which was synthesized prior to use. All protected intermediates and final products were characterized via NMR and will serve as reference materials for further investigations in the fields of toxicology and bioanalytics of mycotoxins. PMID:25170180
Hydrolysis of aluminum dross material to achieve zero hazardous waste.
David, E; Kopac, J
A simple method with high efficiency for generating high pure hydrogen by hydrolysis in tap water of highly activated aluminum dross is established. Aluminum dross is activated by mechanically milling to particles of about 45 ?m. This leads to removal of surface layer of the aluminum particles and creation of a fresh chemically active metal surface. In contact with water the hydrolysis reaction takes place and hydrogen is released. In this process a Zero Waste concept is achieved because the other product of reaction is aluminum oxide hydroxide (AlOOH), which is nature-friendly and can be used to make high quality refractory or calcium aluminate cement. For comparison we also used pure aluminum powder and alkaline tap water solution (NaOH, KOH) at a ratio similar to that of aluminum dross content. The rates of hydrogen generated in hydrolysis reaction of pure aluminum and aluminum dross have been found to be similar. As a result of the experimental setup, a hydrogen generator was designed and assembled. Hydrogen volume generated by hydrolysis reaction was measured. The experimental results obtained reveal that aluminum dross could be economically recycled by hydrolysis process with achieving zero hazardous aluminum dross waste and hydrogen generation. PMID:22326245
Enzymatic hydrolysis of fractionated products from oil thermally oxidated
Yashida, H.; Alexander, J.C.
Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180/sup 0/C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether in n-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products. (33 Refs.)
Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis.
Koski, G; Klee, W A
Specific, GTP hydrolysis catalyzed by membranes prepared from neuroblastoma--glioma (NG108-15) hybrid cells can be measured in the presence of adenosine-5'-[beta, gamma-imido] triphosphate (p[NH]ppA), ATP, and a nucleotide triphosphate-regenerating system. Opiates and opioid peptides stimulate low Km GTP hydrolysis when measured in the presence of Na+ and Mg2+. Opiate stimulation is rapid, stereospecific, and reserved by the antagonist naloxone. Potencies of opiates as stimulators of GTP hydrolysis and as inhibitors of adenylate cyclase are closely correlated. Agents that stimulate adenylate cyclase, including prostaglandin E1, 2-Cl-adenosine, secretin, and NaF, have little or no effect upon the rate of GTP hydrolysis. Opiates have no effect upon either adenylate cyclase or GTPase activity in membranes prepared from C6-BU1 glioma cells, which lack opiate receptors. In view of the pivotal role of GTP in the activation of adenylate cyclase, we conclude that receptor-mediated stimulation of GTP hydrolysis is the mechanism by which opiates and other inhibitory hormones lower adenylate cyclase activity in NG108-15 cell membranes. PMID:6117072
Hydrolysis and photolysis of flumioxazin in aqueous buffer solutions.
Kwon, Jeong-Wook; Armbrust, Kevin L; Grey, Timothy L
To determine the degradation rates and degradation products of the herbicide flumioxazin in aqueous buffer solutions (pH 5, 7 and 9), its hydrolysis and photolysis were investigated at 30 degrees C in the dark, and in a growth chamber fitted with fluorescent lamps simulating the UV output of sunlight. The rate of hydrolysis of flumioxazin was accelerated by increasing pH. The t(1/2) values at pH 5, 7 and 9 were 16.4, 9.1 and 0.25 h, respectively. Two degradation products were detected and their structural assignments were made on the basis of LC-MS data. Degradation product I was detected in all buffer solutions while degradation product II was detected in acidic buffer only. Both degradation products appeared to be stable to further hydrolysis. After correcting for the effects of hydrolysis, the photolytic degradation rate also increased as a function of pH and was approximately 10 times higher at pH 7 than that at pH 5, showing t(1/2) values of 4.9 and 41.5 h, respectively. Degradation products formed by photolysis were the same as those formed by hydrolysis. Flumioxazin was degraded more extensively at high pH and should degrade in surface water. PMID:15382510
Benefits from Tween during enzymic hydrolysis of corn stover
Kaar, W.E.; Holtzapple, M.T. [Texas A and M Univ., College Station, TX (United States). Dept. of Chemical Engineering] [Texas A and M Univ., College Station, TX (United States). Dept. of Chemical Engineering
Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution. The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector.
Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).
Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian
Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions. PMID:25680958
Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis.
Liu, Fengying; Yang, Mingkun; Wang, Xude; Yang, Shanshan; Gu, Jing; Zhou, Jie; Zhang, Xian-En; Deng, Jiaoyu; Ge, Feng
The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen. PMID:25180227
O-Acetylation of Peptidoglycan in Gram-negative Bacteria
Moynihan, Patrick J.; Clarke, Anthony J.
The ape2 gene encoding a hypothetical O-acetylpeptidoglycan esterase was amplified from genomic DNA of Neisseria gonorrhoeae FA1090 and cloned to encode either the full-length protein or a truncated version lacking its hypothetical signal sequence. Expression trials revealed that production of the full-length version possessing either an N-terminal or C-terminal His6 tag was toxic to Escherichia coli transformants and that the host rapidly degraded the small amount of protein that was produced. An N-terminally truncated protein could be produced in sufficient yields for purification only if it possessed an N-terminal His6 tag. This form of the protein was isolated and purified to apparent homogeneity, and its enzymatic properties were characterized. Whereas the protein could bind to insoluble peptidoglycan, it did not function as an esterase. Phenotypic characterization of E. coli transformants producing various forms of the protein revealed that it functions instead to O-acetylate peptidoglycan within the periplasm, and it was thus renamed peptidoglycan O-acetyltransferase B. This activity was found to be dependent upon a second protein, which functions to translocate acetate from the cytoplasm to the periplasm, demonstrating that the O-acetylation of peptidoglycan in N. gonorrhoeae, and other Gram-negative bacteria, requires a two component system. PMID:20178982
Investigation of acetyl chloride photodissociation by photofragment imaging
Deshmukh, S.; Myers, J.D.; Xantheas, S.S.; Hess, W.P. (Pacific Northwest Lab., Richland, WA (United States))
The photofragment ion imaging technique is used to measure the velocity distributions of atomic chlorine, methyl, and carbon monoxide fragments generated in the photodissociation of acetyl chloride at 236 nm. The fragments are selectively ionized by (2 + 1) multiphoton ionization and projected onto a two-dimensional position-sensitive detector to obtain speed and angular distributions. The Cl images display anisotropic angular distributions, characteristic of a prompt, impulsive dissociation of the C-Cl bond. A fraction of the CH[sub 3]CO, produced as a primary photoproduct, subsequently decomposes to form CH[sub 3] and CO. The CH[sub 3] and CO images are isotropic, suggesting that rotation of the acetyl radical intermediate occurs prior to the secondary dissociation. The internal state distribution of CO is probed using (2 + 1) multiphoton ionization via the B[sup 1][Sigma][sup +] state near 230 nm. The rotational state distribution of CO extends to J[double prime] = 30, while no vibrational excitation is observed. The transition state structure of the CH[sub 3]CO intermediate, leading to dissociation into CH[sub 3] and CO, is computed via ab initio quantum mechanical methods. 42 refs., 7 figs., 4 tabs.
Protein acetylation sites mediated by Schistosoma mansoni GCN5
Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David [Instituto de Bioquimica Medica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Fantappie, Marcelo Rosado [Instituto de Bioquimica Medica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)], E-mail: firstname.lastname@example.com
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.
Two Arabidopsis proteins synthesize acetylated xylan in vitro.
Urbanowicz, Breeanna R; Peńa, Maria J; Moniz, Heather A; Moremen, Kelley W; York, William S
Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties. PMID:25141999
Dicarboxylic aciduria due to medium chain acyl CoA dehydrogenase defect. A cause of hypoglycemia in childhood.
Divry, P; David, M; Gregersen, N; Křlvraa, S; Christensen, E; Collet, J P; Dellamonica, C; Cotte, J
Dicarboxylic aciduria was found during hypoglycemic episode in a 14 months old girl. Her brother had died at the age of 4 years during febrile illness. A ketogenic diet induced in this patient a severe hypoglycemia. Urinary organic acid profile exhibited abnormal excretion of the C6-C10 dicarboxylic acids (adipic-suberic-sebacic) and related metabolites (5 hydroxyhexanoic, hexanoylglycine, suberyl glycine). This pattern suggested a defect in fatty acids beta oxidation. Plasma carnitine values was within control limits. Similar clinical findings and urinary organic acids excretion have been described in 6 patients since the initial case of Gregersen. Enzymatic studies on cultivated fibroblasts from our patient showed a defect in medium chain CoA dehydrogenase. The treatment of this disease consists of glucose infusion during attacks and prevention of fasting. This rare disease must be considered in a child with non ketotic hypoglycemia or Reye's syndrome. PMID:6673498
Gas-phase hydrolysis of SOF2 and SOF4
NASA Astrophysics Data System (ADS)
Van Brunt, R. J.; Sauers, I.
The rates for gas-phase hydrolysis of SOF2 (thionylfluoride) and SOF4 (thionyl tetrafluoride) have been measured at a temperature of 298 K. The second order rate constant for SOF2 hydrolysis in SF6 buffer gas was found to have the value (1.2±0.3)×10-23 cm3/s which agrees with previous estimates of Sauers et al., but is three orders of magnitude lower than the value obtained by Rüegsegger et al. at 340 K. The rate constant for SOF4 hydrolysis has not previously been measured and its value in both SF6 and N2 buffer gases was found here to be (1.0±0.3)×10-21 cm3/s.
Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.
Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben
Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries. PMID:24755261
Bacterial hydrolysis and methane fermentation of lignocellulosic materials
Tong, X.
In order for methane fermentation of lignocellulosic materials to be an effective method for providing both a renewable energy source and a means to reduce the volume of municipal solids wastes, a better understanding of the fermentation process is required, because this process has been generally observed to be a slow and incomplete one. This dissertation focused on understanding of the rate-limiting mechanisms of methane fermentation, including the influence of the type of lignocellulosic materials, bacterial culture characteristics, bacterial concentration, pH, and temperature. Lignocellulosic materials selected for this study were: corn stover, wheat straw, napier grass, wood grass, newspaper, and white fir. Four methanogenic cultures grown on monosaccharides, purified holocellulose, wheat straw, and mixed municipal sludge, respectively, were developed at 35[degrees]C and neutral pH. Each of the four bacterial cultures developed a glucose and cellobiose consumption potential higher than the methanogenic potential, which in turn was higher than the lignocellulosic hydrolysis potential. Further examination of lignocellulosic hydrolysis revealed that it is the step in which bacteria and enzymes can have access to holocellulosic polymers that limits the hydrolysis rate. Microscopic examination revealed that hydrolysis appears to have occurred only at the points of physical contact between the hydrolytic bacteria and the particle surface. Both fermentation rate and extent were greatly influenced by the lignocellulosic material used and by pH and temperature, but they were much less affected by the bacterial culture employed. Lignocellulosic hydrolysis reached a maximum rate at relatively low bacterial concentrations. Both hydrolysis and fermentation processes can be adequately modeled by a first-order rate equation. Linear correlations between lignin content and biodegradability or methane conversion rate were very poor.
Microwave-assisted hydrolysis of polysaccharides over polyoxometalate clusters.
Tsubaki, Shuntaro; Oono, Kiriyo; Ueda, Tadaharu; Onda, Ayumu; Yanagisawa, Kazumichi; Mitani, Tomohiko; Azuma, Jun-ichi
Polyoxometalate (POM) clusters were utilized as recyclable acid catalysts and microwave-absorbing agents for the microwave-assisted hydrolysis of corn starch and crystalline cellulose. Phosphotungstic (PW) and silicotungstic (SiW) acids showed high hydrolyzing activity, while phosphomolybdic acid (PMo) showed lower glucose stability. The PW catalyst could be recycled by ether extraction at least 4 times without changing its catalytic activity. The addition of PW could reduce the energy demand required for running the hydrolysis by 17-23%. The dielectric property of the aqueous PW solution was important for increasing the microwave-absorption capability of the reaction system and reducing the energy consumption. PMID:23859983
Benzene/nitrous oxide flammability in the precipitate hydrolysis process
Jacobs, R A [Du Pont de Nemours (E.I.) and Co., Aiken, SC (USA). Savannah River Lab.
The HAN (hydroxylamine nitrate) process for destruction of nitrite in precipitate hydrolysis produces nitrous oxide (N2O) gas as one of the products. N2O can form flammable mixtures with benzene which is also present due to radiolysis and hydrolysis of tetraphenylborate. Extensive flame modeling and explosion testing was undertaken to define the minimum oxidant for combustion of N2O/benzene using both nitrogen and carbon dioxide as diluents. The attached memorandum interprets and documents the results of the studies.
The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis
Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus
Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741
Protein acetylation in prokaryotes increases stress resistance Qun Ma, Thomas K. Wood
Wood, Thomas K.
Protein acetylation in prokaryotes increases stress resistance Qun Ma, Thomas K. Wood Department of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we of a specific environmental role of acetylation in prokaryotes. Ó 2011 Elsevier Inc. All rights reserved. 1
A dysregulated acetyl/SUMO switch of FXR promotes hepatic inflammation in obesity.
Kim, Dong-Hyun; Xiao, Zhen; Kwon, Sanghoon; Sun, Xiaoxiao; Ryerson, Daniel; Tkac, David; Ma, Ping; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Zhou, Edward; Xu, H Eric; Palvimo, Jorma J; Chen, Lin-Feng; Kemper, Byron; Kemper, Jongsook Kim
Acetylation of transcriptional regulators is normally dynamically regulated by nutrient status but is often persistently elevated in nutrient-excessive obesity conditions. We investigated the functional consequences of such aberrantly elevated acetylation of the nuclear receptor FXR as a model. Proteomic studies identified K217 as the FXR acetylation site in diet-induced obese mice. In vivo studies utilizing acetylation-mimic and acetylation-defective K217 mutants and gene expression profiling revealed that FXR acetylation increased proinflammatory gene expression, macrophage infiltration, and liver cytokine and triglyceride levels, impaired insulin signaling, and increased glucose intolerance. Mechanistically, acetylation of FXR blocked its interaction with the SUMO ligase PIASy and inhibited SUMO2 modification at K277, resulting in activation of inflammatory genes. SUMOylation of agonist-activated FXR increased its interaction with NF-?B but blocked that with RXR?, so that SUMO2-modified FXR was selectively recruited to and trans-repressed inflammatory genes without affecting FXR/RXR? target genes. A dysregulated acetyl/SUMO switch of FXR in obesity may serve as a general mechanism for diminished anti-inflammatory response of other transcriptional regulators and provide potential therapeutic and diagnostic targets for obesity-related metabolic disorders. PMID:25425577
The Effect of Acetyl-L-Carnitine Administration on Persons with Down Syndrome
ERIC Educational Resources Information Center
Pueschel, Siegfried M.
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…
Swelling of acetylated wood in organic liquids Eiichi OBATAYA* and Joseph GRIL**
Paris-Sud XI, Université de
Civil, Université Montpellier 2 Abstract To investigate the affinity of acetylated wood for organic and alcohols remained unchanged or decreased by the acetylation. Consequently the maximum volume of wood researchers have dealt with the swelling of wood in various organic liquids for comprehensive understanding
Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIalpha.
Syrovets, T; Büchele, B; Gedig, E; Slupsky, J R; Simmet, T
Acetyl-boswellic acids (acetyl-BA) are pentacyclic triterpenes derived from the gum resin of frankincense. We have previously shown that these compounds are effective cytotoxic agents, acting through a mechanism that appears to involve the inhibition of topoisomerase activity. We have now investigated the mechanism of action of acetyl-BA and show that these compounds are more potent inhibitors of human topoisomerases I and IIalpha than camptothecin, and amsacrine or etoposide, respectively. Our data demonstrate that acetyl-BA and, to a lesser extent, some other pentacyclic triterpenes, such as betulinic acid, ursolic acid, and oleanolic acid, inhibit topoisomerases I and IIalpha through a mechanism that does not involve stabilization of the cleavable complex or the intercalation of DNA. Surface plasmon resonance analysis revealed that topoisomerases I and IIalpha bind directly to an immobilized derivative of acetyl-BA. This acetyl-BA derivative interacts with human topoisomerases through high-affinity binding sites yielding K(D) values of 70.6 nM for topoisomerase I and 7.6 nM for topoisomerase IIalpha. Based on our data, we propose that acetyl-BA inhibit topoisomerases I and IIalpha through competition with DNA for binding to the enzyme. Thus, acetyl-BA are a unique class of dual catalytic inhibitors of human topoisomerases I and IIalpha. PMID:10860928
Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.
Gyorffy, S; Clarke, A J
A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed. Images PMID:1629161
The effect of acetylation on interfacial shear strength between plant fibres and various matrices
H. P. S. A Khalil; H Ismail; H. D Rozman; M. N Ahmad
Interfacial shear strength between acetylated fibres and various matrices was studied. The chemical modification of oil palm empty fruit bunch (EFB) and coconut fibre (coir) using non-catalysed acetic anhydride were investigated. Proof of acetylation of EFB and coir was indicated by the increase in weight percent gain, and was confirmed by Fourier transform infrared spectra. Pull-out tests were employed to
The Role of Histone Acetylation in Cocaine-Induced Neural Plasticity and Behavior
Wood, Marcelo A.
The Role of Histone Acetylation in Cocaine-Induced Neural Plasticity and Behavior George A Rogge1 of abuse, such as cocaine, cause stable changes in neural plasticity that in turn drive long-term changes regulation via histone acetylation in cocaine action. Neuropsychopharmacology Reviews advance online
Structural analysis of brain ganglioside acetylation patterns in mice with altered ganglioside biosynthesis.
Mlinac, Kristina; Fabris, Dragana; Vukeli?, Zeljka; Rožman, Marko; Heffer, Marija; Bognar, Svjetlana Kalanj
Gangliosides are sialylated membrane glycosphingolipids especially abundant in mammalian brain tissue. Sialic acid O-acetylation is one of the most common structural modifications of gangliosides which considerably influences their chemical properties. In this study, gangliosides extracted from brain tissue of mice with altered ganglioside biosynthesis (St8sia1 null and B4galnt1 null mice) were structurally characterized and their acetylation pattern was analyzed. Extracted native and alkali-treated gangliosides were resolved by high performance thin layer chromatography. Ganglioside mixtures as well as separated individual ganglioside fractions were further analyzed by tandem mass spectrometry. Several O-acetylated brain ganglioside species were found in knockout mice, not present in the wild-type mice. To the best of our knowledge this is the first report on the presence of O-acetylated GD1a in St8sia1 null mice and O-acetylated GM3 species in B4galnt1 null mice. In addition, much higher diversity of abnormally accumulated brain ganglioside species regarding the structure of ceramide portion was observed in knockout versus wild-type mice. Obtained findings indicate that the diversity of brain ganglioside structures as well as acetylation patterns in mice with altered ganglioside biosynthesis, is even higher than previously reported. Further investigation is needed in order to explore the effects of acetylation on ganglioside interactions with other molecules and consequently the physiological role of acetylated ganglioside species. PMID:24140892
Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene
Technology Transfer Automated Retrieval System (TEKTRAN)
Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene, ae1, encoding the acetyl esterase (Ae1) of Hypocrea jecorina was identified by amino terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino ...
LAceP: Lysine Acetylation Site Prediction Using Logistic Regression Classifiers
Hou, Ting; Zheng, Guangyong; Zhang, Pingyu; Jia, Jia; Li, Jing; Xie, Lu; Wei, Chaochun; Li, Yixue
Background Lysine acetylation is a crucial type of protein post-translational modification, which is involved in many important cellular processes and serious diseases. However, identification of protein acetylated sites through traditional experiment methods is time-consuming and laborious. Those methods are not suitable to identify a large number of acetylated sites quickly. Therefore, computational methods are still very valuable to accelerate lysine acetylated site finding. Result In this study, many biological characteristics of acetylated sites have been investigated, such as the amino acid sequence around the acetylated sites, the physicochemical property of the amino acids and the transition probability of adjacent amino acids. A logistic regression method was then utilized to integrate these information for generating a novel lysine acetylation prediction system named LAceP. When compared with existing methods, LAceP overwhelms most of state-of-the-art methods. Especially, LAceP has a more balanced prediction capability for positive and negative datasets. Conclusion LAceP can integrate different biological features to predict lysine acetylation with high accuracy. An online web server is freely available at http://www.scbit.org/iPTM/. PMID:24586884
Copper deficiency results in AMP-activated protein kinase activation and acetylCoA carboxylase phosphorylation in rat cerebellum
Gybina, Anna A.; Prohaska, Joseph R.
Copper (Cu) deficiency impairs cerebellar development including biosynthetic processes like myelination and synaptogenesis. The activity of cerebellar mitochondrial cuproenzyme cytochrome c oxidase is markedly lower in Cu deficient rat pups and is accompanied by higher lactate levels indicating mitochondrial inhibition. Cu deficiency impaired energy metabolism is thought to contribute to developmental delays, but specific mechanisms linking these phenomena have remained unexplored. AMP activated protein kinase (AMPK) is a cellular energy sensor that is activated during mitochondrial inhibition and shuts down biosynthetic processes to help conserve cellular ATP levels. Activated AMPK phosphorylates and inhibits acetyl CoA carboxylase (ACC), the first enzyme in fatty acid biosynthesis. We hypothesize AMPK is activated and ACC inhibited in Cu deficient cerebella. Perinatal copper deficiency was studied in young rats in rapidly frozen cerebella. Compared to copper adequate (Cu+) pups, copper deficient (Cu?) pups were hypothermic, had lower brain copper levels and markedly higher cerebellar lactate. Concentration of phosphorylated AMPK (pAMPK), indicating AMPK activation, was robustly higher in Cu? cerebella of rat pups at two ages and in two separate experiments. Compared to Cu+ cerebella, pACC content was significantly higher in all Cu? samples. Mechanisms leading to AMPK activation remain elusive. Higher AMP/ATP ratios and increased reactive nitrogen species (RNS) can lead to AMPK activation. ATP and AMP concentrations were unaltered and nitric oxide metabolites and 3-nitrotyrosine peptide levels remained unchanged in Cu? cerebella. AMPK activation may explain how ATP levels can be maintained even with a severe mitochondrial loss of CCO function. PMID:18339363
VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis
Aruni, A. Wilson; Lee, J.; Osbourne, D.; Dou, Y.; Roy, F.; Muthiah, A.; Boskovic, D. S.
The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA-defective mutant in a different P. gingivalis genetic background were evaluated. The vimA-defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA-defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting. PMID:22144476
Mitotic activity of survivin is regulated by acetylation at K129.
Aljaberi, Aysha M; Webster, Jamie Rm; Wheatley, Sally P
Survivin is a cancer-associated protein regulated by multiple factors, including acetylation at K129 within its C-terminal ?-helical tail. Acetylation of survivin is being pursued as a potential prognostic marker in breast cancer. This modification at K129 may cause nuclear accumulation of survivin in interphase cells; however, whether this affects its essential role during mitosis has not been addressed. We posited whether mimicking acetylation of survivin at K129 alters its activity during mitosis. Fluorescence microscopy and time-lapse imaging showed that, mutating this site to an alanine to act as a constitutive acetyl mimetic, K129A, causes defects in chromosome segregation and cytokinesis. As a non-acetylatable version, K129R, also has difficulty during mitotic exit, we conclude that cyclical acetylation and deacetylation is required for fully functional survivin during mitosis. PMID:25928399
Functional, thermal and rheological properties of oat ?-glucan modified by acetylation.
de Souza, Nelisa Lamas; Bartz, Josiane; Zavareze, Elessandra da Rosa; de Oliveira, Patrícia Diaz; da Silva, Wagner Schellin Vieira; Alves, Gabriela Hörnke; Dias, Alvaro Renato Guerra
Fibers of ?-glucan have been added to foods for their thickening properties, their ability to form gel at low concentrations, but mainly for their appeal in health promotion. Current analysis evaluates the influence of acetylation (4% and 6% acetic anhydride for 10 and 20 min) on the functional, thermal, morphological and rheological properties of the concentrate containing 31% of oat ?-glucan. The degree of substitution of the acetylated ?-glucans ranged from 0.03 to 0.12, suitable for use in foods. Acetylation increased the heterogeneity of molecule degradation and promoted a more compacted hole-less microstructure. Functional properties such as the swelling power and bile acid binding capacity were increased by acetylation. The ?-glucan gel showed a reduction in hardness and adhesiveness, which was confirmed by its rheological behavior similar to liquid. The above information is relevant to establish the industrial application of acetylated ?-glucan. PMID:25704708
Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum
The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B- 1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules. PMID:3294857
Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells
Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A.; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S.; Capra, John A.; Schnölzer, Martina; Cole, Philip A.; Geyer, Matthias; Bruneau, Benoit G.; Adelman, Karen; Ott, Melanie
SUMMARY Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes. PMID:24207025
System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases
Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison
Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.
Furry promotes acetylation of microtubules in the mitotic spindle by inhibition of SIRT2 tubulin deacetylase.
Nagai, Tomoaki; Ikeda, Masanori; Chiba, Shuhei; Kanno, Shin-Ichiro; Mizuno, Kensaku
The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of ?-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2. PMID:23886946
40 CFR 721.10152 - Oxirane, substituted silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica...
Code of Federal Regulations, 2013 CFR
...silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica, acetates...silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica, acetates...hydrolysis products with alkanol zirconium(4+) salt and silica,...
40 CFR 721.10152 - Oxirane, substituted silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica...
Code of Federal Regulations, 2012 CFR
...silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica, acetates...silylmethyl-, hydrolysis products with alkanol zirconium(4+) salt and silica, acetates...hydrolysis products with alkanol zirconium(4+) salt and silica,...
Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J.; Cui, Liwang
Summary Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites. PMID:23796209
40 CFR 180.1089 - Poly-N-acetyl-D-glucosamine; exemption from the requirement of tolerance.
Code of Federal Regulations, 2010 CFR
...2010-07-01 false Poly-N-acetyl-D-glucosamine; exemption from the requirement of...1089 Poly-N -acetyl-D -glucosamine; exemption from the requirement of...nematicide poly-N -acetyl-D -glucosamine on a variety of agricultural...
40 CFR 180.1089 - Poly-N-acetyl-D-glucosamine; exemption from the requirement of tolerance.
Code of Federal Regulations, 2011 CFR
...2011-07-01 false Poly-N-acetyl-D-glucosamine; exemption from the requirement of...1089 Poly-N -acetyl-D -glucosamine; exemption from the requirement of...nematicide poly-N -acetyl-D -glucosamine on a variety of agricultural...
Energy Optimization of Bioethanol Production via Hydrolysis of Switchgrass
Grossmann, Ignacio E.
of syngas and planning the construction of commercial plants in the near future. Two different production routes can be used to produce ethanol from lignocellulosic switchgrass, either biomass hydrolysis or biomass gasification. A previous paper by the authors8 proposed an optimized conceptual production process
Effects of hydrolysis and carbonization reactions on hydrochar production.
Fakkaew, K; Koottatep, T; Polprasert, C
Hydrothermal carbonization (HTC) is a thermal conversion process which converts wet biomass into hydrochar. In this study, a low-energy HTC process named "Two-stage HTC" comprising of hydrolysis and carbonization stages using faecal sludge as feedstock was developed and optimized. The experimental results indicated the optimum conditions of the two-stage HTC to be; hydrolysis temperature of 170°C, hydrolysis reaction time of 155min, carbonization temperature of 215°C, and carbonization reaction time of 100min. The hydrolysis reaction time and carbonization temperature had a statistically significant effect on energy content of the produced hydrochar. Energy input of the two-stage HTC was about 25% less than conventional HTC. Energy efficiency of the two-stage HTC for treating faecal sludge was higher than that of conventional HTC and other thermal conversion processes such as pyrolysis and gasification. The two-stage HTC could be considered as a potential technology for treating FS and producing hydrochar. PMID:26051497
Perspective How does ATP hydrolysis control actin's associations?
Spudich, James A.
Perspective How does ATP hydrolysis control actin's associations? Elena P. Sablin*, John F. Dawson in the DNaseI-binding loop from a random coil to a helix in the ADP-bound actin (3) was suggested (4 is similar to that used by other families of nucleotide- hydrolyzing proteins (NTPases). Various families
Synergistic Catalysis of Dimetilan Hydrolysis by Metal Ions and
Huang, Ching-Hua
catalyzed by +II transition metal ions exhibiting strong affinities for nitrogen- and oxygen-donor ligands that dissolved metal ions (1), simple hydrous metal oxides (2-5), and clays (6, 7) can increase hydrolysis rates- chemicalorwiththeattackingnucleophile(e.g.,H2OorOH- ). In other situations, dissolved metal ions and metal oxides decrease
Pancreatic Lipase Hydrolysis of Cow Milk Fat1
E. L. Jack; C. P. Freeman; L. M. Smith; J. B. Mickle
SUMMARY Knowledge of the position of individual fatty acids within the trig'lycerides is necessary to the understanding of the utilization of a fat. Pancreatic lipase hydrolysis to convert triglycerides to 2-monoglycerides has been used to study this type of glyceride structure in roany food fats, but it has recently been claimed that it cannot be used with cow milk fat
Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus
Technology Transfer Automated Retrieval System (TEKTRAN)
Particle size reduction is a crucial factor in transportation logistics as well as cellulosic conversion. The effect of particle size on enzymatic hydrolysis of pretreated Miscanthus x giganteus was determined. Miscanthus was ground using a hammer mill equipped with screens having 0.08, 2.0 or 6.0...
Recycling of cellulose enzyme complex after cellulose hydrolysis. Final report
This report describes the development of a low-cost process for the recycling of enzymes used in cellulose hydrolysis. The enzymes can be used in an advanced technology to convert cellulose, a component of wood and paper, to sugar for the production of fuel ethanol. The market for fuel ethanol, used as an octane booster in gasoline, is growing. New York
[A rate-limiting stage of enteropeptidase hydrolysis].
Mikha?lova, A G; Likhareva, V V; Rumsh, L D
It has been shown for the first time that deacylation is the rate-limiting stage in the enteropeptidase-catalyzed hydrolysis of highly efficient oligopeptide substrates containing four Asp residues in positions P2-P5. On the other hand, the rate-limiting stage in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2-P5 is acylation, as has previously been suggested for all amide and peptide substrates of serine proteases on the basis of the classic studies by Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation stage was used to determine the rate-limiting stage in the enteropeptidase hydrolysis of Nalpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp4-Lys beta-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru. PMID:18522276
Kinetic Modeling of Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass
Technology Transfer Automated Retrieval System (TEKTRAN)
A semimechanistic multi-reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose-to-glucose and two heterogeneous reactions of cellulose-to...
Production of alcohol fuels via acid-hydrolysis extrusion technology
Noon, R.; Hochstetler, T.
Pilot plant data obtained using a modified single screw grain extruder to facilitate the conversion of raw cellulosic materials into fermentable monosaccharides via acid hydrolysis are analyzed. The following are discussed: cellulose availability and cost, cellulose conversion theory, cellulose conversion performance of extrusion technology, system analysis, and economics. (MHR)
Enzymatic hydrolysis of tannery fleshings using chicken intestine proteases
A. Annapurna Raju; C. Rose; N. Muralidhara Rao
Chicken intestine and tannery fleshings, the major wastes from poultry and tannery industries posing wide disposal problems, are used in this study for the recovery of proteins through biodegradation. Chicken intestines have been investigated as a source of proteolytic and autolytic enzymes for the hydrolysis of tannery fleshings. A combination of tannery fleshings and chicken intestines at acidic pH, when
Edinburgh Research Explorer Organotrifluoroborate Hydrolysis: Boronic Acid Release
Millar, Andrew J.
and an AcidBase Paradox in Cross-Coupling Citation for published version: Lennox, AJJ & Lloyd-jones, GC 2012, 'Organotrifluoroborate Hydrolysis: Boronic Acid Release Mechanism and an AcidBase Paradox in Cross-Coupling' Journal: Boronic Acid Release Mechanism and an AcidBase Paradox in Cross-Coupling** Alastair J. J. Lennox1 and Guy
Hydrolysis of oligosaccharides over solid acid catalysts: a review.
Vilcocq, Léa; Castilho, Paula C; Carvalheiro, Florbela; Duarte, Luís C
Mild fractionation/pretreatment processes are becoming the most preferred choices for biomass processing within the biorefinery framework. To further explore their advantages, new developments are needed, especially to increase the extent of the hydrolysis of poly- and oligosaccharides. A possible way forward is the use of solid acid catalysts that may overcome many current drawbacks of other common methods. In this Review, the advantages and limitations of the use of heterogeneous catalysis for the main groups of solid acid catalysts (zeolites, resins, carbon materials, clays, silicas, and other oxides) and their relation to the hydrolysis of model soluble disaccharides and soluble poly- and oligosaccharides are presented and discussed. Special attention is given to the hydrolysis of hemicelluloses and hemicellulose-derived saccharides into monosaccharides, the impact on process performance of potential catalyst poisons originating from biomass and biomass hydrolysates (e.g., proteins, mineral ions, etc.). The data clearly point out the need for studying hemicelluloses in natura rather than in model compound solutions that do not retain the relevant factors influencing process performance. Furthermore, the desirable traits that solid acid catalysts must possess for the efficient hemicellulose hydrolysis are also presented and discussed with regard to the design of new catalysts. PMID:24616436
Radioactive demonstration of the ``late wash`` Precipitate Hydrolysis Process
Bibler, N.E.; Ferrara, D.M.; Ha, B.C.
This report presents results of the radioactive demonstration of the DWPF Precipitate Hydrolysis Process as it would occur in the ``late wash`` flowsheet in the absence of hydroxylamine nitrate. Radioactive precipitate containing Cs-137 from the April, 1983, in-tank precipitation demonstration in Tank 48 was used for these tests.
Radioactive demonstration of the late wash'' Precipitate Hydrolysis Process
Bibler, N.E.; Ferrara, D.M.; Ha, B.C.
This report presents results of the radioactive demonstration of the DWPF Precipitate Hydrolysis Process as it would occur in the late wash'' flowsheet in the absence of hydroxylamine nitrate. Radioactive precipitate containing Cs-137 from the April, 1983, in-tank precipitation demonstration in Tank 48 was used for these tests.
Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis
Harold H. Brownell; John N. Saddler
Pretreatment methods were compared with steam explosion, and differing views on the relative importance of mechanical and chemical effects were outlined. Hydrolysis was desirable; pyrolysis was undesirable. The effects of initial moisture content on steam consumption, mechanism and rate of heat transfer, pentosan solubilization, and subsequent glucose yield were summarized. The insignificant effect, after treatment at 240 degrees C, of
Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis
Technology Transfer Automated Retrieval System (TEKTRAN)
This work is the first report of the successful design, construction, and application of multi-functional, self-assembling biocatalysts for targeted xylan hydrolysis, termed xylanosomes. Using the architecture of cellulosomes found in some anaerobic cellulolytic microbes, four different xylanosomes...
Fractionation and Molecular Characteristics of Cellulose During Enzymatic Hydrolysis
T. Eremeeva; T. Bikova; M. Eisimonte; U. Viesturs; A. Treimanis
The effect of enzymatic hydrolysis (EH) on the molecular characteristics as well as fractional composition of cellulose was studied using the direct size exclusion chromatography (SEC) analysis in sodium hydroxide. Bleached hardwood pulp was subjected to the action of the cellulase complex Celluclast™ supplemented with Novozyme 188™. The residues after the enzymatic treatment were fractionated by dissolution in 10% NaOH
Hydrolysis of methyloleate using immobilized lipase from Chromobacterium viscosum
M. S. R. C. Murthy; Z. Aslam Basha; T. Swaminathan
Various lipases were screened for their hydrolytic efficiency towards methyloleate. Lipase from Chromobacterium viscosum gave highest hydrolysis efficiency of 92% in 24 h. Different cation exchange resins were screened to immobilize lipase from Chromobacterium viscosum. A weakly acidic macroreticular type resin, IRC-50 having carboxyl end group functionality gave highest activity yield of 18.8%. Strongly acidic cation exchange resins with sulphonic
Catalytic hydrolysis of ammonia borane via cobalt palladium nanoparticles.
Sun, Daohua; Mazumder, Vismadeb; Metin, Önder; Sun, Shouheng
Monodisperse 8 nm CoPd nanoparticles (NPs) with controlled compositions were synthesized by the reduction of cobalt acetylacetonate and palladium bromide in the presence of oleylamine and trioctylphosphine. These NPs were active catalysts for hydrogen generation from the hydrolysis of ammonia borane (AB), and their activities were composition dependent. Among the 8 nm CoPd catalysts tested for the hydrolysis of AB, the Co(35)Pd(65) NPs exhibited the highest catalytic activity and durability. Their hydrolysis completion time and activation energy were 5.5 min and 27.5 kJ mol(-1), respectively, which were comparable to the best Pt-based catalyst reported. The catalytic performance of the CoPd/C could be further enhanced by a preannealing treatment at 300 °C under air for 15 h with the hydrolysis completion time reduced to 3.5 min. This high catalytic performance of Co(35)Pd(65) NP catalyst makes it an exciting alternative in pursuit of practical implementation of AB as a hydrogen storage material for fuel cell applications. PMID:21766875
Hydrolysis of hydroxybenzoate saxitoxin analogues originating from Gymnodinium catenatum
Paulo Vale
The paralytic shellfish poisoning (PSP) toxin producer Gymnodinium catenatum produces several hydrophobic analogues of saxitoxin (STX). These are poorly studied due to their recent discovery and lack of standards. It was previously observed these hydrophobic analogues could be partially hydrolysed, loosing its benzoate moiety during alkaline oxidation to obtain fluorescent products measurable by HPLC analysis. The hydrolysis reaction was further
Evaluation of Cation Hydrolysis Schemes with a Pocket Calculator.
ERIC Educational Resources Information Center
Clare, Brian W.
Described is the use of two models of pocket calculators. The Hewlett-Packard HP67 and the Texas Instruments TI59, to solve problems arising in connection with ionic equilibria in solution. A three-parameter regression program is described and listed as a specific example, the hydrolysis of hexavalent uranium, is provided. (BT)
Non-ionic surfactants do not consistently improve the enzymatic hydrolysis of pure cellulose.
Zhou, Yan; Chen, Hongmei; Qi, Feng; Zhao, Xuebing; Liu, Dehua
Non-ionic surfactants have been frequently reported to improve the enzymatic hydrolysis of pretreated lignocellulosic biomass and pure cellulose. However, how the hydrolysis condition, substrate structure and cellulase formulation affect the beneficial action of surfactants has not been well elucidated. In this work, it was found that the enzymatic hydrolysis of pure cellulose was not consistently improved by surfactants. Contrarily, high surfactant concentration, e.g. 5 g/L, which greatly improved the hydrolysis of dilute acid pretreated substrates, actually showed notable inhibition to pure cellulose conversion in the late phase of hydrolysis. Under an optimal hydrolysis condition, the improvement by surfactant was limited, but under harsh conditions surfactant indeed could enhance cellulose conversion. It was proposed that non-ionic surfactants could interact with substrates and cellulases to impact the adsorption behaviors of cellulases. Therefore, the beneficial action of surfactants on pure cellulose hydrolysis is influenced by hydrolysis condition, cellulose structural features and cellulase formulation. PMID:25689307
On a Three Step Model of Anaerobic Digestion Including the Hydrolysis of
Paris-Sud XI, Université de
On a Three Step Model of Anaerobic Digestion Including the Hydrolysis of Particulate Matter R degradation, chemostat, models, growth rate, equilibrium, bistability. 1. INTRODUCTION Anaerobic digestion, the anaerobic digestion is generally considered as a three step process: hydrolysis and liquefaction
Review: Continuous hydrolysis and fermentation for cellulosic ethanol production Simone Brethauer, Charles E. Wyman *
California at Riverside, University of
Review: Continuous hydrolysis and fermentation for cellulosic ethanol production Simone Brethauer Available online 14 December 2009 Keywords: Continuous fermentation Enzymatic hydrolysis Fuel ethanol Lignocellulosic biomass Simultaneous saccharification and fermentation (SSF) a b s t r a c t Ethanol made
Acetylation of barnyardgrass starch with acetic anhydride under iodine catalysis.
Bartz, Josiane; Goebel, Jorge Tiago; Giovanaz, Marcos Antônio; Zavareze, Elessandra da Rosa; Schirmer, Manoel Artigas; Dias, Alvaro Renato Guerra
Barnyardgrass (Echinochloa crus-galli) is an invasive plant that is difficult to control and is found in abundance as part of the waste of the paddy industry. In this study, barnyardgrass starch was extracted and studied to obtain a novel starch with potential food and non-food applications. We report some of the physicochemical, functional and morphological properties as well as the effect of modifying this starch with acetic anhydride by catalysis with 1, 5 or 10mM of iodine. The extent of the introduction of acetyl groups increased with increasing iodine levels as catalyst. The shape of the granules remained unaltered, but there were low levels of surface corrosion and the overall relative crystallinity decreased. The pasting temperature, enthalpy and other gelatinisation temperatures were reduced by the modification. There was an increase in the viscosity of the pastes, except for the peak viscosity, which was strongly reduced in 10mM iodine. PMID:25704707
Toxoplasma histone acetylation remodelers as novel drug targets
Vanagas, Laura; Jeffers, Victoria; Bogado, Silvina S; Dalmasso, Maria C; Sullivan, William J; Angel, Sergio O
Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite. PMID:23199404
Effect of dietary di-2-ethylhexyl phthalate on oxidation of 14 C-palmitoyl CoA by mitochondria from mammalian heart and liver
Frank P. Bell; Peter J. Gillies
Oxidation of [1-14C] palmitoyl CoA by heart and liver mitochondria from rats fed dietary di-2-ethylhexyl phthalate (DEHP) was investigated in\\u000a vitro. Oxidation of14C-palmitoyl CoA to14CO2 increased two- to threefold in hepatic mitochondria from rats fed 0.1% DEHP for 2 to 3 days; this increase appeared to be\\u000a a maximum response since similar data were obtained using hepatic mitochondria from rats
Cross sections for production of the CO(A 1 Pi)-(X 1 Sigma) fourth positive band system and O(3 S) by photodissociation of CO2
NASA Technical Reports Server (NTRS)
Gentieu, E. P.; Mentall, J. E.
The CO(A 1 Pi) cross sections reported here, along with previously determined electron impact results, establish the basis for calculating CO fourth positive system volume emission rates in the Martian dayglow. Calculated volume emission rates in turn determine relative distribution of photon vs. electron impact as mechanisms for producing CO(A 1 Pi) in the Mars atmosphere. The smallness of the O(1304) cross section confirms previous indirect evidence that photodissociative excitation of CO2 is not an important source of O(3 S) in the upper atmosphere of Mars.
Acetyl-L-carnitine treatment in minimal hepatic encephalopathy.
Malaguarnera, Mariano; Gargante, Maria Pia; Cristaldi, Erika; Vacante, Marco; Risino, Corrado; Cammalleri, Lisa; Pennisi, Giovanni; Rampello, Liborio
Minimal hepatic encephalopathy (MHE) is characterized by disturbance of mental state and neuromuscular function. To assess the clinical efficacy of acetyl-L: -carnitine (ALC) in the treatment of MHE, we performed a randomized, double-blind, placebo-controlled study administering ALC in cirrhotic patients with this disease and evaluating their cognitive functions. One hundred and twenty-five cirrhotic patients, of whom 21 were infected by hepatitis B virus, 75 by hepatitis C virus and 29 with cryptogenic cirrhosis, were enrolled in our study. Patients were randomly divided into two groups, and using double-blind administration, group A was treated with ALC and group B with placebo for 90 days. The two groups were similar in demographic characteristics, aetiology of cirrhosis, duration and Child-Pugh grade. Minimal hepatic encephalopathy was diagnosed with the Trail Making Test (TMT), Symbol Digit Modalities Test (SDMT) and Auditory Verbal Learning Test (AVL) and cognitive function with the Mini Mental State Examination (MMSE). After 90 days in group A treated with ALC, we observed a significant decrease in prothrombin time (P < 0.001), bilirubin serum levels (P < 0.01), AST (P < 0.001), fasting NH(4) serum levels (P < 0.001), Trail Making Test-A (P < 0.001) and Trail Making Test-B (P < 0.001), and a significant increase in albumin serum levels (P < 0.005), MMSE test (P < 0.001), Symbol Digit Modalities Test (P < 0.001), BDT (P < 0.001), AVL long-term test (P < 0.001) and AVL total test (P < 0.001). No significant differences were observed in EEG in either group of patients treated with ALC or placebo. The benefits of ALC in comparison with placebo are demonstrated in greater reductions in serum ammonia levels, as well as in improvements of neuropsychological functioning. PMID:18357530
Acetylated lysozyme as impurity in lysozyme crystals: constant distribution coefficient
NASA Astrophysics Data System (ADS)
Thomas, B. R.; Chernov, A. A.
Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A and B impurities added were 0.76, 0.38 and 0.1 mg/ml and 0.43, 0.22, 0.1 mg/ml, respectively. The HEWL concentration were 20, 30 and 40 mg/ml. The crystals grown in 18 experiments for each impurity concentration and supersaturation were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K=2.15±0.13 for A and K=3.42±0.25 for B. According to definition of K by Eq. (1) in the text, the condition K=const is equivalent to a decrease of impurity amount in the crystal as the supersaturation increases. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that the impurity adsorption and incorporation rates are proportional to the impurity concentration and that the growth rate is proportional to the concentration of crystallizing protein in solution. The frequency at which an impurity molecules irreversibly join the crystal was estimated to be 3 s -1, much higher than such frequency for regular crystal molecules 5×10 -2 s -1 at 30 mg/ml lysozyme concentration. Reasons for this inequality are discussed.
A reactivity-selectivity study of the Friedel-Crafts acetylation of 3,3?-dimethylbiphenyl and the oxidation of the acetyl derivatives
Background Friedel-Crafts acetylation is an important route to aromatic ketones, in research laboratories and in industry. The acetyl derivatives of 3,3?-dimethylbiphenyl (3,3?-dmbp) have applications in the field of liquid crystals and polymers and may be oxidized to the dicarboxylic acids and derivatives that are of interest in cancer treatment. Findings The effect of solvent and temperature on the selectivity of monoacetylation of 3,3'-dmbp by the Perrier addition procedure was studied using stoichiometric amounts of reagents. 4-Ac-3,3?-dmbp was formed almost quantitatively in boiling 1,2-dichloroethane and this is almost twice the yield hitherto reported. Using instead a molar ratio of substrate:AcCl:AlCl3 equal to 1:4:4 or 1:6:6 in boiling 1,2-dichloroethane, acetylation afforded 4,4?- and 4,6?-diacetyl-3,3?-dmbp in a total yield close to 100%. The acetyl derivatives were subsequently converted to the carboxylic acids by hypochlorite oxidation. The relative stabilities of the isomeric products and the corresponding ?-complexes were studied by DFT calculations and the data indicated that mono- and diacetylation followed different mechanisms. Conclusions Friedel-Crafts acetylation of 3,3?-dmbp using the Perrier addition procedure in boiling 1,2-dichloroethane was found to be superior to other recipes. The discrimination against the 6-acetyl derivative during monoacetylation seems to reflect a mechanism including an AcCl:AlCl3 complex or larger agglomerates as the electrophile, whereas the less selective diacetylations of the deactivated 4-Ac-3,3?-dmbp are suggested to include the acetyl cation as the electrophile. The DFT data also showed that complexation of intermediates and products with AlCl3 does not seem to be important in determining the mechanism. PMID:22682296
A new class of inhibitors of 2-arachidonoylglycerol hydrolysis and invasion of prostate cancer cells q
Hammock, Bruce D.
A new class of inhibitors of 2-arachidonoylglycerol hydrolysis and invasion of prostate cancer-independent prostate cancer cells. Blocking cellular hydrolysis of 2-AG to increase its endogenous concentration to inhibit 2-AG hydrolysis and prostate cancer cell invasion. Compounds containing a thioether b to a TFK
Kinetic study on hydrolysis of oils by lipase with ultrasonic emulsification
K. B. Ramachandran; Sulaiman Al-Zuhair; C. S. Fong; C. W. Gak
The hydrolysis of oil by lipase takes place at the interface between the oil and the aqueous solution containing the enzyme. For such systems, interfacial area between the oil phase and the aqueous phase influences the rate of hydrolysis. In this study, to enhance the hydrolysis rates of lipids, ultrasonication instead of mechanical agitation was used for interfacial area generation.
FANCJ/BACH1 acetylation at lysine 1249 regulates the DNA damage response.
Xie, Jenny; Peng, Min; Guillemette, Shawna; Quan, Steven; Maniatis, Stephanie; Wu, Yuliang; Venkatesh, Aditya; Shaffer, Scott A; Brosh, Robert M; Cantor, Sharon B
BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance. PMID:22792074
Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae
Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the acetyl-CoA metabolism by co-transformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10) and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB) and poly(3-hydroxybutyrate) polymerase (phaC) from Ralstonia eutropha H16. Introduction of the acetyl-CoA plasmid together with the PHB plasmid, improved the productivity of PHB more than 16 times compared to the reference strain used in this study, as well as it reduced the specific product formation of side products. PMID:23009357
Effect of acetyl esterification on physicochemical properties of chick pea (Cicer arietinum L.) starch.
Yadav, Dev Kumar; Patki, Prakash Eknatharao
Acetyl esterification of isolated Bengal gram starch was carried out using acetic anhydride as reactant. Modification of native starch at variant concentrations of acetic anhydride (6, 8 and 10 %, w/w) resulted in modified starch with 2.14, 3.35, 4.47% acetyl content and 0.082, 0.130 and 0.176° of substitution (DS) respectively. The acetyl esterification of native starch brought significant changes in physicochemical properties with respect to pasting behavior, granule morphology, thermal properties and retrogradation profile. Acetyl modifications of native starch increased swelling capacity, water absorption power and oil absorption capability by 17, 13 and 20 % respectively. Acetylation has decreased pasting temperature, pasting time, final viscosity and set back viscosity due to increase in amylsoe content, hydrogen bonding and porosity of starch granule. The acetyl modification was confirmed by IR spectra with the presence of an ester carbonyl group (C = O) at 1720.3 cm(-1) and absorption band at 174.8 cm(-1). In DSC evaluation there was decrease in To, Tp, Tc and ?H of acetylated starch than native starch which resulted in reduced retrogradation by 56 %. PMID:26139882
Acetylation of amikacin, a new semisynthetic antibiotic, by Pseudomonas aeruginosa carrying an R factor.
Kawabe, H; Naito, T; Mitsuhashi, S
A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), a new semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C(1). The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH(2) group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-d-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide. PMID:806257
Neuroprotective Effects of N-Acetyl-Cysteine and Acetyl-L-Carnitine after Spinal Cord Injury in Adult Rats
Karalija, Amar; Novikova, Liudmila N.; Kingham, Paul J.; Wiberg, Mikael; Novikov, Lev N.
Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4–L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury. PMID:22815926
Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays
NASA Technical Reports Server (NTRS)
Komoszynski, M.; Bandurski, R. S.
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.
Acetylation of human hemoglobins by cord blood ribosomal and postribosomal acetyltransferases
Qiu, C.C.; Kasten-Jolly, J.; Abraham, E.C.
The authors have been interested in human fetal hemoglobin acetylation and have focused on search of an acetyltransferase that can acetylate this hemoglobin. Cord blood ribosomal and postribosomal acetyltransferases have been partially purified by histone-Sepharose 4B affinity chromatography. Analysis of Dextran 40 fractionated cells has indicated that the enzyme is present only in reticulocytes and young erythrocytes, and mature erythrocytes are devoid of this enzyme. Enzymes prepared from the ribosomal and postribosomal sources showed similar temperature, pH, and cation dependence. In vitro acetylation of Hb F0, Hb A0 and native el, US , and chains with ( UC)acetyl-CoA in the presence of these enzyme preparations failed to show any specificity to any one of the hemoglobins. No significant differences in hemoglobin acetylation were seen between the two types of enzymes. However, acetylation of Hb F0 and el chains did produce Hb F/sub Iac/ (Hb F/sub I/ or Hb F/sub Ic/) and el/sub Iac/ (el/sub I/ or el/sub Ic/), respectively. Peptide analyses showed that the el chain NH2-terminus and other sites were being acetylated. Thus, site specificity is seen only when the ribosomal enzyme acts on ribosome-bound nascent chains. It is also possible that the presence of free enzyme, acetyle-CoA and potential hemoglobin substrates in the cytoplasm may lead to the formation of some Hb F/sub Iac/ and small amounts of other unidentified acetylated hemoglobins in red cells.
The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase
Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. DOI: http://dx.doi.org/10.7554/eLife.05560.001 PMID:25742604
Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.
Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M
Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. PMID:23688416
Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR).
Wadenbäck, Johan; von Arnold, Sara; Egertsdotter, Ulrika; Walter, Michael H; Grima-Pettenati, Jacqueline; Goffner, Deborah; Gellerstedt, Göran; Gullion, Terry; Clapham, David
An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway. PMID:17610137
Polymorphisms in cinnamoyl CoA reductase (CCR) are associated with variation in microfibril angle in Eucalyptus spp.
Thumma, Bala R; Nolan, Maureen F; Evans, Robert; Moran, Gavin F
Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of marker-trait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees. PMID:16085705
Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa
Guo, Dianjing; Chen, Fang; Inoue, Kentaro; Blount, Jack W.; Dixon, Richard A.
Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in ?-5 linked dimers of G units. Accumulation of soluble caffeic acid ?-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis. PMID:11158530
The influence of solid\\/liquid separation techniques on the sugar yield in two-step dilute acid hydrolysis of softwood followed by enzymatic hydrolysis
Sanam Monavari; Mats Galbe; Guido Zacchi
BACKGROUND: Two-step dilute acid hydrolysis of softwood, either as a stand-alone process or as pretreatment before enzymatic hydrolysis, is considered to result in higher sugar yields than one-step acid hydrolysis. However, this requires removal of the liquid between the two steps. In an industrial process, filtration and washing of the material between the two steps is difficult, as it should
Coordinate Regulation of the Nuclear and Plastidic Genes Coding for the Subunits of the Heteromeric Acetyl-Coenzyme A Carboxylase1
Ke, Jinshan; Wen, Tuan-Nan; Nikolau, Basil J.; Wurtele, Eve Syrkin
Plastidic acetyl-coenzyme A (CoA) carboxylase (ACCase) catalyzes the first committed reaction of de novo fatty acid biosynthesis. This heteromeric enzyme is composed of one plastid-coded subunit (?-carboxyltransferase) and three nuclear-coded subunits (biotin carboxy-carrier, biotin carboxylase, and ?-carboxyltransferase). We report the primary structure of the Arabidopsis ?-carboxyltransferase and ?-carboxyltransferase subunits deduced from nucleotide sequences of the respective genes and/or cDNA. Co-immunoprecipitation experiments confirm that the ?-carboxyltransferase and ?-carboxyltransferase subunits are physically associated. The plant ?-carboxyltransferases have gained a C-terminal domain relative to eubacteria, possibly via the evolutionary acquisition of a single exon. This C-terminal domain is divergent among plants and may have a structural function rather than being essential for catalysis. The four ACCase subunit mRNAs accumulate to the highest levels in tissues and cells that are actively synthesizing fatty acids, which are used either for membrane biogenesis in rapidly growing tissues or for oil accumulation in developing embryos. Development coordinately affects changes in the accumulation of the ACCase subunit mRNAs so that these four mRNAs maintain a constant molar stoichiometric ratio. These data indicate that the long-term, developmentally regulated expression of the heteromeric ACCase is in part controlled by a mechanism(s) that coordinately affects the steady-state concentrations of each subunit mRNA. PMID:10759501
How to Determine the Sponsored Award Chartstring(s) Contracts & Grants Accounting COA-232 03/27/2014 Page 1 of 2
Klein, Ophir
Government Contract 25000-28999 4003 Fed Government Grant, ARRA 21501-21594, 33162-33337 4004 Fed GovernmentHow to Determine the Sponsored Award Chartstring(s) Contracts & Grants Accounting COA-232 03 when they have completed award setup. #12;How to Determine the Sponsored Award Chartstring(s) Contracts
RNA-Catalyzed CoA, NAD, and FAD Synthesis from Phosphopantetheine, NMN, Faqing Huang,*, Charles Walter Bugg, and Michael Yarus
Huang, Faqing
RNA-Catalyzed CoA, NAD, and FAD Synthesis from Phosphopantetheine, NMN, and FMN Faqing HuangA, NAD, and FAD, from their precursors, 4-phosphopantetheine, NMN, and FMN, respectively. Both ribozymesA), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD), carry out a variety of acyl
Acetylation at Lysine 183 of Progesterone Receptor by p300 Accelerates DNA Binding Kinetics and Transactivation of Direct Target Genes
Chung, Hwa Hwa; Sze, Siu Kwan; Tay, Alvin Shun Long; Lin, Valerie C.-L.
The identification of lysine acetylation of steroid hormone receptors has previously been based on the presence of consensus motif (K/R)XKK. This study reports the discovery by mass spectrometry of a novel progesterone receptor acetylation site at Lys-183 that is not in the consensus motif. In vivo acetylation and mutagenesis experiments revealed that Lys-183 is a primary site of progesterone receptor (PR) acetylation. Lys-183 acetylation is enhanced by p300 overexpression and abrogated by p300 gene silencing, suggesting that p300 is the major acetyltransferase for Lys-183 acetylation. Furthermore, p300-mediated Lys-183 acetylation is associated with heightened PR activity. Accordingly, the acetylation-mimicking mutant PRB-K183Q exhibited accelerated DNA binding kinetics and greater activity compared with the wild-type PRB on genes containing progesterone response element. In contrast, Lys-183 acetylation had no influence on PR tethering effect on the nuclear factor ?-light chain enhancer of activated B cells (NF?B). Additionally, increases of Lys-183 acetylation by p300 overexpression or inhibition of deacetylation resulted in increases of Ser-294 phosphorylation levels. In conclusion, PR acetylation at Lys-183 by p300 potentiates PR activity through accelerated binding of its direct target genes without affecting PR tethering on other transcription factors. The effect may be mediated by enhancing Ser-294 phosphorylation. PMID:24302725
Acetylation of Vertebrate H2A.Z and Its Effect on the Structure of the Nucleosome†
Ishibashi, Toyotaka; Dryhurst, Deanna; Rose, Kristie L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Ausió, Juan
Purified histone H2A.Z from chicken erythrocytes and a sodium butyrate-treated chicken erythroleukemic cell line was used as a model system to identify the acetylation sites (K4, K7, K11, K13, and K15) and quantify their distribution in this vertebrate histone variant. To understand the role played by acetylation in the modulation of the H2A.Z nucleosome core particle (NCP) stability and conformation, an extensive analysis was conducted on NCPs reconstituted from acetylated forms of histones, including H2A.Z and recombinant H2A.Z (K/Q) acetylation mimic mutants. Although the overall global acetylation of core histones destabilizes the NCP, we found that H2A.Z stabilizes the NCP regardless of its state of acetylation. Interestingly and quite unexpectedly, we found that the change in NCP conformation induced by global histone acetylation is dependent on H2A/H2A.Z acetylation. This suggests that acetylated H2A variants act synergistically with the acetylated forms of the core histone complement to alter the particle conformation. Furthermore, the simultaneous occurrence of H2A.Z and H2A in heteromorphic NCPs that most likely occurs in vivo slightly destabilizes the NCP, but only in the presence of acetylation. PMID:19385636
Modifications of cell signalling and redox balance by targeting protein acetylation using natural and engineered molecules: implications in cancer therapy.
Venkateswaran, Kavya; Verma, Amit; Bhatt, Anant N; Agrawala, Paban K; Raj, Hanumantharao G; Malhotra, Shashwat; Prasad, Ashok K; Wever, Olivier De; Bracke, Marc E; Saso, Luciano; Parmar, Virinder S; Shrivastava, Anju; Dwarakanath, B S
Acetylation of proteins with the addition of an acetyl group on the lysine residue is one of the vital posttranslational modifications that regulate protein stability, function and intracellular compartmentalization. Like other posttranslational modifications, protein acetylation influences many if not all vital functions of the cell. Protein acetylation has been originally associated with histone acetylation regulated by Histone Acetyl Transferase (HAT) and Histone Deacetylase (HDAC) and was mainly considered to be involved in epigenetic regulation through chromatin remodelling. It is now widely referred to as lysine acetylation orchestrated by lysine acetyl transferase (KAT) and lysine deacetylase (KDAC) and influences many cellular functions. Protein acetylation fine tunes the redox balance and cell signalling in the context of cancer by exerting its control on expression of two very important redox sensors viz. Nrf2 and NF-?B. Accumulating evidences show that inhibitors of deacetylase (KDACi), responsible for cytotoxic effects in cancer cells, mediate their actions by inhibiting the deacetylases, thereby simulating an hyperacetylation state of histone as well as non-histone proteins, similar to the one created by KATs. Emergence of calreticulin (CRT) mediated protein acetylation system using polyphenolic acetates as donors coupled with over expression of CRT has opened new avenues for targeting protein acetylation for improving cancer therapy. Modifiers of protein acetylation are therefore, emerging as a class of anticancer therapeutics and adjuvant as they inhibit growth, induce differentiation and death (apoptosis) differentially in cancer cells and also exhibit chemo-radiation sensitizing potential. Although pre-clinical investigations with many natural and synthetic KDAC inhibitors have been very promising, their clinical utility has so far been limited to certain types of cancers of the hematopoietic system. The future of protein acetylation modifiers appears to depend on the development of newer engineered molecules and their rational combinations that can exploit the differences in the regulation of protein acetylation between tumor and normal cells/tissues. PMID:25478886
Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.
Rastogi, V K; DeFrank, J J; Cheng, T C; Wild, J R
The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX. PMID:9425265
Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis.
Domanico, P L; Rahil, J F; Benkovic, S J
The stereochemical course of rabbit liver fructose bisphosphatase (EC 22.214.171.124) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water. PMID:2988606
[Enhancement of sewage sludge anaerobic digestibility by thermal hydrolysis pretreatment].
Wang, Zhi-jun; Wang, Wei
Biochemical methane potential (BMP) experiments of thermo-hydrolyzed sewage sludge are carried out to investigate the effects of thermal hydrolysis on the digestibility of sewage sludge. The results show that thermal hydrolysis pretreatment can facilitate the dissolving of organic solid in sludge, and soluble organics hydrolyzed into low molecular organics, in which volatile fat acids accounted for 30% - 40 % of soluble COD, so the digestibility of sewage sludge remarkably improved. The optimum pretreatment temperature and holding time were 170 degrees C and 30 minutes, under which the total COD removal rate enhanced from original 38.11% to 56.78%, and biogas production rate of COD in feeding sludge from 160mL/g to 250mL/g. PMID:15859411
Investigation of the Polymorphs and Hydrolysis of Uranium Trioxide
Sweet, Lucas E.; Blake, Thomas A.; Henager, Charles H.; Hu, Shenyang Y.; Johnson, Timothy J.; Meier, David E.; Peper, Shane M.; Schwantes, Jon M.
This work focuses on progress in gaining a better understanding of the polymorphic nature of the UO3-water system, one of several important materials associated with the nuclear fuel cycle. The UO3-water system is complex and has not been fully characterized, even though these species are common throughout the fuel cycle. Powder x-ray diffraction, Raman and fluorescence characterization was performed on polymorphic forms of UO3 and UO3 hydrolysis products for the purpose of developing some predictive capability of estimating process history and utility, e.g. for polymorphic phases of unknown origin. Specifically, we have investigated three industrially relevant production pathways of UO3 and discovered a previously unknown low temperature route to ?-UO3. Pure phases of UO3, hydrolysis products and starting materials were used to establish optical spectroscopic signatures for these compounds.
Kinetics of the surface hydrolysis of raw starch by glucoamylase.
Tatsumi, Hirosuke; Katano, Hajime
The hydrolysis of raw starch catalyzed by glucoamylase has been studied with starch granules of different sizes by use of an amperometric glucose sensor by which the direct and continuous observation of the concentration of glucose can be achieved even in a thick raw starch suspension. The initial rate of the enzymatic hydrolysis in the raw starch suspension increased with increasing concentration of the enzyme to approach a saturation value and was proportional to the amount of substrate. Also, the rate was proportional to the specific surface area of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism, which consists of adsorption of the free enzyme onto the surface of the substrate, reaction of the adsorbed enzyme with the substrate, and liberation of the product. PMID:16218653
Kinetics of the alkaline hydrolysis of flavonoid glycosides
M. D. Alaniya
Summary 1. In a study of the kinetics of the alkaline hydrolysis of flavone glycosides it has been found that derivatives of 3,3?,4?,5,7-pentahydroxyflavone\\u000a hydrolyze faster than derivatives of 3,4?,5,7-tetrahydroxyflavone and of 3,4?,5,7-tetrahydroxy-3?-methoxyflavone.\\u000a \\u000a 2. In the hydrolysis of diglycosides of 3,3?,4?,5,7-pentahydroxyflavones the maximum amount of intermediate product is formed\\u000a after 2 min (3,4?,5,7-tetrahydroxyflavone glycoside), and in the case of 3,4?,5,7-tetrahydroxy-3?-methoxyflavone glycosides\\u000a after
Enzymatic hydrolysis of cellulosic materials: a kinetic study
Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Sarto, V.
A kinetic study of the enzymatic hydrolysis of two celluloses with different structural features was performed at various temperatures (26-50/sup 0/C). The enzymatic system consisted of three types of enzymes: E/sub 1/-..beta..-1,4-glucan glucanohydrolase; E/sub 2/-..beta..-1,4-glucan cellobiohydrolase; and E/sub 3/-..beta..-glucosidase. A mathematical model for the mechanism of the hydrolysis of cellulosic materials catalyzed by a multienzymatic system was checked and a good rationalization of the experimental results was achieved. Uncompetitive and competitive glucose inhibition on E/sub 1/ and E/sub 2/, respectively, appeared to occur for both substrates. Inhibition by cellobiose was checked at 34/sup 0/C on one substrate. The V/sub max/, K/sub m/, and glucose inhibition constants were optimized and their dependence on temperature determined.
Redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis
Khare, Sagar D.; Kipnis, Yakov; Greisen, Per; Takeuchi, Ryo; Ashani, Yacov; Goldsmith, Moshe; Song, Yifan; Gallaher, Jasmine L.; Silman, Israel; Leader, Haim; Sussman, Joel L.; Stoddard, Barry L.; Tawfik, Dan S.; Baker, David
The ability to redesign enzymes to catalyze non-cognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of active site functional groups of metalloenzymes to catalyze new reactions. Using this method, we engineered a zinc-containing murine adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency kcat/Km ~104 M?1s?1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzed the hydrolysis of the RP-isomer of a coumarinyl analog of the nerve agent cyclosarin, and showed striking substrate selectivity for coumarinyl leaving-groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities. PMID:22306579
Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.
Khare, Sagar D; Kipnis, Yakov; Greisen, Per; Takeuchi, Ryo; Ashani, Yacov; Goldsmith, Moshe; Song, Yifan; Gallaher, Jasmine L; Silman, Israel; Leader, Haim; Sussman, Joel L; Stoddard, Barry L; Tawfik, Dan S; Baker, David
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities. PMID:22306579
Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors.
Cao, Guangli; Ximenes, Eduardo; Nichols, Nancy N; Frazer, Sarah E; Kim, Daehwan; Cotta, Michael A; Ladisch, Michael
A stepwise removal of inhibitory compounds by bioabatement combined with hemicellulase supplementation was conducted to enhance cellulose hydrolysis of liquid hot water-pretreated corn stover. Results showed that the fungus Coniochaeta ligniaria NRRL30616 eliminated most of the enzyme and fermentation inhibitors from liquid hot water-pretreated corn stover hydrolysates. Moreover, addition of hemicellulases after bioabatement and before enzymatic hydrolysis of cellulose achieved 20% higher glucose yields compared to non-treated samples. This work presents the mechanisms by which supplementation of the fungus with hemicellulase enzymes enables maximal conversion, and confirms the inhibitory effect of xylo-oligosaccharides in corn stover hydrolysates once the dominant inhibitory effect of phenolic compounds is removed. PMID:25958134
Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.
Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun
Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ?90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose. PMID:23553108
Hydrolysis of black soybean isoflavone glycosides by Bacillus subtilis natto
Lun-Cheng Kuo; Wei-Yi Cheng; Ren-Yu Wu; Ching-Jang Huang; Kung-Ta Lee
Hydrolysis of isoflavone glycosides by Bacillus subtilis natto NTU-18 in black soymilk is reported. At the concentration of 3–5% (w\\/v), black soymilk in flask cultures, the isoflavones, daidzin, and genistin were highly deglycosylated within 24 h. Deglycosylation of isoflavones was further carried out in a 7-l fermenter with 5% black soymilk. During the fermentation, viable cells increased from 103 to 109 CFU
Novel Penicillium cellulases for total hydrolysis of lignocellulosics.
Marjamaa, Kaisa; Toth, Karolina; Bromann, Paul Andrew; Szakacs, George; Kruus, Kristiina
The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and ?-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35°C while at 45°C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases. PMID:23608505
Preparation of icariside II from icariin by enzymatic hydrolysis method.
Xia, Quan; Xu, Dujuan; Huang, Zhaogang; Liu, Jianjun; Wang, Xinqun; Wang, Xiu; Liu, Shangquan
It has been reported that icariin and icariside II, two flavonoid glycosides coming from herba epimedii, which have a closely structural relationship, show some pharmacological effects such as preventing osteoporosis, cancer and depression. The content of natural icariside II is very low in herba epimedii, but it is the main component in vivo after the administration of herba epimedii. More icariside II can be obtained from icariin by enzymatic hydrolysis method than by traditional isolation method. This study focuses on finding a simple and feasible method to prepare icariside II from icariin by enzymatic hydrolysis, so as to meet the request for further pharmacologic actions study. Icariin was obtained successively with 90% ethanol extraction, isolation on macroporous resin and purification on silica gel chromatography. Enzymatic hydrolysis conditions were tested for the bioconversion of icariin into icariside II by orthogonal array design. The structures of isolated icariin and produced icariside II were identified by UV, IR, ESIMS, (1)H NMR, (13)C NMR, and DEPT spectroscope. Enzymatic hydrolysis experiment showed that icariin could be transformed into icariside II with the action of beta-glucosidase and the optimum reaction conditions were determined as follows: 50 degrees C, 0.2 M disodium hydrogen phosphate and citric acid buffer system (pH6.0), the ratio of icariin/enzyme is 1:1 and reaction time 5 h. By using this enzymatic condition, 95.5 mg icariside II (with the purity of 99.1%) was obtained eventually by transforming 200 mg icariin. PMID:20026390
Endo-exo synergism in cellulose hydrolysis revisited.
Jalak, Jürgen; Kurašin, Mihhail; Teugjas, Hele; Väljamäe, Priit
Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios. PMID:22733813
Pt Catalysed Hydrogen Generation by Hydrolysis of Sodium Tetrahydroborate
U. B. Demirci; F. Garin
The hydrolysis of sodium tetrahydroborate is a promising way for generating hydrogen. The key element of this process is the catalyst. The present study reports the performances of Pt-based catalysts (supported over various metal oxides like TiO2 or ZrO2, with different Pt loadings, or doped with metal-'impurities' like Ag or Pd). TiO2 is the best support and the optimal Pt
Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate
D. Jain; T. K. Ghose
The rate of cellulose degradation, limited by inhibition by cellobiose, can be increased by hydrolysis of cellobiose to glucose using immobilized ..beta..-glucosidase. Production of ..beta..-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU\\/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. Immobilization of ..beta..-glucosidase containing cells
Management of spent solvents by alkaline hydrolysis process
Smitha Manohar; C. Srinivas; Tessy Vincent; P. K. Wattal
This paper deals with the treatment method for the management of spent solvents of reprocessing origin (30% tri-n-butyl phosphate in n-dodecane) using the 'alkaline hydrolysis process'. The consolidated work reported herein has established total conversion of TBP to aqueous soluble reaction products and transfer of near total radioactivity associated with the waste into this aqueous phase. It was also observed
Calcium stimulation of glutamine hydrolysis in synaptosomes from rat brain
E. Kvamme; G. Svenneby; I. Aa. Torgner
Calcium stimulates the hydrolysis of glutamine in synaptosomes prepared from rat brain both by the sucrose- (12) and the Ficoll\\/sucrose-gradient techniques (13). The calcium activation is phosphate-dependent and maximal effect is obtained at a calcium concentration of 0.5–1.0 mM. It is reduced by increasing the numbers of synaptosomes in the incubation mixture, and abolished by the product inhibitors of glutaminase,
Supercritical CO 2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis
Kyoung Heon Kim; Juan Hong
The supercritical carbon dioxide (SC–CO2) pretreatment of lignocellulose for enzymatic hydrolysis of cellulose was investigated. Aspen (hardwood) and southern yellow pine (softwood) with moisture contents in the range of 0–73% (w\\/w) were pretreated with SC–CO2 at 3100 and 4000 psi and at 112–165°C for 10–60 min. Each pretreated lignocellulose was hydrolyzed with commercial cellulase to assess its enzymatic digestibility. Untreated
Biological pretreatment of lignocellulose for enzymatic hydrolysis of cellulose
A. I. Hatakka
Pretreatment of lignocellulosic materials is considered as the rate-limiting step in an economically feasible process for\\u000a enzymatic hydrolysis of cellulose. Biological delignification techniques have not been developed as intensively as physical\\u000a and chemical methods. However, white-rot fungi are effective degraders of lignin, and some of them even preferentially remove\\u000a lignin from wood compared with carbohydrates, and therefore might be suitable
Pancreatic lipase hydrolysis of triglycerides by a semimicro technique
F. E. Luddy; R. A. Barford; S. F. Herb; P. Magidman; R. W. Riemenschneider
Procedures are described for rapid lipase hydrolysis of triglycerides, isolation of the hydrolytic products by TLC and their\\u000a conversion to methyl esters and fatty acid analysis by GLC. The techniques are applicable to a few mg of triglycerides or\\u000a fats. Examples of data obtained with purified triglycerides indicate that the specific action of pancreatic lipase for the\\u000a 1,3 ester groups
Application of immobilized lipase to hydrolysis of triacylglyceride
Yoshiharu Kimura; Atsuo Tanaka; Kenji Sonomoto; Takuya Nihira; Saburo Fukui
Lipase from Candida cylindracea was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers, and by covalent binding or by adsorption to different types of porous inorganic or organic supports. All of the immobilized lipase preparations thus obtained showed some activity for hydrolysis of olive oil. Lipase entrapped with a hydrophobic photo-cross-linkable resin prepolymer exhibited the highest activity, which
Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood
Charlotte Tengborg; Mats Galbe; Guido Zacchi
Softwood constitutes the main source of lignocellulosic material in Sweden which can be used for ethanol production from renewable resources. To make the biomass-to-ethanol process more economically feasible, it is preferable to include the sugar-rich prehydrolysate, i.e. the liquid obtained after the pretreatment step, in the enzymatic hydrolysis of the solid fraction. This study shows that the prehydrolysate inhibits cellulose
Further studies on the pancreatic hydrolysis of some natural fats
M. H. Coleman
A series of animal and vegetable fats has been subjected to hydrolysis with pancreatic lipase. From the results obtained,\\u000a the triglyceride compositions of the original fats have been calculated by the method previously proposed by Coleman and Fulton.\\u000a \\u000a These compositions show substantial agreement with those obtained by other methods. Similarities and differences between fats\\u000a are shown to be reflections of
Molecular Mechanism of Slow Acetylation of Drugs and Carcinogens in Humans
Martin Blum; Anne Demierre; Denis M. Grant; Markus Heim; Urs A. Meyer
The acetylation polymorphism is one of the most common genetic variations in the transformation of drugs and chemicals. More than 50% of individuals in Caucasian populations are homozygous for a recessive trait and are of the \\
Histone Acetylation Modifiers in the Pathogenesis of Alzheimer's Disease
Lu, Xi; Wang, Li; Yu, Caijia; Yu, Daohai; Yu, Gang
It is becoming more evident that histone acetylation, as one of the epigenetic modifications or markers, plays a key role in the etiology of Alzheimer's disease (AD). Histone acetylases and histone deacetylases (HDACs) are the well-known covalent enzymes that modify the reversible acetylation of lysine residues in histone amino-terminal domains. In AD, however, the roles of these enzymes are controversial. Some recent studies indicate that HDAC inhibitors are neuroprotective by regulating memory and synaptic dysfunctions in cellular and animal models of AD; while on the other hand, increase of histone acetylation have been implicated in AD pathology. In this review, we focus on the recent advances on the roles of histone acetylation covalent enzymes in AD and discuss how targeting these enzymes can ultimately lead to therapeutic approaches for treating AD. PMID:26136662
Solid-state structure of methyl 2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-?-D-glucopyranosyl)-?-D-galactopyranoside and methyl 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-?-D-glucopyranosyl)-?-D-galactopyranoside
NASA Astrophysics Data System (ADS)
Gubica, Tomasz; Bukowicki, Jaros?aw; St?pie?, Dorota K.; Ostrowski, Andrzej; Pisklak, Dariusz M.; Cyra?ski, Micha? K.
Comprehensive structural analyses of methyl 2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-?-D-glucopyranosyl)-?-D-galactopyranoside (1) and methyl 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-?-D-glucopyranosyl)-?-D-galactopyranoside (2) have been performed. The analyses combined experimental and theoretical methods: single-crystal and powder X-ray diffraction and 13C CP/MAS NMR spectroscopy served as experimental techniques, whereas the grid search of the conformers of disaccharides implemented by genetic algorithm was chosen as the theoretical method. One of the calculated conformers of 1 fits the experimental structure very well. Although the two best conformations of 2 are of similar energy, their structures are significantly different. Probably therefore 2 cannot form any crystallographic forms and is amorphous.
Tri3, Which Controls Trichothecene C-15 Acetylation, is Functional in 3ADON Chemotype
Technology Transfer Automated Retrieval System (TEKTRAN)
Three different trichothecene chemotypes have been identified in U.S. strains of Fusarium graminearum: 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV), although grain is typically contaminated with deoxynivalenol (DON) or nivalenol rather than the acetylated ...
Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth
Sun, Ting; Li, Xuan; Zhang, Peng; Chen, Wen-Dan; Zhang, Hai-liang; Li, Dan-Dan; Deng, Rong; Qian, Xiao-Jun; Jiao, Lin; Ji, Jiao; Li, Yun-Tian; Wu, Rui-Yan; Yu, Yan; Feng, Gong-Kan; Zhu, Xiao-Feng
Beclin 1, a protein essential for autophagy, regulates autophagy by interacting with Vps34 and other cofactors to form the Beclin 1 complex. Modifications of Beclin 1 may lead to the induction, inhibition or fine-tuning of the autophagic response under a variety of conditions. Here we show that Beclin 1 is acetylated by p300 and deacetylated by SIRT1 at lysine residues 430 and 437. In addition, the phosphorylation of Beclin 1 at S409 by CK1 is required for the subsequent p300 binding and Beclin 1 acetylation. Beclin 1 acetylation inhibits autophagosome maturation and endocytic trafficking by promoting the recruitment of Rubicon. In tumour xenografts, the expression of 2KR mutant Beclin 1 (substitution of K430 and K437 to arginines) leads to enhanced autophagosome maturation and tumour growth suppression. Therefore, our study identifies an acetylation-dependent regulatory mechanism governing Beclin 1 function in autophagosome maturation and tumour growth. PMID:26008601
Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast
NASA Technical Reports Server (NTRS)
Satyanarayana, T.; Mandel, A. D.; Klein, H. P.
Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.
In vivo treatment by diallyl disulfide increases histone acetylation in rat colonocytes
Druesne-Pecollo, Nathalie [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France)]. E-mail: email@example.com; Chaumontet, Catherine [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Pagniez, Anthony [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Vaugelade, Pierre [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Bruneau, Aurelia [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Thomas, Muriel [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Cherbuy, Claire [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Duee, Pierre-Henri [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France); Martel, Paule [Laboratoire de Nutrition et Securite Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas cedex (France)
Diallyl disulfide (DADS) is an organosulfur compound from garlic which exhibits various anticarcinogenic properties including inhibition of tumor cell proliferation. DADS antiproliferative effects were previously associated with an increase in histone acetylation in two human tumor colon cell lines, suggesting that DADS-induced histone hyperacetylation could be one of the mechanisms involved in its protective properties on colon carcinogenesis. The effects of DADS on histone H4 and H3 acetylation levels were investigated in vivo in colonocytes isolated from non-tumoral rat. Administrated by intracaecal perfusion or gavage, DADS increases histone H4 and H3 acetylation in colonocytes. Moreover, data generated using cDNA expression arrays suggest that DADS could modulate the expression of a subset of genes. These results suggest the involvement of histone acetylation in modulation of gene expression by DADS in normal rat colonocytes, which might play a role in its biological effects as well as in its anticarcinogenic properties in vivo.
Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change
Welsch, D.J.; Nelsestuen, G.L.
Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 /sup 0/C or with 0.2 M hydroxylamine at 50 /sup 0/C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. The major radiolabeled product contained Asn/sup 101/-Ser/sup 102/ along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue. Mass spectral analysis showed the correct mass for this glycopeptide plus a single added acetyl group. Amide /sup 1/H NMR analysis showed only three amide protons rather than the anticipated four. On the basis of these several observations, it is postulated that the site of acetylation is the ..beta..-amide nitrogen of Asn-101. Consequently, these studies showed an unusual chemical reactivity in prothrombin fragment 1. They further show that metal ion binding to prothrombin fragment 1 and subsequent protein fluorescence quenching involve sites ion the kringle region of the protein.
Reaction pathways and free energy profiles for spontaneous hydrolysis of urea and tetramethylurea: Unexpected substituent effects
Yao, Min; Tu, Wenlong; Chen, Xi; Zhan, Chang-Guo
It has been difficult to directly measure the spontaneous hydrolysis rate of urea and, thus, 1,1,3,3-tetramethylurea (Me4U) was used as a model to determine the "experimental" rate constant for urea hydrolysis. The use of Me4U was based on an assumption that the rate of urea hydrolysis should be 2.8 times that of Me4U hydrolysis because the rate of acetamide hydrolysis is 2.8 times that of N,N-dimethyl-acetamide hydrolysis. The present first-principles electronic-structure calculations on the competing non-enzymatic hydrolysis pathways have demonstrated that the dominant pathway is the neutral hydrolysis via the CN addition for both urea (when pH<~11.6) and Me4U (regardless of pH), unlike the non-enzymatic hydrolysis of amides where alkaline hydrolysis is dominant. Based on the computational data, the substituent shift of free energy barrier calculated for the neutral hydrolysis is remarkably different from that for the alkaline hydrolysis, and the rate constant for the urea hydrolysis should be ~1.3×109-fold lower than that (4.2×10?12 s?1) measured for the Me4U hydrolysis. As a result, the rate enhancement and catalytic proficiency of urease should be 1.2×1025 and 3×1027 M?1, respectively, suggesting that urease surpasses proteases and all other enzymes in its power to enhance the rate of reaction. All of the computational results are consistent with available experimental data for Me4U, suggesting that the computational prediction for urea is reliable. PMID:24097048
Mutation analysis of methylmalonyl CoA mutase gene exon 2 in Egyptian families: Identification of 25 novel allelic variants
Ghoraba, Dina A.; Mohammed, Magdy M.; Zaki, Osama K.
Methylmalonic aciduria (MMA) is an autosomal recessive disorder of methylmalonate and cobalamin (cbl; vitamin B12) metabolism. It is an inborn error of organic acid metabolism which commonly results from a defect in the gene encoding the methylmalonyl-CoA mutase (MCM) apoenzyme. Here we report the results of mutation study of exon 2 of the methylmalonyl CoA mutase (MUT) gene, coding MCM residues from 1 to 128, in ten unrelated Egyptian families affected with methylmalonic aciduria. Patients were presented with a wide-anion gap metabolic acidosis. The diagnosis has established by the measurement of C3 (propionylcarnitine) and C3:C2 (propionylcarnitine/acetylcarnitine) in blood by using liquid chromatography–tandem mass spectrometry (LC/MS–MS) and was confirmed by the detection of an abnormally elevated level of methylmalonic acid in urine by using gas chromatography–mass spectrometry (GC/MS) and isocratic cation exchange high-performance liquid-chromatography (HPLC). Direct sequencing of gDNA of the MUT gene exon 2 has revealed a total of 26 allelic variants: ten of which were intronic, eight were located upstream to the exon 2 coding region, four were novel modifications predicted to affect the splicing region, three were novel mutations within the coding region: c.15G > A (p.K5K), c.165C > A (p.N55K) and c.7del (p.R3EfsX14), as well as the previously reported mutation c.323G > A (p.R108H). PMID:25750861
Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production
Background During cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis. Results Cellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the hydrolysis and degree of synergism among enzymes. Conclusions The model was effective in capturing the dynamic behavior of cellulose hydrolysis during action of individual as well as multiple cellulases. Simulations were in qualitative and quantitative agreement with experimental data. Several experimentally observed phenomena were simulated without the need for any additional assumptions or parameter changes and confirmed the validity of using the stochastic molecular modeling approach to quantitatively and qualitatively describe the cellulose hydrolysis. PMID:23638989
Pretreatment for cellulose hydrolysis by carbon dioxide explosion
Zheng, Y.; Lin, H.M.; Tsao, G.T. [Purdue Univ., West Lafayette, IN (United States). Lab of Renewable Resources Engineering] [Purdue Univ., West Lafayette, IN (United States). Lab of Renewable Resources Engineering
Cellulosic materials were treated with supercritical carbon dioxide to increase the reactivity of cellulose, thereby to enhance the rate and the extent of cellulose hydrolysis. In this pretreatment process, the cellulosic materials such as Avicel, recycled paper mix, sugarcane bagasse and the repulping waste of recycled paper are placed in a reactor under pressurized carbon dioxide at 35 C for a controlled time period. Upon an explosive release of the carbon dioxide pressure, the disruption of the cellulosic structure increases the accessible surface area of the cellulosic substrate to enzymatic hydrolysis. Results indicate that supercritical carbon dioxide is effective for pretreatment of cellulose. An increase in pressure facilitates the faster penetration of carbon dioxide molecules into the crystalline structures, thus more glucose is produced from cellulosic materials after the explosion as compared to those without the pretreatment. This explosion pretreatment enhances the rate of cellulosic material hydrolysis as well as increases glucose yield by as much as 50%. Results from the simultaneous saccharification and fermentation tests also show the increase in the available carbon source from the cellulosic materials for fermentation to produce ethanol. As an alternative method, this supercritical carbon dioxide explosion has a possibility to reduce expense compared with ammonia explosion, and since it is operated at the low temperature, it will not cause degradation of sugars such as those treated with steam explosion due to the high-temperature involved.
Enzymatic hydrolysis of defatted mackerel protein with low bitter taste
NASA Astrophysics Data System (ADS)
Hou, Hu; Li, Bafang; Zhao, Xue
Ultrasound-assisted solvent extraction was confirmed as a novel, effective method for separating lipid from mackerel protein, resulting in a degreasing rate (DR) of 95% and a nitrogen recovery (NR) of 88.6%. To obtain protein hydrolysates with high nitrogen recovery and low bitter taste, enzymatic hydrolysis was performed using eight commercially available proteases. It turned out that the optimum enzyme was the `Mixed enzymes for animal proteolysis'. An enzyme dosage of 4%, a temperature of 50°, and a hydrolysis time of 300 min were found to be the optimum conditions to obtain high NR (84.28%) and degree of hydrolysis (DH, 16.18%) by orthogonal experiments. Glutamic acid was the most abundant amino acid of MDP (defatted mackerel protein) and MDPH (defatted mackerel protein hydrolysates). Compared with the FAO/WHO reference protein, the essential amino acid chemical scores (CS) were greater than 1.0 (1.0-1.7) in MDPH, which is reflective of high nutritional value. This, coupled with the light color and slight fishy odor, indicates that MDPH would potentially have a wide range of applications such as nutritional additives, functional ingredients, and so on.
Enzymatic hydrolysis: a method in alleviating legume allergenicity.
Kasera, Ramkrashan; Singh, A B; Lavasa, S; Prasad, Komarla Nagendra; Arora, Naveen
Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes - kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase(®) and Flavourzyme(®). Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2?±?7.7%, 87.1?±?9.6% and 91.8?±?7.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (p?0.01). The release of histamine was decreased significantly when sensitized basophils were challenged with hydrolysates as compared to raw extracts. Significant reduction in the biopotency of hydrolysates was also observed in SPT where only 1/10 kidney bean-sensitive individuals, 2/6 black gram-sensitive individuals and 1/7 peanut-sensitive individuals were found positive to their respective hydrolysates. In conclusion, enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts. PMID:25481434
Programmed Hydrolysis in Designing Paclitaxel Prodrug for Nanocarrier Assembly
Fu, Q.; Wang, Y.; Ma, Y.; Zhang, D.; Fallon, J. K.; Yang, X.; Liu, D.; He, Z.; Liu, F.
Nanocarriers delivering prodrugs are a way of improving in vivo effectiveness and efficiency. For therapeutic efficacy, the prodrug must hydrolyze to its parent drug after administration. Based on the fact that the hydrolysis is impeded by steric hindrance and improved by sufficient polarity, in this study, we proposed the PTX-S-S-VE, the conjugation of paclitaxel (PTX) to vitamin E (VE) through a disulfide bridge. This conjugate possessed the following advantages: first, it can be encapsulated in the VE/VE2-PEG2000/water nanoemulsions because of favorable hydrophobic interactions; second, the nanoemulsions had a long blood circulation time; finally, the concentrated glutathione in the tumor microenvironment could cleave the disulfide bond to weaken the steric hindrance and increase the polarity, promoting the hydrolysis to PTX and increasing the anticancer activity. It was demonstrated in vitro that the hydrolysis of PTX-S-S-VE was enhanced and the cytotoxicity was increased. In addition, PTX-S-S-VE had greater anticancer activity against the KB-3-1 cell line tumor xenograft and the tumor size was smaller after the 4th injection. The present result suggests a new way, use of reduction, to improve the in vivo anticancer activity of a prodrug for nanocarrier delivery by unshielding the ester bond and taking off the steric block. PMID:26166066
Hydrolysis of organonitrate functional groups in aerosol particles
Liu, Shang; Shilling, John E.; Song, Chen; Hiranuma, Naruki; Zaveri, Rahul A.; Russell, Lynn M.
Organonitrate (ON) groups are important substituents in secondary organic aerosols. Model simulations and laboratory studies indicate a large fraction of ON groups in aerosol particles, but much lower quantities are observed in the atmosphere. Hydrolysis of ON groups in aerosol particles has been proposed recently. To test this hypothesis, we simulated formation of ON molecules in a reaction chamber under a wide range of relative humidity (0% to 90%). The mass fraction of ON groups (5% to 20% for high-NOx experiments) consistently decreased with increasing relative humidity, which was best explained by hydrolysis of ON groups at a rate of 4 day-1 (lifetime of 6 hours) for reactions under relative humidity greater than 20%. In addition, we found that secondary nitrogen-containing molecules absorb light, with greater absorption under dry and high-NOx conditions. This work provides the first evidence for particle-phase hydrolysis of ON groups, a process that could substantially reduce ON group concentration in the atmosphere.
Enteric nerve fibers of holothurians are recognized by an antibody to acetylated alpha-tubulin.
García-Arrarás, J E; Viruet, E
The distribution of immunoreactivity to 6-11B-1, a monoclonal antibody that labels acetylated alpha-tubulin, was studied in the radial nerve and intestinal system of holothurians. As shown previously for other species, this antibody recognizes cilia and nerve fibers in Holothuria glaberrima and Holothuria mexicana. Thus, anti-acetylated alpha-tubulin can be used as a marker for nerve fibers in the enteric nervous system. PMID:8233047
Characterization of acetylated wood decayed by brown-rot and white-rot fungi
Makoto Ohkoshi; Atsushi Kato; Kentaro Suzuki; Noriko Hayashi; Mitsuro Ishihara
The objective of this study was to characterize the decay of acetylated wood due to brown-rot and white-rot fungi by analysis\\u000a of chemical composition, X-ray measurements, and13C-NMR spectroscopy. The decay by brown-rot fungus became inhibited at a weight percent gain (WPG) due to acetylation of more\\u000a than 10%, and the mass loss (LOSS) due to decay became zero at a
N-Acetyl cysteine blunts proteotoxicity in a heat shock protein-dependent manner.
Jiang, Y; Rumble, J L; Gleixner, A M; Unnithan, A S; Pulugulla, S H; Posimo, J M; Choi, H J H; Crum, T S; Pant, D B; Leak, R K
N-Acetyl cysteine, a glutathione precursor, has been shown to benefit patients with Alzheimer's disease and reduce the symptoms of traumatic brain injury in soldiers. Parkinson's and Alzheimer's disease are both characterized by stress from protein misfolding, or proteotoxicity. We have developed a high-throughput model of proteotoxicity by treating neuroblastoma N2a cells with the proteasome inhibitor MG132 and performing three independent assays for viability. Our previous study showed that N-acetyl cysteine protects N2a cells against two sequential treatments of MG132 and raises glutathione levels in a two-hit model of synergistic neurodegeneration. In the present study, however, N-acetyl cysteine was found to reduce the toxicity of a single hit of MG132 independent of its effect on glutathione. All three viability assays confirmed this protection. We measured heat shock protein 70 (Hsp70) levels because Hsp70 is a protective chaperone that helps refold proteins or guides ubiquitinated proteins toward degradation by the proteasome. Hsp70 levels were higher in MG132-treated cells when N-acetyl cysteine was applied. No parallel change in heat shock cognate 70 (Hsc70) was elicited. Inhibition of Hsp70/Hsc70 activity with VER 155008 attenuated the protection afforded by N-acetyl cysteine in a dose-responsive manner. MG132 induced a large rise in ubiquitinated proteins and N-acetyl cysteine reduced this effect. Consistent with the chaperone functions of Hsp70, VER 155008 also prevented the reduction in ubiquitin-conjugated proteins by N-acetyl cysteine. These data reveal a new role for N-acetyl cysteine: this compound may reduce misfolded protein levels and ameliorate proteotoxicity through heat shock proteins. These findings broaden the potential mechanisms of action for this dietary supplement in neurodegenerative proteinopathies. PMID:24096134
Inhibition of corn oil oxidation by N-acetyl-cysteine and glutathione
Despina Papadopoulou; Ioannis G. Roussis
The ability of N-acetyl-cysteine and glutathione to inhibit the oxidation of corn oil was evaluated.The absorbances at 234nm and 270nm, and p-anisidine value were monitored during storage of corn oil at 50°C, 120°C and 180°C for up to 30 days, 12h and 180min, respectively. Both thiols exhibited inhibitory action that was dose dependent in the range 0–40mg\\/L, while N-acetyl-cysteine was
Isolation and characterization of ganglioside 9-O-acetyl-GD3 from bovine buttermilk.
Bonafede, D M; Macala, L J; Constantine-Paton, M; Yu, R K
Bovine buttermilk contains a unique ganglioside, 9-O-acetyl-GD3. In order to isolate large quantities of this ganglioside, a simplified isolation scheme which consists of several ion-exchange and silica gel column chromatographic procedures was devised. The isolated 9-O-acetyl-GD3 was characterized on the basis of its thin-layer chromatographic behavior, its immunoreactivity with a specific monoclonal antibody, JONES, and by conversion to authentic GD3 by mild base treatment. PMID:2685488
Purification and Characterization of a Bovine Acetyl Low Density Lipoprotein Receptor
Tatsuhiko Kodama; Pranhitha Reddy; Chiharu Kishimoto; Monty Krieger
The acetyl low density lipoprotein (LDL) receptor is expressed on macrophages and some endothelial cells and mediates macrophage--foam cell formation in culture. A 220-kDa acetyl LDL binding protein was partially purified from bovine liver membranes and was used to make a specific monoclonal antibody. The 220-kDa protein immunoprecipitated by this antibody retained binding activity, and the antibody was used to
Structure of a Sir2 Enzyme Bound to an Acetylated p53 Peptide
Jose L Avalos; Ivana Celic; Shabazz Muhammad; Michael S Cosgrove; Jef D Boeke; Cynthia Wolberger
Sir2 proteins are NAD+-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate ? sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a
Horseshoe Crab Acetyl Group-Recognizing Lectins Involved in Innate Immunity are Structurally Related to Fibrinogen
Soutaro Gokudan; Tatsushi Muta; Ryoko Tsuda; Kumiko Koori; Takeshi Kawahara; Noriaki Seki; Yoshimitsu Mizunoe; Sun N. Wai; Sadaaki Iwanaga; Shun-Ichiro Kawabata
We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity
Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF
The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcrip- tional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation
BET acetyl-lysine binding proteins control pathological cardiac hypertrophy.
Spiltoir, Jessica I; Stratton, Matthew S; Cavasin, Maria A; Demos-Davies, Kim; Reid, Brian G; Qi, Jun; Bradner, James E; McKinsey, Timothy A
Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure. PMID:23939492
Lysine Acetylation Controls Local Protein Conformation by Influencing Proline Isomerization
Howe, Françoise S.; Boubriak, Ivan; Sale, Matthew J.; Nair, Anitha; Clynes, David; Grijzenhout, Anne; Murray, Struan C.; Woloszczuk, Ronja; Mellor, Jane
Summary Gene transcription responds to stress and metabolic signals to optimize growth and survival. Histone H3 (H3) lysine 4 trimethylation (K4me3) facilitates state changes, but how levels are coordinated with the environment is unclear. Here, we show that isomerization of H3 at the alanine 15-proline 16 (A15-P16) peptide bond is influenced by lysine 14 (K14) and controls gene-specific K4me3 by balancing the actions of Jhd2, the K4me3 demethylase, and Spp1, a subunit of the Set1 K4 methyltransferase complex. Acetylation at K14 favors the A15-P16trans conformation and reduces K4me3. Environmental stress-induced genes are most sensitive to the changes at K14 influencing H3 tail conformation and K4me3. By contrast, ribosomal protein genes maintain K4me3, required for their repression during stress, independently of Spp1, K14, and P16. Thus, the plasticity in control of K4me3, via signaling to K14 and isomerization at P16, informs distinct gene regulatory mechanisms and processes involving K4me3. PMID:25127513
Resolving Acetylated and Phosphorylated Proteins by Neutral Urea Triton-Polyacrylamide Gel Electrophoresis, NUT-PAGE
Buehl, Christopher J.; Deng, Xiexiong; Liu, Mengyu; Hovde, Stacy; Xu, Xinjing; Kuo, Min-Hao
Protein acetylation and phosphorylation can be key modifications that regulate both normal and pathological protein functions. Current gel systems used to analyze modified proteins require either expensive reagents or time–consuming second dimension electrophoresis. In this manuscript, we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. This neutral pH urea Triton-polyacrylamide gel electrophoresis system, or NUT-PAGE, separates proteins based on their charge at pH 7 and generates discrete bands from each acetylated and phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents, and requires only a single dimension of electrophoresis. We are able to demonstrate the effectiveness of this system by analyzing phosphorylated species of an acidic protein, ?-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative to resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge. Method Summary Here we present a single-dimension neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system affording high-resolution separation of acetylated and phosphorylated proteins. PMID:25109292
Histone acetylation inhibitors promote axon growth in adult dorsal root ganglia neurons.
Lin, Shen; Nazif, Kutaiba; Smith, Alexander; Baas, Peter W; Smith, George M
Intrinsic mechanisms that guide damaged axons to regenerate following spinal cord injury remain poorly understood. Manipulation of posttranslational modifications of key proteins in mature neurons could reinvigorate growth machinery after injury. One such modification is acetylation, a reversible process controlled by two enzyme families, the histone deacetylases (HDACs) and the histone acetyl transferases (HATs), acting in opposition. Whereas acetylated histones in the nucleus are associated with upregulation of growth-promoting genes, deacetylated tubulin in the axoplasm is associated with more labile microtubules, conducive to axon growth. This study investigates the effects of HAT and HDAC inhibitors on cultured adult dorsal root ganglia (DRG) neurons and shows that inhibition of HATs by anacardic acid or CPTH2 improves axon outgrowth, whereas inhibition of HDACs by TSA or tubacin inhibits axon growth. Anacardic acid increased the number of axons able to cross an inhibitory chondroitin sulfate proteoglycan border. Histone acetylation but not tubulin acetylation level was affected by HAT inhibitors, whereas tubulin acetylation levels were increased in the presence of the HDAC inhibitor tubacin. Although the microtubule-stabilizing drug taxol did not have an effect on the lengths of DRG axons, nocodazole decreased axon lengths. Determining the mechanistic basis will require future studies, but this study shows that inhibitors of HAT can augment axon growth in adult DRG neurons, with the potential of aiding axon growth over inhibitory substrates produced by the glial scar. © 2015 Wiley Periodicals, Inc. PMID:25702820
Determination of DNA damage in experimental liver intoxication and role of N-acetyl cysteine.
Aksit, Hasan; Bildik, Aysegül
The present study aimed at detecting DNA damage and fragmentation as well as histone acetylation depending on oxidative stress caused by CCl4 intoxication. Also, the protective role of N-acetyl cysteine, a precursor for GSH, in DNA damage is investigated. Sixty rats were used in this study. In order to induce liver toxicity, CCl4 in was dissolved in olive oil (1/1) and injected intraperitoneally as a single dose (2 ml/kg). N-acetyl cysteine application (intraperitoneal, 50 mg/kg/day) was started 3 days prior to CCl4 injection and continued during the experimental period. Control groups were given olive oil and N-acetyl cysteine. After 6 and 72 h of CCl4 injection, blood and liver tissue were taken under ether anesthesia. Nuclear extracts were prepared from liver. Changes in serum AST and ALT activities as well as MDA, TAS, and TOS levels showed that CCl4 caused lipid peroxidation and liver damage. However, lipid peroxidation and liver damage were reduced in the N-acetyl cysteine group. Increased levels in 8-hydroxy-2-deoxy guanosine and histone acetyltransferase activities, decreased histone deacetylase activities, and DNA breakage detected in nuclear extracts showed that CCl4 intoxication induces oxidative stress and apoptosis in rat liver. The results of the present study indicate that N-acetyl cysteine has a protective effect on CCl4-induced DNA damage. PMID:24819310
Combinatorial regulation of a signal-dependent activator by phosphorylation and acetylation
Paz, Jose C.; Park, Sangho; Phillips, Naomi; Matsumura, Shigenobu; Tsai, Wen-Wei; Kasper, Lawryn; Brindle, Paul K.; Zhang, Guangtao; Zhou, Ming-Ming; Wright, Peter E.; Montminy, Marc
In the fasted state, increases in catecholamine signaling promote adipocyte function via the protein kinase A-mediated phosphorylation of cyclic AMP response element binding protein (CREB). CREB activity is further up-regulated in obesity, despite reductions in catecholamine signaling, where it contributes to the development of insulin resistance. Here we show that obesity promotes the CREB binding protein (CBP)-mediated acetylation of CREB at Lys136 in adipose. Under lean conditions, CREB acetylation was low due to an association with the energy-sensing NAD+-dependent deacetylase SirT1; amounts of acetylated CREB were increased in obesity, when SirT1 undergoes proteolytic degradation. Whereas CREB phosphorylation stimulated an association with the KIX domain of CBP, Lys136 acetylation triggered an interaction with the CBP bromodomain (BRD) that augmented recruitment of this coactivator to the promoter. Indeed, coincident Ser133 phosphorylation and Lys136 acetylation of CREB stimulated the formation of a ternary complex with the KIX and BRD domains of CBP by NMR analysis. As disruption of the CREB:BRD complex with a CBP-specific BRD inhibitor blocked effects of CREB acetylation on target gene expression, our results demonstrate how changes in nutrient status modulate cellular gene expression in response to hormonal signals. PMID:25404345
Combinatorial regulation of a signal-dependent activator by phosphorylation and acetylation.
Paz, Jose C; Park, Sangho; Phillips, Naomi; Matsumura, Shigenobu; Tsai, Wen-Wei; Kasper, Lawryn; Brindle, Paul K; Zhang, Guangtao; Zhou, Ming-Ming; Wright, Peter E; Montminy, Marc
In the fasted state, increases in catecholamine signaling promote adipocyte function via the protein kinase A-mediated phosphorylation of cyclic AMP response element binding protein (CREB). CREB activity is further up-regulated in obesity, despite reductions in catecholamine signaling, where it contributes to the development of insulin resistance. Here we show that obesity promotes the CREB binding protein (CBP)-mediated acetylation of CREB at Lys136 in adipose. Under lean conditions, CREB acetylation was low due to an association with the energy-sensing NAD(+)-dependent deacetylase SirT1; amounts of acetylated CREB were increased in obesity, when SirT1 undergoes proteolytic degradation. Whereas CREB phosphorylation stimulated an association with the KIX domain of CBP, Lys136 acetylation triggered an interaction with the CBP bromodomain (BRD) that augmented recruitment of this coactivator to the promoter. Indeed, coincident Ser133 phosphorylation and Lys136 acetylation of CREB stimulated the formation of a ternary complex with the KIX and BRD domains of CBP by NMR analysis. As disruption of the CREB:BRD complex with a CBP-specific BRD inhibitor blocked effects of CREB acetylation on target gene expression, our results demonstrate how changes in nutrient status modulate cellular gene expression in response to hormonal signals. PMID:25404345
The oncoprotein HBXIP promotes migration of breast cancer cells via GCN5-mediated microtubule acetylation.
Li, Leilei; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong
We have documented that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. A subset of acetylated microtubules that accumulates in the cell leading edge is necessary for cell polarization and directional migration. In this study, we explored the hypothesis that HBXIP contributes to migration of breast cancer cells by supporting microtubule acetylation in breast cancer cells. We found that HBXIP could induce acetylated microtubules accumulating into the leading protrusion in wound-induced directional migration in breast cancer cells by immunofluorescence staining analysis. Interestingly, HBXIP was able to increase the acetylation of ?-tubulin in the cells by immunofluorescence staining and Western blot analysis. Furthermore, we observed that acetyltransferase GCN5 was involved in the event that HBXIP induced increase of acetylated microtubules and their expansion in protrusions in breast cancer cells by Western blot analysis and immunofluorescence staining. Moreover, GCN5 was required for the HBXIP-enhanced migration of breast cancer cells by wound healing assay. Thus, we conclude that HBXIP promotes the migration of breast cancer cells through modulating microtubule acetylation mediated by GCN5. Therapeutically, HBXIP may serve as a novel target in breast cancer. PMID:25686500
Physico-chemical properties of acetylated starches from Indian black gram (Phaseolus mungo L.) cultivars.
Wani, Idrees Ahmed; Sogi, Dalbir Singh; Gill, Balmeet Singh
Starches separated from three black gram cultivars were modified by acetylation and compared to their native starches. Acetylation was carried out by treating starches with 0.04 and 0.08 g of acetic anhydride/g of starch dry weight basis (db) at 25 °C. The extent of acetylation increased proportionally with the concentration of acetic anhydride used. Retrogradation of acetylated starch pastes decreased significantly (p???0.05) as revealed by significant decrease in syneresis, increased freeze thaw stability and increased light transmittance. The pasting curves of 10.7 % starch slurries showed that acetylation decreased the setback viscosity values by 51.2-82.8 % and pasting temperature by 3.1-5.6 °C than respective native starches. Differential scanning calorimetry observations also revealed significant decrease in gelatinisation temperature of acetylated starches than native starches. Hardness and adhesiveness of starch gels varied between 10.3 and 32.6 g and 4.6-82.3gs, respectively which were significantly lower than corresponding native starch gels. PMID:26139873
Significant enhancement of hPrx1 chaperone activity through lysine acetylation.
Pan, Yanchao; Jin, Jing-Hua; Yu, Yang; Wang, Jiangyun
The reversible acetylation of proteins plays a key role in regulating biological processes, including chromatin remodeling, progression of the cell cycle, and actin nucleation. Human peroxiredoxin 1(hPrx1), one of the most abundant proteins in the cytoplasm, has been shown to be acetylated in human liver-carcinoma tissues. However, little is known about what function(s) the acetylation serves for hPrx1. Herein, using the method of genetic code expansion, we incorporated N(?)-acetyllysine (AcK) site-specifically into hPrx1. Our data showed that acetylation the K(27) residue promotes oligomerization of hPrx1 at low concentrations. In addition, K(27)-acetylated hPrx1(hPrx1-AcK27) exhibited greatly enhanced chaperone activity (e.g. protecting the protein malate dehydrogenase (MDH) from thermally induced aggregation and assisting the refolding of denatured citrate synthase (CS)). These findings suggest that the site-specific acetylation of hPrx1 may change its biological role in response to environmental changes. PMID:25082442
PPAR? activation induces N(?)-Lys-acetylation of rat liver peroxisomal multifunctional enzyme type 1.
Contreras, Miguel A; Alzate, Oscar; Singh, Avtar K; Singh, Inderjit
Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPAR?-agonist treated rat liver screened with an anti-N(?)-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (Ką??, Ką?ł, Ką??, and K??ł). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPAR?-dependent proliferation of peroxisomes in rat liver. PMID:24092543
Lack of association between slow acetylator status and spontaneous lupus erythematosus.
Kumana, C R; Chan, M M; Wong, K L; Wong, R W; Kou, M; Lauder, I J
The Chinese in Southeast Asia are recognized as a population group that has a relatively high prevalence of rapid "acetylators" and a relatively high incidence of systemic lupus erythematosus. This study was designed to evaluate the possibility that there were environmental lupus erythematosus provocative substances eliminated by acetylation that resulted in a preponderance of slow acetylators among patients with the disease. We compared acetylator status in 36 Chinese women with mild, stable, and confirmed lupus erythematosus and 36 healthy control subjects matched for age, sex, and ethnic origin. Acetylator status was determined by use of HPLC to assay 5-acetylamino-6-formylamino-3-methyluracil/methylxanthine (AFMU/MX) and AFMU/(AFMU + MX) ratios in urine 1 to 4 hours after drinking a strong cup of coffee (caffeine). By use of parametric and nonparametric methods of analysis, the frequency distribution of AFMU/MX and AFMU/(AFMU + MX) ratios in both the patients and control subjects were determined to be very similar. Thus there was no association between slow acetylator status and lupus erythematosus in the study subjects. PMID:2379389
Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis
Zhao, Hua [Savannah State University; Jones, Cecil L [Savannah State University; Baker, Gary A [ORNL; Xia, Shuqian [Tianjin University, Tianjin, China; Olubajo, Olarongbe [Savannah State University; Person, Vernecia [Savannah State University
The efficient conversion of lignocellulosic materials into fuel ethanol has become a research priority in producing affordable and renewable energy. The pretreatment of lignocelluloses is known to be key to the fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs)were found capable of dissolving more than 10 wt% cellulose. Preliminary investigations [Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904 910; Liu, L., Chen, H., 2006. Enzymatic hydrolysis of cellulose materials treated with ionic liquid [BMIM]Cl. Chin. Sci. Bull. 51, 2432 2436; Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Appl. Biochem. Biotechnol. 137 140, 407 421] suggest that celluloses regenerated from IL solutions are subject to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and nonvolatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is a corrosive, toxic and extremely hygroscopic solid (m.p. 70 C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several ILs newly developed in our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirm that all regenerated celluloses are less crystalline (58 75% lower) and more accessible to cellulase (>2 times) than untreated substrates. As a result, regenerated Avicel cellulose, filter paper and cottonwere hydrolyzed 2 10 times faster than the respective untreated celluloses. A complete hydrolysis of Avicel cellulose could be achieved in 6 h given the Trichoderma reesei cellulase/substrate ratio (w/w) of 3:20 at 50 C. In addition,we observed that cellulase is more thermally stable (up to 60 C) in the presence of regenerated cellulose. Furthermore, our systematic studies suggest that the presence of various ILs during the hydrolysis induced different degrees of cellulase inactivation. Therefore, a thorough removal of IL residues after cellulose regeneration is highly recommended, and a systematic investigation on this subject is much needed.
Lysine Acetylation Is Widespread on Proteins of Diverse Function and Localization in the Protozoan Parasite Toxoplasma gondii
Jeffers, Victoria
While histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes, Arabidopsis thaliana, Drosophila melanogaster, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite Toxoplasma gondii. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on Toxoplasma histones and ?-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to Apicomplexa, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites. PMID:22544907
Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis
Technology Transfer Automated Retrieval System (TEKTRAN)
Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced soli...
FTIR analysis of the hydrolysis rate in the sol–gel formation of gadolinia-doped ceria with acetylacetonate precursors
Chunhui Shen; Leon L. Shaw
This study reveals that Fourier transform infrared is an effective analytical tool in probing the extent of hydrolysis of\\u000a cerium and gadolinium acetylacetonates dissolved in methanol. It is found that these acetylacetonates have relatively fast\\u000a hydrolysis rates at the early stage of hydrolysis (<6 h). However, their hydrolysis rates become very slow beyond 6-h of hydrolysis\\u000a and decrease to near zero
Origin of acetyl strophanthidin-induced ventricular arrhythmias.
Ettinger, P O; Calabro, J; Regan, T J; Oldewurtel, H A
To examine the origin of digitalis-induced ventricular tachycardia (VT), acetyl strophanthidin (AS) (25 mug/min) was perfused into a limited zone of myocardium in intact anesthetized dogs through a catheter placed fluoroscopically in the left anterior descending artery without ischemia. A second catheter in the great cardiac vein sampled venous effluent from this region. His and left bundle branch depolarizations were recorded and bipolar intramural electrograms from endocardial and epicardial sites within the anterior descending region were obtained. No conduction alterations preceded arrhythmia. Cardiac venous K+ rose from 3.3 +/- to 4.4 +/- 0.2 meq/liter (P less than 0.001), indicating egress from the perfused zone. 10 animals (Group 1) were sacrificed 2 min after onset of VT while 11 (Group 2) continued until fibrillation (4-14 min). All showed normal (endocardial leads to epicardial) transmural depolarization during sinus rhythm, but 10/21 demonstrated reversal, usually late during VT, including 8/11 in Group 2. Epicardial activation preceded fascicular activation and QRS. Recordings from the border and circumflex regions in 10 additional dogs (Group 3) demonstrated activation reversal only in the border zone. Myocardial K+ was reduced (mean 63 +/- 1 mueq/g) and Na+ increased (mean 41 +/- 2 mueq/g) in the perfused zone (nonperfused circumflex area K+ 72 +/- 1, Na+ 33 +/- 1 mueq/g, P less than 0.001 for both); changes were similar in inner and outer ventricular wall. In related experiments, subepicardial injections of AS induced activation reversal within the immediate area, similar to recordings during coronary infusion. Reversed transmural activation with early epicardial depolarization suggest VT arises within myocardium; electrolyte gradients between adjacent regions may be causative. PMID:833270
Acetylated Tau Neuropathology in Sporadic and Hereditary Tauopathies
Irwin, David J.; Cohen, Todd J.; Grossman, Murray; Arnold, Steven E.; McCarty-Wood, Elisabeth; Van Deerlin, Vivianna M.; Lee, Virginia M.-Y.; Trojanowski, John Q.
We have recently shown acetylation of tau at lysine residue 280 (AC-K280) to be a disease-specific modification in Alzheimer disease (AD), corticobasal degeneration, and progressive supranuclear palsy, likely representing a major regulatory tau modification. Herein, we extend our observations using IHC with a polyclonal antibody specific for AC-K280. Thirty brain regions were examined in argyrophilic grain disease (AGD; n = 5), tangle-predominant senile dementia (TPSD; n = 5), Pick disease (n = 4), familial AD (FAD; n = 2; PSEN1 p.G206A and p.S170P), and frontotemporal dementia with parkinsonism linked to chromosome-17 (FTDP-17; n = 2; MAPT p.P301L and IVS10 + 16). All AGD, TPSD, FAD, and FTDP-17 cases had significant AC-K280 reactivity that was similar in severity and distribution to phosphorylated tau. AC-K280 robustly labeled grain pathological characteristics in AGD and was predominantly associated with thioflavin-S–positive neurofibrillary tangles and less reactive in neuropil threads and extracellular tangles in TPSD and FAD. Thioflavin-S–negative neuronal and glial inclusions of patients with FTDP-17 were robustly AC-K280 reactive. A low degree of AC-K280 was found in a subset of 4-repeat tau-containing lesions in Pick disease. AC-K280 is a prominent feature of both neuronal and glial tau aggregations in tauopathies of various etiologies. The close association of AC-K280 with amyloid and pre-amyloid conformations of tau suggests a potential role in tangle maturation and, thus, could serve as a useful biomarker or therapeutic target in a variety of tauopathies. PMID:23885714
Expression of ?-methylacyl-coa racemase (p504s) in various malignant neoplasms and normal tissues: a study of 761 cases
Zhong Jiang; Gary R Fanger; Bruce A Woda; Barbara F Banner; Paul Algate; Karen Dresser; Jiangchun Xu; Peiguo G Chu
?-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In
The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM1
Lihong Zhou; Trevor S. Marks; Roy P. C. Poh; Rodney J. Smith; Babur Z. Chowdhry; Anthony R. W. Smith
4-Chlorobenzoate:CoA ligase, the first enzyme in the pathwayfor 4-chlorobenzoate dissimilation, has been partially purifiedfrom Arthrobacter sp. strain TM-1, by sequential ammoniumsulphate precipitation and chromatography on DEAE-Sepharoseand Sephacryl S-200. The enzyme, a homodimer of subunitmolecular mass approximately 56 kD, is dependent onMg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory,
Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal ?-Oxidation of Unsaturated Fatty Acids
Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie (Nankai) [Nankai; (Chinese Aca. Sci.)
Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via ?-oxidation, differences exist between the peroxisomal and mitochondrial ?-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 Ĺ 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 C? 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.
Änderungen von Enzymaktivitäten während des Wachstums von Zellsuspensionskulturen von Glycine max : Phenylalanin Ammonium-Lyase und p-Cumarat: CoA Ligase
K. Hahlbrock; E. Kuhlen; T. Lindl
1.A typical growth curve including a lag phase, phases of cell division and cell expansion and a stationary phase could be demonstrated for batch propagated cells ofGlycine max. in synthetic liquid medium.2.Shortly before the stationary phase was reached, dramatic changes in the activities of two enzymes of "secondary plant metabolism", phenylalanine ammonia-lyase and p-coumarate: CoA ligase, were observed. In contrast,
Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase is regulated by acetylation.
Ventura, Mireia; Mateo, Francesca; Serratosa, Joan; Salaet, Ignasi; Carujo, Sonia; Bachs, Oriol; Pujol, María Jesús
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping glycolitic enzyme that recently has been implicated in cell signaling. Under apoptotic stresses, cells activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The GAPDH-Siah interaction depends on the integrity of lysine 227 in human GAPDH, being the mutant K227A unable to associate with Siah. As lysine residues are susceptible to be modified by acetylation, we aimed to analyze whether acetylation could mediate transport of GAPDH from cytoplasm to the nucleus. We observed that the acetyltransferase P300/CBP-associated factor (PCAF) interacts with and acetylates GAPDH. We also found that over-expression of PCAF induces the nuclear translocation of GAPDH and that for this translocation its intact acetylase activity is needed. Finally, the knocking down of PCAF reduces nuclear translocation of GAPDH induced by apoptotic stimuli. By spot mapping analysis we first identified Lys 117 and 251 as the putative GAPDH residues that could be acetylated by PCAF. We further demonstrated that both Lys were necessary but not sufficient for nuclear translocation of GAPDH after apoptotic stimulation. Finally, we identified Lys 227 as a third GAPDH residue whose acetylation is needed for its transport from cytoplasm to the nucleus. Thus, results reported here indicate that nuclear translocation of GAPDH is mediated by acetylation of three specific Lys residues (117, 227 and 251 in human cells). Our results also revealed that PCAF participates in the GAPDH acetylation that leads to its translocation to the nucleus. PMID:20601085
Study of enzymatic hydrolysis of fructans from Agave salmiana characterization and kinetic assessment.
Michel-Cuello, Christian; Ortiz-Cerda, Imelda; Moreno-Vilet, Lorena; Grajales-Lagunes, Alicia; Moscosa-Santillán, Mario; Bonnin, Johanne; González-Chávez, Marco Martín; Ruiz-Cabrera, Miguel
Fructans were extracted from Agave salmiana juice, characterized and subjected to hydrolysis process using a commercial inulinase preparation acting freely. To compare the performance of the enzymatic preparation, a batch of experiments were also conducted with chicory inulin (reference). Hydrolysis was performed for 6 h at two temperatures (50, 60 °C) and two substrate concentrations (40, 60 mg/ml). Hydrolysis process was monitored by measuring the sugars released and residual substrate by HPLC. A mathematical model which describes the kinetics of substrate degradation as well as fructose production was proposed to analyze the hydrolysis assessment. It was found that kinetics were significantly influenced by temperature, substrate concentration, and type of substrate (P < 0.01). The extent of substrate hydrolysis varied from 82 to 99%. Hydrolysis product was mainly constituted of fructose, obtaining from 77 to 96.4% of total reducing sugars. PMID:22629216
Study of Enzymatic Hydrolysis of Fructans from Agave salmiana Characterization and Kinetic Assessment
Michel-Cuello, Christian; Ortiz-Cerda, Imelda; Moreno-Vilet, Lorena; Grajales-Lagunes, Alicia; Moscosa-Santillán, Mario; Bonnin, Johanne; González-Chávez, Marco Martín; Ruiz-Cabrera, Miguel
Fructans were extracted from Agave salmiana juice, characterized and subjected to hydrolysis process using a commercial inulinase preparation acting freely. To compare the performance of the enzymatic preparation, a batch of experiments were also conducted with chicory inulin (reference). Hydrolysis was performed for 6?h at two temperatures (50, 60°C) and two substrate concentrations (40, 60?mg/ml). Hydrolysis process was monitored by measuring the sugars released and residual substrate by HPLC. A mathematical model which describes the kinetics of substrate degradation as well as fructose production was proposed to analyze the hydrolysis assessment. It was found that kinetics were significantly influenced by temperature, substrate concentration, and type of substrate (P < 0.01). The extent of substrate hydrolysis varied from 82 to 99%. Hydrolysis product was mainly constituted of fructose, obtaining from 77 to 96.4% of total reducing sugars. PMID:22629216
Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase.
Aubert, Sarah D; Li, Yingchun; Raushel, Frank M
Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed. PMID:15134445
Hydroxypropylation of cellulose as a pretreatment for enzymatic hydrolysis
Brix, Scott Tyson
hours using Trichoderma cellulase (Allen, 1983). Optimal hydrolysis conditions for Trirhoderme cellulase are at a pH of 4. 8 and a temperature of 50'C. It is also reported that approximately 14mg of enzyme is needed per gram of cellulosic substrate... oxide (Fig. 3). This step was done to achieve a consistent amount of swelling of the pulp by the alkali. Reaction times reported in Table 1 are based on the amount of time the reactor was held at the indicated temperature. The heating time between...
Nanocrystalline hydroxyapatite ceramics prepared by hydrolysis in polyol medium
NASA Astrophysics Data System (ADS)
Mechay, Abderrahmen; Feki, Hafed E. L.; Schoenstein, Fréderic; Jouini, Noureddine
This Letter describes a new approach for the synthesis of hydroxyapatite nanoparticles, which involves precipitation and hydrolysis reactions conducted in polyol medium. In fact, ammonium-hydrogen phosphate and calcium nitrate were dissolved in polyol, and then heated at the boiling point of the polyol (ethane1, 2diol or propane1, 2diol). Besides, the phase and composition of the polycrystalline were studied by TGA/DTA, FT-IR, TEM and XRD techniques. The nanoparticles thus obtained present interesting morphological characters varying from needle to very thin platelet. Moreover, the hydroxyapatite prepared in ployol shows higher cristallinity in comparison with that obtained by other 'chimie douce' methods.
Factors affecting hydrolysis of condensed phosphates in soils
Stewart, William M.
in two soils (pH 8. 40 and 7. 56) 24 to 96 hours after application of 2 x 10 4 M sodium PP. This increase in OP concentration was attributed to hydrolysis of the sodium PP. As previously mentioned, the half-life of TPP in soils has been found to range..., found PP to be less effective than OP on an acid soil (pH 4. 0), while there were only very slight differences i. n the effectiveness of the two sources in a soil with a pH value of 7. 2. Reported results have shown that increasing the pH of acid...
Enhanced attrition bioreactor for enzyme hydrolysis or cellulosic materials
Scott, T.C.; Scott, C.D.; Faison, B.D.; Davison, B.H.; Woodward, J.
A process is described for converting cellulosic materials, such as waste paper, into fuels and chemicals, such as sugars and ethanol, utilizing enzymatic hydrolysis of the major carbohydrate of paper: cellulose. A waste paper slurry is contacted by cellulase in an agitated hydrolyzer. An attritor and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. Additionally, microfiltration, ultrafiltration and reverse osmosis steps are included to further increase reaction efficiency. The resulting sugars are converted to a dilute product in a fluidized-bed bioreactor utilizing a biocatalyst, such as microorganisms. The dilute product is then concentrated and purified. 1 fig.
Enhanced attrition bioreactor for enzyme hydrolysis of cellulosic materials
Scott, T.C.; Scott, C.D.; Faison, B.D.; Davison, B.H.; Woodward, J.
A process is described for converting cellulosic materials, such as waste paper, into fuels and chemicals, such as sugars and ethanol, utilizing enzymatic hydrolysis of the major carbohydrate of paper: cellulose. A waste paper slurry is contacted by cellulase in an agitated hydrolyzer. An attritor and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. Additionally, microfiltration, ultrafiltration and reverse osmosis steps are included to further increase reaction efficiency. The resulting sugars are converted to a dilute product in a fluidized-bed bioreactor utilizing a biocatalyst, such as microorganisms. The dilute product is then concentrated and purified. 1 fig.
Enhanced attrition bioreactor for enzyme hydrolysis or cellulosic materials
Scott, Timothy C. (Knoxville, TN); Scott, Charles D. (Oak Ridge, TN); Faison, Brendlyn D. (Knoxville, TN); Davison, Brian H. (Knoxville, TN); Woodward, Jonathan (Oak Ridge, TN)
A process for converting cellulosic materials, such as waste paper, into fuels and chemicals, such as sugars and ethanol, utilizing enzymatic hydrolysis of the major carbohydrate of paper: cellulose. A waste paper slurry is contacted by cellulase in an agitated hydrolyzer. An attritor and a cellobiase reactor are coupled to the agitated hydrolyzer to improve reaction efficiency. Additionally, microfiltration, ultrafiltration and reverse osmosis steps are included to further increase reaction efficiency. The resulting sugars are converted to a dilute product in a fluidized-bed bioreactor utilizing a biocatalyst, such as microorganisms. The dilute product is then concentrated and purified.
Hydrolysis of synthetic triacylglycerols by pancreatic and lipoprotein lipase
N. H. Morley; A. Kuksis; D. Buchnea
The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate\\u000a and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies\\u000a of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1?1 molar ratio\\u000a to the substrate as emulsifiers and were limited to short time intervals which
The Hydrolysis of Di-Isopropyl Methylphosphonate in Ground Water
Sega, G.A., Tomkins, B.A., Griest, W.H., Bayne, C.K.
Di-isopropyl methylphosphonate (DIMP) is a byproduct from the manufacture of the nerve agent Sarin. The persistence of DIMP in the ground water is an important question in evaluating the potential environmental impacts of DIMP contamination. The half-life of DIMP in ground water at 10 deg C was estimated to be 500 years with a 95% confidence interval of 447 to 559 years from measurements of the hydrolysis rates at temperatures between 70 to 98 deg C.Extrapolation of the kinetics to 10 deg C used the Arrhenius equation, and calculation of the half-life assumed first-order kinetics. Inorganic phosphate was not detected.
Flow injection spectrophotometric determination of methamidophos using online hydrolysis.
Jan, Mohammad Rasul; Shah, Jasmin; Bashir, Nadia; Salman, M
A new protocol for the online spectrophotometric determination of methamidophos has been developed. The method is based on online hydrolysis of methamidophos, and the resulting hydrolyzed product is reacted with sodium nitroproside to form a colored complex. The calibration curve is linear over the concentration range of 0.5-20 microgml(-1), with a molar absorptivity of 2.5x10(4) L mol(-1) cm(-1). The method is fast and reproducible with a sample throughput of 90 samples/h. The method is successfully applied to formulations and real samples. PMID:19609695
Towards zero discharge of chromium-containing leather waste through improved alkali hydrolysis
Changdao Mu; Wei Lin; Mingrang Zhang; Qingshi Zhu
The treatment of chromium-containing leather waste (CCLW), the major solid waste generated at the post-tanning operations of leather processing, has the potential to generate value-added leather chemicals. Various alkali and enzymatic hydrolysis were compared, and calcium oxide was found to be important for effective (but still incomplete) hydrolysis. Three possible reasons are given for the incomplete hydrolysis under alkaline conditions.
Xylose Monomer and Oligomer Yields for Uncatalyzed Hydrolysis of Sugarcane Bagasse Hemicellulose at Varying Solids Concentration
California at Riverside, University of
Xylose Monomer and Oligomer Yields for Uncatalyzed Hydrolysis of Sugarcane Bagasse Hemicellulose of varying sugarcane bagasse concentrations on xylose monomer and oligomer yields was experimentally measured
Acetylation curtails nucleosome binding, not stable nucleosome remodeling, by FoxO1
Hatta, M.; Liu, F. [Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (United States); Cirillo, L.A. [Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (United States)], E-mail: email@example.com
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.
The acetylation of tau inhibits its function and promotes pathological tau aggregation
Cohen, Todd J.; Guo, Jing L.; Hurtado, David E.; Kwong, Linda K.; Mills, Ian P.; Trojanowski, John Q.; Lee, Virginia M. Y.
The microtubule associated protein tau promotes neuronal survival through binding and stabilization of MTs. Phosphorylation regulates tau–microtubule interactions and hyperphosphorylation contributes to the aberrant formation of insoluble tau aggregates in Alzheimer's disease (AD) and related tauopathies1. However, other pathogenic post-translational tau modifications have not been well characterized. Here we demonstrate that tau acetylation inhibits tau function via impaired tau–microtubule interactions and promotes pathological tau aggregation. Mass spectrometry analysis identified specific lysine residues, including lysine 280 (K280) within the microtubule-binding motif as the major sites of tau acetylation. Immunohistochemical and biochemical studies of brains from tau transgenic mice and patients with AD and related tauopathies showed that acetylated tau pathology is specifically associated with insoluble, Thioflavin-positive tau aggregates. Thus, tau K280 acetylation in our studies was only detected in diseased tissue, suggesting it may have a role in pathological tau transformation. This study suggests that tau K280 acetylation is a potential target for drug discovery and biomarker development for AD and related tauopathies. PMID:21427723
Asymmetric distribution of glucose and indole-3-acetyl-myo-inositol in geostimulated Zea mays seedlings
NASA Technical Reports Server (NTRS)
Momonoki, Y. S.; Bandurski, R. S. (Principal Investigator)
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.
Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus
Chuang, Peter Y.; Dai, Yan; Liu, Ruijie; He, Helen; Kretzler, Matthias; Jim, Belinda; Cohen, Clemens D.; He, John C.
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. PMID:21858169
Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects
Myklebust, Line M.; Van Damme, Petra; Střve, Svein I.; Dörfel, Max J.; Abboud, Angčle; Kalvik, Thomas V.; Grauffel, Cedric; Jonckheere, Veronique; Wu, Yiyang; Swensen, Jeffrey; Kaasa, Hanna; Liszczak, Glen; Marmorstein, Ronen; Reuter, Nathalie; Lyon, Gholson J.; Gevaert, Kris; Arnesen, Thomas
The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease. PMID:25489052
Purification and characterization of acetyl-CoA carboxylase from the Diatom Cyclotella cryptica
Roessler, P.G. (Solar Energy Research Institute, Golden, CO (USA))
Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a molecular weight of approximately 740 kilodaltons and appears to be composed of four identical biotin-containing subunits. The enzyme has maximal activity at pH 8.2, but enzyme stability is greater at pH 6.5 K{sub m} values for MgATP, acetyl-CoA, and HCO{sub 3} were determined to be 65, 233, and 750 micromolar, respectively. The purified enzyme is strongly inhibited by palmitoyl-CoA, and is inhibited to a lesser extent by malonyl-CoA, ADP, and phosphate. Pyruvate stimulates enzymatic activity to a slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar clethodim and 100 micromolar haloxyfop, respectively.
The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation
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)] [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: email@example.com [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)
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.
Physicochemical and drug release characteristics of acetylated starches of five Lagenaria siceraria cultivars.
Kulkarni, Sameer D; Sinha, Barij N; Kumar, K Jayaram
Modified starches play a crucial role in the pharmaceutical industries in controlling the drug release at a pre-determined rate. The effect of acetylation on the physicochemical and drug release characteristics of the starches from five different Indian L. siceraria cultivars was investigated. Starches isolated from the seeds of L. siceraria were subjected to varying degrees of acetylation. Using a range of characterization methods including amylose content, elemental analysis, light transmittance, swelling power, scanning electron microscopy, FT-IR and X-ray diffraction, the effect of acetylation was determined. The swelling power of starch acetates improved significantly (P < 0.05) with the increase in degree of substitution. The increase in swelling shows that acetylation improved the accessibility of an amorphous area to the water. The formation of V-type of complex crystalline structures confirmed the acetylation of L. siceraria starch. Modification in the crystalline structure of starch acetate retarded the drug release, which is controlled by water uptake. The starch acetates from all the cultivars showed better sustained release properties with the increase in degree of substitution. Drug release through the swellable matrix was found to be controlled by fickian diffusion from the gel layer as indicated by Korsmeyer-Peppas models (R(2)) 0.9885-0.9984. PMID:25453280
Proteomic Analysis Reveals Differentially Regulated Protein Acetylation in Human Amyotrophic Lateral Sclerosis Spinal Cord
Azadzoi, Kazem; Yang, Yun; Fei, Zhou; Dou, Kefeng; Kowall, Neil W.; Choi, Han-Pil; Vieira, Fernando; Yang, Jing-Hua
Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC) inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE) analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identifies glial fibrillary acidic protein (GFAP) as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS. PMID:24312501
[The role of histone acetylation in the basolateral amygdala in morphine-associated memory in rats].
Xiaomeng, Qiao; Fangyuan, Yin; Yunxiao, Li; Shuguang, Wei; Jianghua, Lai
To examine the regulatory effect of histone acetylation on memory related molecules, 34 healthy male SD rats were randomly divided into control and basolateral amygdala (BLA) intracranial positioning operation groups. In the process of conditioned place preference (CPP) training, Trichostafin A (TSA) was administrated by the route of BLA and morphine was injected into enterocoelia with dimethyl sulfoxide or saline as control. Expression levels of H3K14 acetylation and brain-derived neurotrophic factor (BDNF) in BLA were evaluated by Western blotting.The results showed that CPP could be established by intraperitoneal injection of morphine. Compared with control groups, a stronger place preference was established and expression of H3K14 acetylation and BDNF was significantly increased in the group treated with TSA and morphine. In addition, there was a synergistic effect between morphine and TSA. Our results suggested that the level of histone acetylation in BLA is associated with the formation of morphine memory in rats. Inhibition of the activity of histone deacetylases in BLA can promote the formation of cue-associated memory induced by morphine and the involvement of BDNF in BLA maybe was regulated by histone acetylation. PMID:25881704
A sensitive protein-based sensor for quantifying histone acetylation levels.
Sanchez, Oscar F; Williamson, Drew; Cai, Lutong; Yuan, Chongli
H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigenetic modifications in cells. Aberrant changes in H3K14ac are commonly found in various types of cancers and neurological disorders. Current detection approaches for histone modifications, however, require either tedious sample pre-treatments or lack the quantitative accuracy required for biochemical and biomedical applications. In this study, we engineered a protein sensor using the amino acid sequences derived from the bromodomain of human polybromo-1 (PB1). The protein sensor was conjugated to a fluorescent dye for sensitive detection of H3K14ac. Different detection conditions, such as additive concentrations and probe concentrations, were optimally selected by balancing signal strength (IRel) and signal-to-noise ratio (SNR). The protein sensor was verified using histone H3 peptides containing different H3K14 acetylation levels. The detection signal was found to be linearly dependent on acetylation levels of H3K14 ranging from 5% to 100%. The designed platform can be used for screening epigienetic drugs regulating H3K14 acetylation levels as well as monitoring H3K14 acetylation level of circulating nucleosomes for disease progression. PMID:26048844
Parameter and Process Significance in Mechanistic Modeling of Cellulose Hydrolysis
NASA Astrophysics Data System (ADS)
Rotter, B.; Barry, A.; Gerhard, J.; Small, J.; Tahar, B.
The rate of cellulose hydrolysis, and of associated microbial processes, is important in determining the stability of landfills and their potential impact on the environment, as well as associated time scales. To permit further exploration in this field, a process-based model of cellulose hydrolysis was developed. The model, which is relevant to both landfill and anaerobic digesters, includes a novel approach to biomass transfer between a cellulose-bound biofilm and biomass in the surrounding liquid. Model results highlight the significance of the bacterial colonization of cellulose particles by attachment through contact in solution. Simulations revealed that enhanced colonization, and therefore cellulose degradation, was associated with reduced cellulose particle size, higher biomass populations in solution, and increased cellulose-binding ability of the biomass. A sensitivity analysis of the system parameters revealed different sensitivities to model parameters for a typical landfill scenario versus that for an anaerobic digester. The results indicate that relative surface area of cellulose and proximity of hydrolyzing bacteria are key factors determining the cellulose degradation rate.
Spectroscopy techniques for analyzing the hydrolysis of PLGA and PLLA.
Tan, Hwee Yun; Widjaja, Effendi; Boey, Freddy; Loo, Say Chye Joachim
The in vitro hydrolytic degradation of irradiated biodegradable polymers was studied. Poly(L-lactide), poly(lactide-co-glycolic acid) (PLGA)(80:20), and PLGA(50:50) polymers were first electron beam irradiated at 5 Mrad before hydrolytic degradation. Hydrolysis of these films was characterized through their physical properties--mass loss, average molecular weight, and thermal properties. Changes to the chemical structures of these polyesters were also analyzed using Fourier-transformed infrared (FTIR) and Raman spectroscopy, and the spectra results were correlated to their physical properties. The results showed that an increase in hydroxyl (OH) group, observed from the FTIR spectroscopy, indicates that the polymer is degrading through hydrolysis--first-stage degradation. Subsequently, a decrease in C==O group, observed from Raman spectroscopy, indicates that the polymer is experiencing mass loss--second-stage degradation. Therefore, a good correlation exists in determining the extent of polymer degradation through the use of FTIR and Raman spectroscopy by observing changes to the OH and C==O groups from the spectra of these nondestructive techniques. PMID:19489010
[Municipal biowaste thermal-hydrolysis and ASBR anaerobic digestion].
Hou, Hua-hua; Wang, Wei; Hu, Song; Xu, Yi-xian
Thermal-hydrolysis can remarkably improve the solid organics dissolving efficiency of urban biomass waste, and anaerobic sequencing batch reactor (ASBR) was used to improve the efficiency of urban biomass waste anaerobic digestion. The optimum thermal-hydrolysis temperature and holding time was 175 degrees C and 60 min, the volatile suspended solid (VSS) dissolving ratio of kitchen waste, fruit-and-vegetable waste and sludge were 31.3%, 31.9% and 49.7%, respectively. Two ASBR and one continuous-flow stirred tank reactor (CSTR) were started at hydraulic retention time (HRT) = 20 d, COD organic loading rate (OLR) = 3.2-3.6 kg/(m3 x d). The biogas production volumes were 5656 mL/d(A1), 6335 mL/d(A2) and 3 103 mL/d(CSTR), respectively; VSS degradation ratios were 45.3% (A1), 50.87% (A2), 20.81% (CSTR), and the total COD (TCOD) removal rates were 88.1% (A1), 90% (A2), 72.6% (CSTR). In ASBR, organic solid and anaerobic microorganism were remained in the reactor during settling period. When HRT was 20 d, the solid retention time (SRT) was over 130 d, which made ASBR higher efficiency than CSTR. PMID:20391728
Aqueous fractionation of biomass based on novel carbohydrate hydrolysis kinetics
Torget, Robert W. (Littleton, CO)
A multi-function process for hydrolysis and fractionation of lignocellulosic biomass to separate hemicellulosic sugars from other biomass components comprising extractives and proteins; a portion of a solubilized lignin; cellulose; glucose derived from cellulose; and insoluble lignin from said biomass comprising: a) introducing either solid fresh biomass or partially fractioned lignocellulosic biomass material with entrained acid or water into a reactor and heating to a temperature of up to about 185.degree. C.-205.degree. C. b) allowing the reaction to proceed to a point where about 60% of the hemicellulose has been hydrolyzed in the case of water or complete dissolution in case of acid; c) adding a dilute acid liquid at a pH below about 5 at a temperature of up to about 205.degree. C. for a period ranging from about 5 to about 10 minutes; to hydrolyze the remaining 40% of hemicellulose if water is used. d) quenching the reaction at a temperature of up to about 140.degree. C. to quench all degradation and hydrolysis reactions; and e) introducing into said reaction chamber and simultaneously removing from said reaction chamber, a volumetric flow rate of dilute acid at a temperature of up to about 140.degree. C. to wash out the majority of the solubilized biomass components, to obtain improved hemicellosic sugar yields.
Catalytic hydrolysis of a phosphate triester by tetracoordinated zinc complexes
Gellman, S.H.; Petter, R.; Breslow, R.
A zinc complex of a tetraaza macrocycle catalyzes the hydrolysis of diphenyl p-nitrophenyl phosphate is aqueous acetonitrile. The principal products derive from loss of p-nitrophenol, but some alternative hydrolysis with loss of phenol is also observed. Kinetic studies show that the catalytic zinc species is a zinc hydroxide complex with a pK/sub a/ of 8.7 (or its kinetic equivalent). The greater kinetic effectiveness of this weak base than of free hydroxide ion itself, on a molar basis, indicates a bifunctional mechanism in which bound hydroxide acts as a nucleophile, while zinc acts as a electrophilic catalyst. This process is relatively strainless at phosphorus, in contrast to reactions at carbon with the same species in which the bifunctional mechanism would be strained and is not seen. A derivative of the zinc macrocycle complex carrying a long alkyl chain was examined as the catalyst for the same substrate in a Brij micelle. This lipophilic complex is even more effective, acting as a zinc hydroxide species with pK/sub a/ = 9.1, but in the micellar reaction there are contributions from kinetic terms higher than first order in the zinc complex. Thus, in this system, catalysis by aggregates is apparently also occurring.
Hydrolytic depolymerization of hydrolysis lignin: Effects of catalysts and solvents.
Mahmood, Nubla; Yuan, Zhongshun; Schmidt, John; Xu, Chunbao Charles
Hydrolytic depolymerization of hydrolysis lignin (HL) in water and water-ethanol co-solvent was investigated at 250°C for 1h with 20% (w/v) HL substrate concentration with or without catalyst (H2SO4 or NaOH). The obtained depolymerized HLs (DHLs) were characterized with GPC-UV, FTIR, GC-MS, (1)H NMR and elemental analyzer. In view of the utilization of depolymerized HL (DHL) for the preparation of rigid polyurethane foams/resins un-catalyzed depolymerization of HL employing water-ethanol mixture appeared to be a viable route with high yield of DHL ?70.5wt.% (SR yield of ?9.8wt.%) and with Mw as low as ?1000g/mole with suitable aliphatic (227.1mgKOH/g) and phenolic (215mgKOH/g) hydroxyl numbers. The overall % carbon recovery under the selected best route was ?87%. Acid catalyzed depolymerization of HL in water and water-ethanol mixture lead to slightly increased Mw. Alkaline hydrolysis helped in reducing Mw in water and opposite trend was observed in water-ethanol mixture. PMID:25936442
Epigenetic regulation of the NR4A orphan nuclear receptor NOR1 by histone acetylation.
Zhao, Yue; Nomiyama, Takashi; Findeisen, Hannes M; Qing, Hua; Aono, Jun; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis
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. PMID:25451221
Roles of dynamic and reversible histone acetylation in plant development and polyploidy
Chen, Z. Jeffrey; Tian, Lu
Transcriptional regulation in eukaryotes is not simply determined by the DNA sequence, but rather mediated through dynamic chromatin modifications and remodeling. Recent studies have shown that reversible and rapid changes in histone acetylation play an essential role in chromatin modification, induce genome-wide and specific changes in gene expression, and affect a variety of biological processes in response to internal and external signals, such as cell differentiation, growth, development, light, temperature, and abiotic and biotic stresses. Moreover, histone acetylation and deacetylation are associated with RNA interference and other chromatin modifications including DNA and histone methylation. The reversible changes in histone acetylation also contribute to cell cycle regulation and epigenetic silencing of rDNA and redundant genes in response to interspecific hybridization and polyploidy. PMID:17556080
Effects of acute doxorubicin treatment on hepatic proteome lysine acetylation status and the apoptotic environment
Dirks-Naylor, Amie J; Kouzi, Samir A; Bero, Joseph D; Tran, Ngan TK; Yang, Sendra; Mabolo, Raean
AIM: To determine if doxorubicin (Dox) alters hepatic proteome acetylation status and if acetylation status was associated with an apoptotic environment. METHODS: Doxorubicin (20 mg/kg; Sigma, Saint Louis, MO; n = 8) or NaCl (0.9%; n = 7) was administered as an intraperitoneal injection to male F344 rats, 6-wk of age. Once animals were treated with Dox or saline, all animals were fasted until sacrifice 24 h later. RESULTS: Dox treatment decreased proteome lysine acetylation likely due to a decrease in histone acetyltransferase activity. Proteome deacetylation may likely not be associated with a proapoptotic environment. Dox did not increase caspase-9, -8, or -3 activation nor poly (adenosine diphosphate-ribose) polymerase-1 cleavage. Dox did stimulate caspase-12 activation, however, it likely did not play a role in apoptosis induction. CONCLUSION: Early effects of Dox involve hepatic proteome lysine deacetylation and caspase-12 activation under these experimental conditions. PMID:25225604
MicroRNA-29a promotion of nephrin acetylation ameliorates hyperglycemia-induced podocyte dysfunction.
Lin, Chun-Liang; Lee, Pei-Hsien; Hsu, Yung-Chien; Lei, Chen-Chou; Ko, Jih-Yang; Chuang, Pei-Chin; Huang, Yu-Ting; Wang, Shao-Yu; Wu, Shin-Long; Chen, Yu-Shan; Chiang, Wen-Chih; Reiser, Jochen; Wang, Feng-Sheng
Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose-induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose-stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may protect against diabetic podocytopathy. PMID:24578127
N-acetyl cysteine prevents synergistic, severe toxicity from two hits of oxidative stress.
Unnithan, Ajay S; Jiang, Yiran; Rumble, Jennifer L; Pulugulla, Sree H; Posimo, Jessica M; Gleixner, Amanda M; Leak, Rehana K
The two hit hypothesis of neurodegeneration states that cells that have been severely stressed once are more vulnerable to the negative impact of a second hit. In other words, the toxicity of two hits of severe stress may be synergistic in neurons. We previously developed a two hit model of proteotoxic neurodegeneration using the proteasome inhibitor MG132. In that study, we found that the potent antioxidant N-acetyl cysteine was able to protect against the toxicity associated with dual MG132 hits. N-acetyl cysteine has been shown to ameliorate cognitive deficits in Alzheimer's patients and to reduce the symptoms of blast injury in soldiers. These studies and many others in experimental models of neurodegeneration suggest that N-acetyl cysteine can protect neurons even when they are severely injured. In the present study, we tested the hypotheses that dual hits of hydrogen peroxide and paraquat would elicit synergistic neurodegeneration and that this extreme toxicity would be prevented by N-acetyl cysteine. The findings reveal for the first time that neuronal N2a cells are much more sensitive to oxidative stress from hydrogen peroxide treatment when they have been exposed previously to the same toxin. Two hits of hydrogen peroxide also caused severe loss of glutathione. N-acetyl cysteine attenuated the loss of glutathione and reduced the near-complete loss of cells after exposure to dual hydrogen peroxide hits. The present study supports the notion that N-acetyl cysteine can robustly protect against severe, unremitting oxidative stress in a glutathione-dependent manner. PMID:24361774
Id4 dependent acetylation restores mutant-p53 transcriptional activity
Background The mechanisms that can restore biological activity of mutant p53 are an area of high interest given that mutant p53 expression is observed in one third of prostate cancer. Here we demonstrate that Id4, an HLH transcriptional regulator and a tumor suppressor, can restore the mutant p53 transcriptional activity in prostate cancer cells. Methods Id4 was over-expressed in prostate cancer cell line DU145 harboring mutant p53 (P223L and V274F) and silenced in LNCaP cells with wild type p53. The cells were used to quantitate apoptosis, p53 localization, p53 DNA binding and transcriptional activity. Immuno-precipitation/-blot studies were performed to demonstrate interactions between Id4, p53 and CBP/p300 and acetylation of specific lysine residues within p53. Results Ectopic expression of Id4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, the transcriptional targets of p53. Mutant p53 gained DNA binding and transcriptional activity in the presence of Id4 in DU145 cells. Conversely, loss of Id4 in LNCaP cells abrogated wild type p53 DNA binding and transactivation potential. Gain of Id4 resulted in increased acetylation of mutant p53 whereas loss of Id4 lead to decreased acetylation in DU145 and LNCaP cells respectively. Id4 dependent acetylation of p53 was in part due to a physical interaction between Id4, p53 and acetyl-transferase CBP/p300. Conclusions Taken together, our results suggest that Id4 regulates the activity of wild type and mutant p53. Id4 promoted the assembly of a macromolecular complex involving CBP/P300 that resulted in acetylation of p53 at K373, a critical post-translational modification required for its biological activity. PMID:24330748
Functional characterization of two new members of the caffeoyl CoA O -methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O -methyltransferases
Thomas Widiez; Thomas G. Hartman; Nativ Dudai; Qing Yan; Michael Lawton; Daphna Havkin-Frenkel; Faith C. Belanger
Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in\\u000a lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well\\u000a characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells\\u000a of pods of
Conformational studies of bacterial peptidoglycan: structure and stereochemistry of N-acetyl-?- D-glucosamine and N-acetyl-?- D-muramic acid
NASA Astrophysics Data System (ADS)
Yadav, P. N. S.; Rai, D. K.; Yadav, J. S.
The energies of various conformations of N-acetyl-?- D-glucosamine (NAG) and its 3-O- D-lactic acid derivative N-acetyl-?- D-muramic acid (NAM) have been calculated by geometry optimization using the molecular mechanics program MM2. The geometries of these systems have been analyzed in the light of ring torsion, bond lengths, bond angles and conformational states of side groups of the pyranosyl ring and compared with available experimental data of similar pyranose derivatives. The present study indicates the presence of hydrogen bonds to stabilize the side group conformations. Discrepancies with experimental data that are seen in a few cases are ascribed to the nature of the side groups and their geometry.
[Canavan disease or N-acetyl aspartic aciduria: a case report].
Boughamoura, L; Chaabane, F; Tilouche, S; Chabchoub, I; Kabachi, N; Tlili, K; Yacoub, M; Essoussi, A-S
Canavan disease or N-acetyl aspartic aciduria, is an autosomal recessive leukodystrophy characterized by spongy degeneration of brain. The disease is an inborn error of metabolism caused by aspartoacylase deficiency resulting from accumulation of N-acetyl aspartic acid in the brain. The authors report a case in a 10-month-old boy who presented with developmental delay and megalencephaly noticeable after 4 months of age. Magnetic resonance imaging of the brain showed diffuse white matter degeneration. The diagnosis of Canavan disease was confirmed by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. PMID:17196380
Electrophoretic and physical properties of N-acetyl-†-D hexosaminidase in normal swine tissues
Barton, Joan Elizabeth
ELECTROPHORETIC AND PHYSICAL PROPERTIES OF N-ACETYL-B-D HEXOSAMINIDASE IN NORMAL SWINE TISSUES A Thesis by JOAN ELIZABETH BARTON Subm1tted to the Graduate College of Texas A 5 M University 1n Partial fulfillment of the requirement... for the degree of MASTER OF SCIENCE Oecember 1979 Major Subject: Biochem1stry ELECTROPHORETIC AND PHYSICAL PROPERTIES OF N-ACETYL-8-D HEXOSAMINIDASE IN NORMAL SWINE TISSUES A Thesis by JOAN ELIZABETH BARTON Approved as to style and content by: 1 WSI...
Electrophoretic and physical properties of N-acetyl-†-D hexosaminidase in normal swine tissues
Barton, Joan Elizabeth
ELECTROPHORETIC AND PHYSICAL PROPERTIES OF N-ACETYL-B-D HEXOSAMINIDASE IN NORMAL SWINE TISSUES A Thesis by JOAN ELIZABETH BARTON Subm1tted to the Graduate College of Texas A 5 M University 1n Partial fulfillment of the requirement... for the degree of MASTER OF SCIENCE Oecember 1979 Major Subject: Biochem1stry ELECTROPHORETIC AND PHYSICAL PROPERTIES OF N-ACETYL-8-D HEXOSAMINIDASE IN NORMAL SWINE TISSUES A Thesis by JOAN ELIZABETH BARTON Approved as to style and content by: 1 WSI...
Structural characterization of the acetylated heteroxylan from the natural hybrid Paulownia elongata\\/ Paulownia fortunei
Virgínia M. F. Gonçalves; Dmitry V. Evtuguin; M. Rosário M. Domingues
The heteroxylan from the hybrid Paulownia elongata\\/Paulownia fortunei is an O-acetyl-(4-O-methylglucurono)xylan with an acetylation degree (DS) of 0.59 and a molecular weight (Mw) of 29kDa. The heteroxylan backbone is composed by (1?4)-linked ?-d-xylopyranosyl units (Xylp) partially ramified with terminal (1?2)-linked 4-O-methyl-?-d-glucuronosyl (MeGlcpA) and a small proportion of ?-d-glucuronosyl (GlcpA) residues in a molar ratio of Xylp:(MeGlcpA+GlcpA) of 20:1. Roughly half
Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase.
Watkins, L M; Mahoney, H J; McCulloch, J K; Raushel, F M
The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon. PMID:9325279