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Sample records for quantify oligopeptide acetylation

  1. Oligopeptide-heavy metal interaction monitoring by hybrid gold nanoparticle based assay.

    PubMed

    Politi, Jane; Spadavecchia, Jolanda; Iodice, Mario; de Stefano, Luca

    2015-01-01

    Phytochelatins are small peptides that can be found in several organisms, which use these oligopeptides to handle heavy metal elements. Here, we report a method for monitoring interactions between lead(ii) ions in aqueous solutions and phytochelatin 6 oligopeptide bioconjugated onto pegylated gold nanorods (PEG-AuNrs). This study is the first step towards a high sensitive label free optical biosensor to quantify heavy metal pollution in water. PMID:25360445

  2. Dynamics of an antibiotic oligopeptide

    NASA Astrophysics Data System (ADS)

    Middendorf, H. D.; Alves, N.; Zanotti, J.-M.; Gomes, P.; Bastos, M.

    2006-11-01

    Neutron time-of-flight spectra were measured for an H 2O-hydrated and a nominally dry sample of a 15-residue antibacterial oligopeptide from 99 to 271 K. Proton mobilities, quasielastic broadenings, and changes in low-frequency inelastic intensities characterise the evolution of the peptide energy landscape as a function of momentum transfer and temperature.

  3. Quantifying solubility enhancement due to particle size reduction and crystal habit modification: case study of acetyl salicylic acid.

    PubMed

    Hammond, Robert B; Pencheva, Klimentina; Roberts, Kevin J; Auffret, Tony

    2007-08-01

    The poor solubility of potential drug molecules is a significant problem in the design of pharmaceutical formulations. It is well known, however, that the solubility of crystalline materials is enhanced when the particle size is reduced to submicron levels and this factor can be expected to enhance drug product bioavailability. Direct estimation of solubility enhancement, as calculated via the Gibbs-Thompson relationship, demands reasonably accurate values for the particle/solution interfacial tension and, in particular, its anisotropy with respect to the crystal product's habit and morphology. In this article, an improved, more molecule-centered, approach is presented towards the calculation of solubility enhancement factors in which molecular modeling techniques are applied, and the effects associated with both crystal habit modification and solvent choice are examined. A case study for facetted, acetyl salicylic acid (aspirin) crystals in equilibrium with saturated aqueous ethanol solution reveals that their solubility will be enhanced in the range (7-58%) for a crystal size of 0.02 microm, with significantly higher enhancement for crystal morphologies in which the hydrophobic crystal faces are more predominant than the hydrophilic faces and for solvents in which the solubility is smaller.

  4. A toolbox of oligopeptide-modified polymers for tailored elastomers.

    PubMed

    Croisier, Emmanuel; Liang, Su; Schweizer, Thomas; Balog, Sandor; Mionić, Marijana; Snellings, Ruben; Cugnoni, Joël; Michaud, Véronique; Frauenrath, Holger

    2014-01-01

    Biomaterials are constructed from limited sets of building blocks but exhibit extraordinary and versatile properties, because hierarchical structure formation lets them employ identical supramolecular motifs for different purposes. Here we exert a similar degree of structural control in synthetic supramolecular elastomers and thus tailor them for a broad range of thermomechanical properties. We show that oligopeptide-terminated polymers selectively self-assemble into small aggregates or nanofibrils, depending on the length of the oligopeptides. This process is self-sorting if differently long oligopeptides are combined so that different nanostructures coexist in bulk mixtures. Blends of polymers with oligopeptides matching in length furnish reinforced elastomers that exhibit shear moduli one order of magnitude higher than the parent polymers. By contrast, novel interpenetrating supramolecular networks that display excellent vibration damping properties are obtained from blends comprising non-matching oligopeptides or unmodified polymers. Hence, blends of oligopeptide-modified polymers constitute a toolbox for tailored elastomers with versatile properties. PMID:25198134

  5. In Silico Approach towards Designing Virtual Oligopeptides for HRSV

    PubMed Central

    Jain, Ruchi; Piramanayagam, Shanmughavel

    2014-01-01

    HRSV (human respiratory syncytial virus) is a serious cause of lower respiratory tract illness in infants and young children. Designing inhibitors from the proteins involved in virus replication and infection process provides target for new therapeutic treatments. In the present study, in silico docking was performed using motavizumab as a template to design motavizumab derived oligopeptides for developing novel anti-HRSV agents. Additional simulations were conducted to study the conformational propensities of the oligopeptides and confirmed the hypothesis that the designed oligopeptide is highly flexible and capable of assuming stable confirmation. Our study demonstrated the best specific interaction of GEKKLVEAPKS oligopeptide for glycoprotein strain A among various screened oligopeptides. Encouraged by the results, we expect that the proposed scheme will provide rational choices for antibody reengineering which is useful for systematically identifying the possible ways to improve efficacy of existing antibody drugs. PMID:25525622

  6. In silico approach towards designing virtual oligopeptides for HRSV.

    PubMed

    Jain, Ruchi; Piramanayagam, Shanmughavel

    2014-01-01

    HRSV (human respiratory syncytial virus) is a serious cause of lower respiratory tract illness in infants and young children. Designing inhibitors from the proteins involved in virus replication and infection process provides target for new therapeutic treatments. In the present study, in silico docking was performed using motavizumab as a template to design motavizumab derived oligopeptides for developing novel anti-HRSV agents. Additional simulations were conducted to study the conformational propensities of the oligopeptides and confirmed the hypothesis that the designed oligopeptide is highly flexible and capable of assuming stable confirmation. Our study demonstrated the best specific interaction of GEKKLVEAPKS oligopeptide for glycoprotein strain A among various screened oligopeptides. Encouraged by the results, we expect that the proposed scheme will provide rational choices for antibody reengineering which is useful for systematically identifying the possible ways to improve efficacy of existing antibody drugs. PMID:25525622

  7. Effect of grafted oligopeptides on friction.

    PubMed

    Iarikov, Dmitri D; Ducker, William A

    2013-05-14

    Frictional and normal forces in aqueous solution at 25 °C were measured between a glass particle and oligopeptide films grafted from a glass plate. Homopeptide molecules consisting of 11 monomers of either glutamine, leucine, glutamic acid, lysine, or phenylalanine and one heteropolymer were each "grafted from" an oxidized silicon wafer using microwave-assisted solid-phase peptide synthesis. The peptide films were characterized using X-ray photoelectron spectroscopy and secondary ion mass spectrometry. Frictional force measurements showed that the oligopeptides increased the magnitude of friction compared to that on a bare hydrophilic silicon wafer but that the friction was a strong function of the nature of the monomer unit. Overall we find that the friction is lower for more hydrophilic films. For example, the most hydrophobic monomer, leucine, exhibited the highest friction whereas the hydrophilic monomer, polyglutamic acid, exhibited the lowest friction at zero load. When the two surfaces had opposite charges, there was a strong attraction, adhesion, and high friction between the surfaces. Friction for all polymers was lower in phosphate-buffered saline than in pure water, which was attributed to lubrication via hydrated salt ions.

  8. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  9. Formation of oligopeptides in high yield under simple programmable conditions

    PubMed Central

    Rodriguez-Garcia, Marc; Surman, Andrew J.; Cooper, Geoffrey J.T.; Suárez-Marina, Irene; Hosni, Zied; Lee, Michael P.; Cronin, Leroy

    2015-01-01

    Many high-yielding reactions for forming peptide bonds have been developed but these are complex, requiring activated amino-acid precursors and heterogeneous supports. Herein we demonstrate the programmable one-pot dehydration–hydration condensation of amino acids forming oligopeptide chains in around 50% yield. A digital recursive reactor system was developed to investigate this process, performing these reactions with control over parameters such as temperature, number of cycles, cycle duration, initial monomer concentration and initial pH. Glycine oligopeptides up to 20 amino acids long were formed with very high monomer-to-oligomer conversion, and the majority of these products comprised three amino acid residues or more. Having established the formation of glycine homo-oligopeptides, we then demonstrated the co-condensation of glycine with eight other amino acids (Ala, Asp, Glu, His, Lys, Pro, Thr and Val), incorporating a range of side-chain functionality. PMID:26442968

  10. Substrates of the human oligopeptide transporter hPEPT2.

    PubMed

    Zhao, Dongxin; Lu, Kui

    2015-08-01

    Oligopeptide transporters serve important functions in nutrition and pharmacology. In particular, these transporters help maintain the homeostasis of peptides. The peptide-transporter PEPT2 is a high-affinity and low-capacity type oligopeptide transporter from the proton-coupled oligopeptide transporter family. PEPT2 has recently received attention because of its potential application in targeted drug delivery. PEPT2 is widely distributed in kidney, central nervous system, and lung of organisms. In general, all dipeptides, tripeptides, and peptide-like drugs such as β-lactam antibiotics and angiotensin-converting enzyme inhibitors could be mediated and transported as a substrate of PEPT2. The design of many extant drugs and prodrugs is based on the substrate structure of PEPT2 to accelerate absorption via peptide transporters. Thus, this paper summarizes the substrate features of PEPT2 to promote the rational design of drugs and prodrugs that target peptide transporters. PMID:26355221

  11. [Theory study on glycine linear oligopeptide vibrational spectrum frequency shift].

    PubMed

    Ye, Zhi-Peng; Li, Xin; Yang, Meng-Shi; Chen, Liang; Xu, Can; Chu, Xiu-Xiang

    2014-04-01

    By using the density functional theory, glycine linear oligopeptide of different lengths was geometrically optimized on the 6-31G (d) basis set level, their growth processes were simulated, and the average binding energy and vibration frequency were calculated with geometry. The results showed that the average binding energies tend to change in a regular pattern and stabilize with the number of residues increasing; With the oligopeptide chain bond length analysis it was found that the chain to the radial direction there is a opposite trend for chain and radial direction, which is anisotropic. It was found by the IR spectrum analysis that red shifts and blue shifts occur respectively when the same group of peptide bond vibrate, which is anisotropic; These phenomena originate from that quasi one-dimensional nanostructures lead to the anisotropy of the bond length; the induced effects, coupling effects and hydrogen bonding etc. between the same groups lead to the vibration frequency red shifts and blue shifts. The authors conclude that the growth of glycine linear oligopeptide is conducive to stability of the structure, and the authors infer that the oligopeptide has the tendency of self-assembled growth; Through the conformation and spectrum, the authors infer that there is a size effect in physical and chemical properties. The physical and chemical properties of peptide chain end group are extremely stable and unaffected by the impact of the oligopeptide chain length The results are significant to measuring the length and the number of residue of peptide, and to manufacturing the special features oligopeptide chain.

  12. Sample pretreatment techniques for oligopeptide analysis from natural sources.

    PubMed

    Poliwoda, Anna; Wieczorek, Piotr P

    2009-02-01

    The analysis of oligopeptides in samples of food, tissues, and body fluids attracts considerable attention. The complexity of such samples requires efficient sample preparation (i.e., concentration and cleanup) procedures to remove interfering endogenous compounds and inorganic or organic salts. The methods of sample pretreatment that enable effective and selective isolation and/or preconcentration of oligopeptides from complex sample matrices have been reviewed. In each case, examples of application were presented and discussed, taking into account selectivity, enrichment, method automation, cleanup, and environmental aspects of the developed methods.

  13. Oligopeptides as biomarkers of cyanobacterial subpopulations. Toward an understanding of their biological role.

    PubMed

    Agha, Ramsy; Quesada, Antonio

    2014-06-23

    Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria.

  14. Oligopeptides as Biomarkers of Cyanobacterial Subpopulations. Toward an Understanding of Their Biological Role

    PubMed Central

    Agha, Ramsy; Quesada, Antonio

    2014-01-01

    Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria. PMID:24960202

  15. Gating Topology of the Proton-Coupled Oligopeptide Symporters

    PubMed Central

    Fowler, Philip W.; Orwick-Rydmark, Marcella; Radestock, Sebastian; Solcan, Nicolae; Dijkman, Patricia M.; Lyons, Joseph A.; Kwok, Jane; Caffrey, Martin; Watts, Anthony; Forrest, Lucy R.; Newstead, Simon

    2015-01-01

    Summary Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport. PMID:25651061

  16. Redox activity and multiple copper(I) coordination of 2His-2Cys oligopeptide.

    PubMed

    Choi, DongWon; Alshahrani, Aisha A; Vytla, Yashodharani; Deeconda, Manogna; Serna, Victor J; Saenz, Robert F; Angel, Laurence A

    2015-02-01

    Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides.

  17. Mechanistic study for immobilization of cysteine-labeled oligopeptides on UV-activated surfaces.

    PubMed

    Ong, Lian Hao; Ding, Xiaokang; Yang, Kun-Lin

    2014-10-01

    In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules.

  18. A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica.

    PubMed

    Kretschmer, R R; Rico, G; Giménez, J A

    2001-02-01

    The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [(35)S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

  19. Mining gut microbiome oligopeptides by functional metaproteome display

    PubMed Central

    Zantow, Jonas; Just, Sarah; Lagkouvardos, Ilias; Kisling, Sigrid; Dübel, Stefan; Lepage, Patricia; Clavel, Thomas; Hust, Michael

    2016-01-01

    Pathogen infections, autoimmune diseases, and chronic inflammatory disorders are associated with systemic antibody responses from the host immune system. Disease-specific antibodies can be important serum biomarkers, but the identification of antigens associated with specific immune reactions is challenging, in particular if complex communities of microorganisms are involved in the disease progression. Despite promising new diagnostic opportunities, the discovery of these serological markers becomes more difficult with increasing complexity of microbial communities. In the present work, we used a metagenomic M13 phage display approach to select immunogenic oligopeptides from the gut microbiome of transgenic mice suffering from chronic ileitis. We constructed three individual metaproteome phage display libraries with a library size of approximately 107 clones each. Using serum antibodies, we selected and validated three oligopeptides that induced specific antibody responses in the mouse model. This proof-of-concept study provides the first successful application of functional metaproteome display for the study of protein-protein interactions and the discovery of potential disease biomarkers. PMID:27703179

  20. Modulation of Biointeractions by Electrically Switchable Oligopeptide Surfaces: Structural Requirements and Mechanism

    PubMed Central

    Yeung, Chun L; Wang, Xingyong; Lashkor, Minhaj; Cantini, Eleonora; Rawson, Frankie J; Iqbal, Parvez; Preece, Jon A; Ma, Jing; Mendes, Paula M

    2014-01-01

    Understanding the dynamic behavior of switchable surfaces is of paramount importance for the development of controllable and tailor-made surface materials. Herein, electrically switchable mixed self-assembled monolayers based on oligopeptides have been investigated in order to elucidate their conformational mechanism and structural requirements for the regulation of biomolecular interactions between proteins and ligands appended to the end of surface tethered oligopeptides. The interaction of the neutravidin protein to a surface appended biotin ligand was chosen as a model system. All the considerable experimental data, taken together with detailed computational work, support a switching mechanism in which biomolecular interactions are controlled by conformational changes between fully extended (“ON” state) and collapsed (“OFF” state) oligopeptide conformer structures. In the fully extended conformation, the biotin appended to the oligopeptide is largely free from steric factors allowing it to efficiently bind to the neutravidin from solution. While under a collapsed conformation, the ligand presented at the surface is partially embedded in the second component of the mixed SAM, and thus sterically shielded and inaccessible for neutravidin binding. Steric hindrances aroused from the neighboring surface-confined oligopeptide chains exert a great influence over the conformational behaviour of the oligopeptides, and as a consequence, over the switching efficiency. Our results also highlight the role of oligopeptide length in controlling binding switching efficiency. This study lays the foundation for designing and constructing dynamic surface materials with novel biological functions and capabilities, enabling their utilization in a wide variety of biological and medical applications. PMID:25793154

  1. Redox activity and multiple copper(I) coordination of 2His-2Cys oligopeptide.

    PubMed

    Choi, DongWon; Alshahrani, Aisha A; Vytla, Yashodharani; Deeconda, Manogna; Serna, Victor J; Saenz, Robert F; Angel, Laurence A

    2015-02-01

    Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides. PMID:25800013

  2. Diversity and evolution of oligopeptide permease systems in staphylococcal species.

    PubMed

    Yu, Dongliang; Pi, Borui; Yu, Meihong; Wang, Yanfei; Ruan, Zhi; Feng, Ye; Yu, Yunsong

    2014-07-01

    Several oligopeptide permease (Opp) systems have been found in staphylococcal species, including Opp1-4, Opp3' and the arginine catabolic mobile element (ACME)-encoded Opp system (ACME-Opp). They confer upon bacteria the increasing fitness, but their evolutionary histories remain unclear. In this work, we performed a genome-wide identification of Opp systems in staphylococcal species. Novel Opp systems were identified, including the duplicate of Opp4 in Staphylococcus pseudintermedius and the ACME-Opp-like systems in coagulase-negative staphylococci (CoNS). Phylogenetic analysis revealed that all of the identified Opp systems were derived from Opp3 system by operon duplication during species divergence, while lateral gene transfer might also confer to the dissemination of Opp in staphylococci. In addition, we proposed an improved theory on evolution of ACME: the Opp and arginine-deiminase systems were firstly transferred from Staphylococcus haemolyticus to Staphylococcus epidermidis independently; in S. epidermidis they were assembled together and then transferred to Staphylococcus aureus. PMID:24793159

  3. Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water.

    PubMed

    Yin, Jie; Rastogi, Sanjay; Terry, Ann E; Popescu, Crisan

    2007-03-01

    Keratins are self-organized proteins that are abundantly available in wool, feather, human hair, etc., making them a potential cheap feedstock for the modification of amino acids. This paper explores the hydrolysis of keratin in water under specific pressure-temperature conditions where the hydrolysis through scission of the protein chain yields oligopeptides. Here we report for the first time that, under appropriate conditions, these oligopeptides self-assemble into a hierarchical architecture, the process being followed in time by optical microscopy. Birefringent needle-like crystals are observed which tend to nucleate heterogeneously. When given sufficient time, these needles become tens of microns in length and act as further nuclei, developing a highly repetitive structure of several hundreds of microns in size. Micro-focus X-ray diffraction studies supported by in situ microscopy reveal that these needles have a crystal structure similar to that of the native protein, although better organized along the ab-plane. Spectroscopic studies on these structures show crystalline bands that disappear above 150 degrees C, coinciding with an endothermic peak in DSC. Amino acid analysis shows that the self-assembled birefringent entities are indeed oligopeptides, consisting of sequences of approximately 40 amino acids. The proposed ecofriendly route provides an effective route for obtaining oligopeptides that can be used as important building blocks for the synthesis of a range of novel polymers. The oligopeptides obtained from the sustainable source can be used as important building blocks for the synthesis of a range of novel polymers.

  4. Stoichiometry of site-specific lysine acetylation in an entire proteome.

    PubMed

    Baeza, Josue; Dowell, James A; Smallegan, Michael J; Fan, Jing; Amador-Noguez, Daniel; Khan, Zia; Denu, John M

    2014-08-01

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

  5. Counting basic sites in oligopeptides via gas-phase ion chemistry

    SciTech Connect

    Stephenson, J.L. Jr.; McLuckey, S.A.

    1997-02-01

    Cations derived from oligopeptides ranging from laminin fragment (5 residues) to {beta}-lactoglobulin (162 residues) have been subjected to gas-phase ion/molecule reactions with hydroiodic acid. The sum of the ion charge state and the maximum number of molecules of hydroiodic acid that attach to the ion is equal to the total number of lysines, arginines, histidines, and N-termini consisting of a primary amine for ions derived from all 21 oligopeptides studied. These results suggest that ion/molecule reactions can provide useful information regarding oligopeptide basic site number, which might be used as a criterion for searching protein data bases instead of, or in conjunction with, use of proteolytic digestion or gas-phase ion dissociation procedures. 31 refs., 3 figs., 1 tab.

  6. Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species.

    PubMed

    Oshiro, Elisa E; Tavares, Milene B; Suzuki, Celso F; Pimenta, Daniel C; Angeli, Claudia B; de Oliveira, Julio C F; Ferro, Maria I T; Ferreira, Luis C S; Ferreira, Rita C C

    2010-04-01

    In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis. PMID:21637492

  7. Synthetic human chorionic gonadotropin-related oligopeptides impair early innate immune responses to Listeria monocytogenes in Mice.

    PubMed

    van der Zee, Marten; Dik, Willem A; Kap, Yolanda S; Dillon, Marilyn J; Benner, Robbert; Leenen, Pieter J M; Khan, Nisar A; Drevets, Douglas A

    2010-04-01

    Background. Synthetic human chorionic gonadotropin (hCG)-related oligopeptides are potent inhibitors of pathogenic inflammatory responses induced by in vivo lipopolysaccharide exposure or hemorrhagic shock-induced injury. In this study, we tested whether hCG-related oligopeptide treatment similarly altered inflammatory responses and innate host defenses in mice during experimental Listeria monocytogenes infection. Methods. Mice were infected with L. monocytogenes and treated with hCG-related oligopeptides (LQGV, VLPALP, or AQGV) or phosphate-buffered saline. Subsequently, mice were analyzed for bacterial loads, cytokine and chemokine responses, and inflammatory cell infiltrates in target organs. Results. Oligopeptide administration increased bacterial numbers in the spleen and liver at 6 h after infection. Simultaneously, CXCL1/KC and CCL2/MCP-1 plasma levels as well as neutrophil numbers in the spleen, blood, and peritoneal cavity decreased. In contrast, at 18 h after infection, systemic tumor necrosis factor alpha, interleukin 12 p70, interleukin 6, and interferon gamma levels increased statistically significantly in oligopeptide-treated mice compared with controls, which correlated with increased bacterial numbers. Conclusion. These data show that treatment with hCG-related oligopeptides (LQGV, VLPALP, and AQGV) inhibits early innate immune activation by reducing initial chemokine secretion following infection. This leads to bacterial overgrowth with subsequent enhanced systemic inflammation. Our data underscore the importance of early innate immune activation and suggest a role for hCG-derived oligopeptides at the placenta that increases the risk of L. monocytogenes infections.

  8. One-step formation of oligopeptide-like molecules from Glu and Asp in hydrothermal environments.

    PubMed

    Kawamura, Kunio; Shimahashi, Masanori

    2008-05-01

    Biopolymer accumulation in the absence of enzymes is an essential step for the chemical evolution of primitive life-like systems, and successful simulation experiments of prebiotic biopolymer formation have suggested that oligopeptides could have formed near submarine hydrothermal vent environments on primitive earth. However, the yield and length of oligopeptides -- typically limited to 6-mers -- seems to be inadequate. One reason is the rapid formation of diketopiperazines (DKPs) from dipeptides. In this study, using a hydrothermal microflow reactor, we show that the one-step synthesis of oligopeptide-like molecules of length up to 20-mers proceeds from Glu and Asp. Yields of up to 0.17-0.57% were obtained in an acidic solution within 183 s at 250-310 degrees C, as evaluated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis and different types of high-performance liquid chromatography (HPLC) analyses. The present study indicates that Glu and Asp may have played important roles in the chemical evolution of oligopeptide-like molecules in hydrothermal vent environments on primitive earth. PMID:18253712

  9. Separation of chiral nanotubes with an opposite handedness by chiral oligopeptide adsorption: A molecular dynamics study.

    PubMed

    Raffaini, Giuseppina; Ganazzoli, Fabio

    2015-12-18

    The separation of enantiomeric chiral nanotubes that can form non-covalent complexes with an unlike stability upon adsorption of chiral molecules is a process of potential interest in different fields and applications. Using fully atomistic molecular dynamics simulations, we report in this paper a theoretical study of the adsorption and denaturation of an oligopeptide formed by 16 chiral amino acids having a helical structure in the native state on both the inner and the outer surface of the chiral (10, 20) and (20, 10) single-walled carbon nanotubes having an opposite handedness, and of the armchair (16, 16) nanotube with a similar diameter for comparison. In the final adsorbed state, the oligopeptide loses in all cases its native helical conformation, assuming elongated geometries that maximize its contact with the surface through all the 16 amino acids. We find that the complexes formed by the two chiral nanotubes and the chosen oligopeptide have a strongly unlike stability both when adsorption takes place on the outer convex surface of the nanotube, and when it occurs on the inner concave surface. Thus, our molecular simulations indicate that separation of chiral, enantiomeric carbon nanotubes for instance by chromatographic methods can indeed be carried out using oligopeptides of a sufficient length.

  10. Molecular characterization of a laminin-derived oligopeptide with implications in biomimetic applications.

    PubMed

    Makohliso, S A; Melchionna, S

    2001-02-15

    The molecular properties of the laminin-derived oligopeptide, H-CDPGYIGSR-NH2, have been investigated with the aid of a tandem computer simulation/experimental approach. The simulation studies placed a particular emphasis on studying the oligopeptide in aqueous media, as well as in a grafted or immobilized state. The simulations revealed the presence of a stable double hydrogen bond between arginine (R) and aspartic acid (D) residues. The mutation of the terminal arginine with lysine, another hydrophilic and positively charged amino acid, resulted in a drastic structural change, thus suggesting a major role of the terminal arginine residue in the overall oligopeptide's conformation and, hence, its bioactivity. In addition, the involvement of the aspartic acid residue in overall peptide structural stabilization also illustrates a previously undetermined role for this region (i.e. CDPG) of the oligopeptide. A subsequent in vitro experiment demonstrated a significant loss of bioactivity upon mutating the terminal residue from arginine to lysine, thereby corroborating the overall findings of the computational model. PMID:11254207

  11. Influence of fermentation level and geographical origin on cocoa bean oligopeptide pattern.

    PubMed

    Caligiani, Augusta; Marseglia, Angela; Prandi, Barbara; Palla, Gerardo; Sforza, Stefano

    2016-11-15

    Peptides and amino acids generated during cocoa bean fermentation are the most important precursors for the development of cocoa aroma, however cocoa oligopeptide fraction is under-investigated. In this study, we perform a deep investigation of the presence of oligopeptides in unfermented, under fermented, and well-fermented cocoa beans from all of the main producing countries, with the aim to obtain a better definition of cocoa quality and a deeper comprehension of biochemical changes occurring during fermentation. Oligopeptides were determined by UPLC/ESI-MS and 35 low-molecular weight peptides were identified and subjected to semi-quantitative analysis. Results showed that fermented cocoas can be differentiated from unfermented cocoas by their possession of a higher total amount of oligopeptides and a lower ratio of vicilin to 21kDa cocoa seed albumin peptides. A variability in the peptide pattern was observed also among well-fermented cocoa samples of different geographical origin, suggesting diversified proteolytic activities. PMID:27283652

  12. Multiple photosynthetic reaction centres using zinc porphyrinic oligopeptide-fulleropyrrolidine supramolecular complexes.

    PubMed

    Fukuzumi, Shunichi; Saito, Kenji; Ohkubo, Kei; Troiani, Vincent; Qiu, Hongjin; Gadde, Suresh; D'Souza, Francis; Solladié, Nathalie

    2011-10-14

    Multiple charge-separation sites have successfully been constructed using supramolecular complexes of multiporphyrinic oligopeptides [P(ZnP)(n), n = 2, 4, 8] with fulleropyrrolidine bearing a pyridine or imidazole coordinating ligand, which are organized by utilizing π-π interaction in addition to the coordination bond. PMID:21897983

  13. Efficacy of an amphipathic oligopeptide to shuttle and release a cis-acting DNA decoy into human cells.

    PubMed

    Citti, L; Rovero, P; Colombo, M G; Mariani, L; Poliseno, L; Rainaldi, G

    2002-01-01

    We investigated the ability of an amphipathic oligopeptide to carry a synthetic dsDNA oligonucleotide inside human cells. The oligonucleotide was designed as a decoy binding site for the transcriptional activator of the methylguanine-DNA methyltransferase (MGMT) gene. The complex oligopeptide and decoy were administered to MCF10A exponentially growing cells, and the uptake was monitored by flow cytometry. After a 1-h exposure, almost all of the MCF10A cells were fluorescent, indicating that all of the cells had been transfected. By increasing the time, the fluorescence intensity per cell rapidly increased to a plateau at the 8-h time point. RT-PCR analysis of the MGMT gene was used as the molecular readout of the intracellular activity of the DNA decoy. MCF10A cells transfected with the oligopeptide/decoy complex showed a strong reduction in MGMT mRNA. Here, we discuss the advantages of using amphipathic oligopeptides as carriers of short DNA sequences.

  14. Acetyl Radical Generation in Cigarette Smoke: Quantification and Simulations.

    PubMed

    Hu, Na; Green, Sarah A

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commerial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass filber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acealdehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke. PMID:25253993

  15. Acetyl radical generation in cigarette smoke: Quantification and simulations

    NASA Astrophysics Data System (ADS)

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  16. Assessing the Indirect Photochemical Transformation of Dissolved Combined Amino Acids through the Use of Systematically Designed Histidine-Containing Oligopeptides.

    PubMed

    Chu, Chiheng; Lundeen, Rachel A; Sander, Michael; McNeill, Kristopher

    2015-11-01

    Photooxidation is an important abiotic transformation pathway for amino acids (AAs) in sunlit waters. Although dissolved free AAs are well studied, the photooxidation of dissolved combined AAs (DCAAs) remains poorly investigated. This study is a systematic investigation of the effect of neighboring photostable AA residues (i.e., aliphatic, cationic, anionic, or aromatic residues) on the environmental indirect photochemical transformation of histidine (His) in His-containing oligopeptides. The pKa values of His residues in the studied oligopeptides were found to be between 4.3 and 8.1. Accordingly, the phototransformation rate constants of the His-containing oligopeptides were highly pH-dependent in an environmentally relevant pH range with higher reactivity for neutral His than for the protonated species. The photostable AA residues significantly modulated the photoreactivity of oligopeptides either through altering the accessibility of His to photochemically produced oxidants or through shifting the pKa values of His residues. In addition, the influence of neighboring photostable AA residues on the sorption-enhanced phototransformation of oligopeptides in solutions containing chromophoric dissolved organic matter (CDOM) was assessed. The constituent photostable AA residues promoted sorption of His-containing oligopeptides to CDOM macromolecules through electrostatic attraction, hydrophobic effects, and/or low-barrier hydrogen bonds, and subsequently increased the apparent phototransformation rate constants by up to 2 orders of magnitude.

  17. Histone acetylation: truth of consequences?

    PubMed

    Choi, Jennifer K; Howe, Leann J

    2009-02-01

    Eukaryotic DNA is packaged into a nucleoprotein structure known as chromatin, which is comprised of DNA, histones, and nonhistone proteins. Chromatin structure is highly dynamic, and can shift from a transcriptionally inactive state to an active form in response to intra- and extracellular signals. A major factor in chromatin architecture is the covalent modification of histones through the addition of chemical moieties, such as acetyl, methyl, ubiquitin, and phosphate groups. The acetylation of the amino-terminal tails of histones is a process that is highly conserved in eukaryotes, and was one of the earliest histone modifications characterized. Since its identification in 1964, a large body of evidence has accumulated demonstrating that histone acetylation plays an important role in transcription. Despite our ever-growing understanding of the nuclear processes involved in nucleosome acetylation, however, the exact biochemical mechanisms underlying the downstream effects of histone acetylation have yet to be fully elucidated. To date, histone acetylation has been proposed to function in 2 nonmutually exclusive manners: by directly altering chromatin structure, and by acting as a molecular tag for the recruitment of chromatin-modifying complexes. Here, we discuss recent research focusing on these 2 potential roles of histone acetylation and clarify what we actually know about the function of this modification.

  18. S-nitrosophytochelatins: investigation of the bioactivity of an oligopeptide nitric oxide delivery system.

    PubMed

    Heikal, Lamia; Aaronson, Philip I; Ferro, Albert; Nandi, Manasi; Martin, Gary P; Dailey, Lea Ann

    2011-06-13

    This study investigates the in vitro bioactivity of S-nitrosophytochelatins (SNOPCs), oligopeptide analogues of S-nitrosoglutathione (GSNO), and their mechanisms of nitric oxide (NO) delivery. SNOPCs were more potent than GSNO in inhibiting platelet aggregation and stimulating vasorelaxation. Their potency was related to the number of S-nitrosated moieties per mole compound. Transnitrosation reactions with cell membrane surface components were shown to be the primary mode of NO delivery to intracellular targets for SNOPCs, while delivery via γ-glutamyl transpeptidase was unique to GSNO. Due to rapid NO release, larger SNOPCs elicited a more transitory effect compared to smaller compounds. The duration of effect was influenced by compound molecular weight, NO release kinetics, ability to undergo transnitrosation, and incubation time with tissues. In summary, a new oligopeptide NO delivery system based on SNOPCs was shown to be biologically active and can be used to investigate the mechanisms of NO delivery to intracellular targets.

  19. Synthetic oligopeptide substrates: their diagnostic application in blood coagulation, fibrinolysis, and other pathologic states

    SciTech Connect

    Huseby, R.M.; Smith, R.E.

    1980-01-01

    This review article with 522 references, focuses on the use of synthetic oligopepide substrates to measure the activity of proteoytic enzymes in human physiology and pathology. A classification of proteinases based on their mechanism of action is presented. The application of these synthetic oligopeptide substrates to understand the disorders of the blood coagulation and fibrinolytic system is reviewed. Intracellular functioning proteinases were also assessed in relation to certain pathologies where their abnormal activity is recognized.

  20. Inclusion of Cu nano-cluster 1D arrays inside a C3-symmetric artificial oligopeptide via co-assembly

    NASA Astrophysics Data System (ADS)

    Gong, Ruiying; Li, Fei; Yang, Chunpeng; Wan, Xiaobo

    2015-12-01

    A peptide sequence N3-GVGV-OMe (G: glycine; V: valine) was attached to a benzene 1,3,5-tricarboxamide (BTA) derivative via ``click chemistry'' to afford a C3-symmetric artificial oligopeptide. The key feature of this oligopeptide is that the binding sites (triazole groups formed by click reaction) are located at the center, while the three oligopeptide arms with a strong tendency to assemble are located around it, which provides inner space to accommodate nanoparticles via self-assembly. The inclusion of Cu nanoclusters and the formation of one-dimensional (1D) arrays inside the nanofibers of the C3-symmetric artificial oligopeptide assembly were observed, which is quite different from the commonly observed nanoparticle growth on the surface of the pre-assembled oligopeptide nanofibers via the coordination sites located outside. Our finding provides an instructive concept for the design of other stable organic-inorganic hybrid 1D arrays with the inorganic nanoparticles inside.A peptide sequence N3-GVGV-OMe (G: glycine; V: valine) was attached to a benzene 1,3,5-tricarboxamide (BTA) derivative via ``click chemistry'' to afford a C3-symmetric artificial oligopeptide. The key feature of this oligopeptide is that the binding sites (triazole groups formed by click reaction) are located at the center, while the three oligopeptide arms with a strong tendency to assemble are located around it, which provides inner space to accommodate nanoparticles via self-assembly. The inclusion of Cu nanoclusters and the formation of one-dimensional (1D) arrays inside the nanofibers of the C3-symmetric artificial oligopeptide assembly were observed, which is quite different from the commonly observed nanoparticle growth on the surface of the pre-assembled oligopeptide nanofibers via the coordination sites located outside. Our finding provides an instructive concept for the design of other stable organic-inorganic hybrid 1D arrays with the inorganic nanoparticles inside. Electronic

  1. Mineral-enhanced hydrothermal oligopeptide formation at the second time scale.

    PubMed

    Kawamura, Kunio; Takeya, Hitoshi; Kushibe, Takao; Koizumi, Yuka

    2011-06-01

    Accumulation of biopolymers should have been an essential step for the emergence of life on primitive Earth. However, experimental simulations for submarine hydrothermal vent systems in which high-temperature water spouts through minerals within a short time scale have not been attempted. Here, we show that enhancement of hydrothermal oligopeptide elongation by naturally occurring minerals was successfully verified for the first time by using a mineral-mediated hydrothermal flow reactor system (MMHF). MMHF consists of a narrow tubular reactor packed with mineral particles, and the enhancement or inhibitory activities of 10 types of naturally occurring minerals were successfully evaluated for an elongation reaction from (Ala)(4) to (Ala)(5) and higher oligopeptides in the absence of condensation reagents. It was unexpected that calcite and dolomite facilitated the elongation from (Ala)(4) to (Ala)(5) and higher oligopeptides with 28% yield at pH 7, while tourmaline, galena, apatite, mica, sphalerite, quartz, chalcopyrite, and pyrite did not show enhancement activities. These facts suggest the importance of carbonate minerals for the accumulation of peptide in primitive Earth environments. PMID:21671764

  2. Polyethylene glycol (PEG) gel arrays for differentiating oligopeptide fragments and on-chip protease assays.

    PubMed

    Zhu, Qingdi; Yang, Kun-Lin

    2016-03-15

    Polyethylene glycol (PEG) hydrogel is permeable to biomolecules, but its permeability depends on the molecular weight of monomers and the concentration of monomer solutions. In this study, we show that PEG hydrogel made from 20% to 30% of PEG700 monomer is permeable to amino acids yet impermeable to oligopeptides. Because of its unique permeability, the gel can be used to detect protease by separating amino acids from oligopeptides when proteases cleave the oligopeptides and release amino acids. Based on this principle, an UV crosslinked gel array is fabricated on a chip for simultaneous detection of protease in up to 40 samples with only 1 µl of volume required for each sample. As a proof of concept, the on-chip protease assays are used to detect trypsin in buffer and serum. The detection limits are 1.2 nM in buffer and 17.7 nM in serum, which are comparable to conventional protease assays. Moreover, because only 1 µl of liquid is required, as little as 1.2 fmol of trypsin can be detected by using the on-chip assay. The protease assay also shows good specificity for trypsin and chymotrypsin. The gel array chip could be a useful miniaturized platform for high-throughput detection of different proteases and screening of their inhibitors. PMID:26569443

  3. Mineral-enhanced hydrothermal oligopeptide formation at the second time scale.

    PubMed

    Kawamura, Kunio; Takeya, Hitoshi; Kushibe, Takao; Koizumi, Yuka

    2011-06-01

    Accumulation of biopolymers should have been an essential step for the emergence of life on primitive Earth. However, experimental simulations for submarine hydrothermal vent systems in which high-temperature water spouts through minerals within a short time scale have not been attempted. Here, we show that enhancement of hydrothermal oligopeptide elongation by naturally occurring minerals was successfully verified for the first time by using a mineral-mediated hydrothermal flow reactor system (MMHF). MMHF consists of a narrow tubular reactor packed with mineral particles, and the enhancement or inhibitory activities of 10 types of naturally occurring minerals were successfully evaluated for an elongation reaction from (Ala)(4) to (Ala)(5) and higher oligopeptides in the absence of condensation reagents. It was unexpected that calcite and dolomite facilitated the elongation from (Ala)(4) to (Ala)(5) and higher oligopeptides with 28% yield at pH 7, while tourmaline, galena, apatite, mica, sphalerite, quartz, chalcopyrite, and pyrite did not show enhancement activities. These facts suggest the importance of carbonate minerals for the accumulation of peptide in primitive Earth environments.

  4. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    PubMed

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-01

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  5. Acetylator phenotype in diabetic neuropathy.

    PubMed

    McLaren, E H; Burden, A C; Moorhead, P J

    1977-07-30

    The proportions of slow and fast acetylators in a group of diabetics with symptomatic peripheral neuropathy were compared with those in a group of diabetics who had had the disease for at least 10 years without developing neuropathy. There was a significantly higher proportion of fast acetylators in the group of diabetics without neuropathy than in those with neuropathy or in the normal population. Hence genetic factors separate from the diabetic diathesis may determine the development of neuropathy in any particular diabetic.

  6. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    PubMed

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. PMID:27216463

  7. Fatal Intoxication with Acetyl Fentanyl.

    PubMed

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. PMID:26389815

  8. Quantifying resilience

    USGS Publications Warehouse

    Allen, Craig R.; Angeler, David G.

    2016-01-01

    Several frameworks to operationalize resilience have been proposed. A decade ago, a special feature focused on quantifying resilience was published in the journal Ecosystems (Carpenter, Westley & Turner 2005). The approach there was towards identifying surrogates of resilience, but few of the papers proposed quantifiable metrics. Consequently, many ecological resilience frameworks remain vague and difficult to quantify, a problem that this special feature aims to address. However, considerable progress has been made during the last decade (e.g. Pope, Allen & Angeler 2014). Although some argue that resilience is best kept as an unquantifiable, vague concept (Quinlan et al. 2016), to be useful for managers, there must be concrete guidance regarding how and what to manage and how to measure success (Garmestani, Allen & Benson 2013; Spears et al. 2015). Ideas such as ‘resilience thinking’ have utility in helping stakeholders conceptualize their systems, but provide little guidance on how to make resilience useful for ecosystem management, other than suggesting an ambiguous, Goldilocks approach of being just right (e.g. diverse, but not too diverse; connected, but not too connected). Here, we clarify some prominent resilience terms and concepts, introduce and synthesize the papers in this special feature on quantifying resilience and identify core unanswered questions related to resilience.

  9. A novel self-assembled oligopeptide amphiphile for biomimetic mineralization of enamel

    PubMed Central

    2014-01-01

    Background Researchers are looking for biomimetic mineralization of ena/mel to manage dental erosion. This study evaluated biomimetic mineralization of demineralized enamel induced by a synthetic and self-assembled oligopeptide amphiphile (OPA). Results The results showed that the OPA self-assembled into nano-fibres in the presence of calcium ions and in neutral acidity. The OPA was alternately immersed in calcium chloride and sodium hypophosphate solutions to evaluate its property of mineralization. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed nucleation and growth of amorphous calcium phosphate along the self-assembled OPA nano-fibres when it was repetitively exposed to solutions with calcium and phosphate ions. Energy dispersive spectrometry (EDS) confirmed that these nano-particles contained calcium and phosphate. Furthermore, electron diffraction pattern suggested that the nano-particles precipitated on OPA nano-fibres were comparable to amorphous calcium phosphate. Acid-etched human enamel slices were incubated at 37°C in metastable calcium phosphate solution with the OPA for biomimetic mineralization. SEM and X-ray diffraction indicated that the OPA induced the formation of hydroxyapatite crystals in organized bundles on etched enamel. TEM micrographs revealed there were 20–30 nm nano-amorphous calcium phosphate precipitates in the biomimetic mineralizing solution. The particles were found separately bound to the oligopeptide fibres. Biomimetic mineralization with or without the oligopeptide increased demineralized enamel microhardness. Conclusions A novel OPA was successfully fabricated, which fostered the biomimetic mineralization of demineralized enamel. It is one of the primary steps towards the design and construction of novel biomaterial for future clinical therapy of dental erosion. PMID:24766767

  10. Acetylator phenotype in diabetic neuropathy.

    PubMed Central

    McLaren, E H; Burden, A C; Moorhead, P J

    1977-01-01

    The proportions of slow and fast acetylators in a group of diabetics with symptomatic peripheral neuropathy were compared with those in a group of diabetics who had had the disease for at least 10 years without developing neuropathy. There was a significantly higher proportion of fast acetylators in the group of diabetics without neuropathy than in those with neuropathy or in the normal population. Hence genetic factors separate from the diabetic diathesis may determine the development of neuropathy in any particular diabetic. PMID:871863

  11. Formation of oligopeptides on the surface of small bodies in solar system by cosmic radiation

    NASA Astrophysics Data System (ADS)

    Simakov, M. B.; Kuzicheva, E. A.; Dodonova, N. Ya.; Antropov, A. E.

    1997-05-01

    The present experiment indicates that oligopeptides are easily produced in solid state from mixtures of simple amino acids by irradiating with high energy charged particles. We investigated such amino acids and their mixtures as tryptophan, tyrosine and glycine. The thin films was irradiated with protons (6.6 MeV). Such dipeptides as Trp-Trp, Gly-Tyr, Tyr-Gly, and Tyr-Tyr have been detected as products of irradiation. Cosmic rays might be an effective energy source for abiotic formation of bioorganic compounds on the surface of small bodies in the solar system on early stage of formation of planets as well as at present day.

  12. Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings 1

    PubMed Central

    Zeiher, Carolyn A.; Randall, Douglas D.

    1990-01-01

    Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a Km for acetyl-CoA of 74 micromolar and a Vmax of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a Kis of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate. PMID:16667687

  13. Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

    PubMed Central

    Castaño-Cerezo, Sara; Bernal, Vicente; Post, Harm; Fuhrer, Tobias; Cappadona, Salvatore; Sánchez-Díaz, Nerea C; Sauer, Uwe; Heck, Albert JR; Altelaar, AF Maarten; Cánovas, Manuel

    2014-01-01

    Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation. PMID:25518064

  14. A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

    PubMed Central

    Damon, Coralie; Vallon, Laurent; Zimmermann, Sabine; Haider, Muhammad Z; Galeote, Virginie; Dequin, Sylvie; Luis, Patricia; Fraissinet-Tachet, Laurence; Marmeisse, Roland

    2011-01-01

    Functional environmental genomics has the potential to identify novel biological functions that the systematic sequencing of microbial genomes or environmental DNA may fail to uncover. We targeted the functions expressed by soil eukaryotes using a metatranscriptomic approach based on the use of soil-extracted polyadenylated messenger RNA to construct environmental complementary DNA expression libraries. Functional complementation of a yeast mutant defective in di/tripeptide uptake identified a novel family of oligopeptide transporters expressed by fungi. This family has a patchy distribution in the Basidiomycota and Ascomycota and is present in the genome of a Saccharomyces cerevisiae wine strain. High throughput phenotyping of yeast mutants expressing two environmental transporters showed that they both displayed broad substrate specificity and could transport more than 60–80 dipeptides. When expressed in Xenopus oocytes one environmental transporter induced currents upon dipeptide addition, suggesting proton-coupled co-transport of dipeptides. This transporter was also able to transport specifically cysteine. Deletion of the two copies of the corresponding gene family members in the genome of the wine yeast strain severely reduced the number of dipeptides that it could assimilate. These results demonstrate that these genes are functional and can be used by fungi to efficiently scavenge the numerous, low concentration, oligopeptides continuously generated in soils by proteolysis. PMID:21654847

  15. Viscoelastic properties and nanoscale structures of composite oligopeptide-polysaccharide hydrogels.

    PubMed

    Hyland, Laura L; Taraban, Marc B; Feng, Yue; Hammouda, Boualem; Yu, Y Bruce

    2012-03-01

    Biocompatible and biodegradable peptide hydrogels are drawing increasing attention as prospective materials for human soft tissue repair and replacement. To improve the rather unfavorable mechanical properties of our pure peptide hydrogels, in this work we examined the possibility of creating a double hydrogel network. This network was created by means of the coassembly of mutually attractive, but self-repulsive oligopeptides within an already-existing fibrous network formed by the charged, biocompatible polysaccharides chitosan, alginate, and chondroitin. Using dynamic oscillatory rheology experiments, it was found that the coassembly of the peptides within the existing polysaccharide network resulted in a less stiff material as compared to the pure peptide networks (the elastic modulus G' decreased from 90 to 10 kPa). However, these composite oligopeptide-polysaccharide hydrogels were characterized by a greater resistance to deformation (the yield strain γ grew from 4 to 100%). Small-angle neutron scattering (SANS) was used to study the 2D cross-sectional shapes of the fibers, their dimensional characteristics, and the mesh sizes of the fibrous networks. Differences in material structures found with SANS experiments confirmed rheology data, showing that incorporation of the peptides dramatically changed the morphology of the polysaccharide network. The resulting fibers were structurally very similar to those forming the pure peptide networks, but formed less stiff gels because of their markedly greater mesh sizes. Together, these findings suggest an approach for the development of highly deformation-resistant biomaterials. PMID:21994046

  16. Directed self-assembly of π-conjugated oligopeptides for supramolecular electronics

    NASA Astrophysics Data System (ADS)

    Li, Bo; Li, Songsong; Zhou, Yuecheng; Tovar, John; Wilson, William; Schroeder, Charles

    The directed mesoscale engineering of nanoscale building blocks holds enormous promise to catalyze a revolution in new functional materials for advanced electronics. Bio-inspired systems can play a key role in this effort due to their inherent ``programmable'' function. In this work, oligopeptide with defined flanking sequences was appended to π-conjugated units, thereby directing their assembly processes in a designed manner. By utilizing custom-designed microfluidic devices and controlled acid vapor diffusion, the self-assembly rate was directed and precisely tuned. Notably, the kinetics was found to play a key role in the morphology of self-assembled π-conjugated oligopeptides. The influence of flanking peptide sequences and π-conjugated core-core interactions on the self-assembly nanostructure was systematically investigated. Importantly, the electronic properties of the synthetic peptide assembly was explored by integration as the active layer of a field effect transistor. The presented study offers insights to the design and fabrication of supramolecular electronics.

  17. Optimization of Enzymatic Production of Oligopeptides from Apricot Almonds Meal with Neutrase and N120P

    PubMed Central

    Wang, Chunyan; Wang, Qiang; Tian, Jinqiang

    2010-01-01

    Neutrase 0.8L and N120P proteases were used for oligopeptide production from apricot almonds meal, and response surface design was carried out to optimize the effect of hydrolysis conditions on hydrolysis degree (DH) and oligopeptide yield rate. Four independent variables were used to optimize the hydrolysis process: hydrolysis temperature (X1), enzyme-to substrate ratio (E/S) (X2), substrate concentration (X3) and reaction time (X4). Statistical analysis indicated that the four variables, quadratic terms of X1, X3, and X4, and the interaction terms with X1 had a significant (p < 0.05) effect on DH. The yield rate was also significantly affected by the four variables and quadratic terms of X1, X2 and X4. Two mathematical models with high determination coefficient were obtained and could be employed to optimize protein hydrolysis. The optimal hydrolysis conditions were determined as follows: hydrolysis temperature 52.5 °C; enzyme-to-substrate ratio (E/S) 7200 U/g; substrate concentration 2%; reaction time 173 min. The initial pH 6.5 and Neutrase-to-N120P dosage ratio 2:1 were fixed in this study according to the preliminary research. Under these conditions, the experimental DH and yield rate were 34.10 ± 5.25% and 72.42 ± 2.27%, respectively. PMID:21614184

  18. Oligopeptide complex for targeted non-viral gene delivery to adipocytes

    NASA Astrophysics Data System (ADS)

    Won, Young-Wook; Adhikary, Partho Protim; Lim, Kwang Suk; Kim, Hyung Jin; Kim, Jang Kyoung; Kim, Yong-Hee

    2014-12-01

    Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

  19. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    PubMed

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

  20. Modelling of the prebiotic synthesis of oligopeptides: silicate catalysts help to overcome the critical stage

    NASA Astrophysics Data System (ADS)

    Zamaraev, Kirill I.; Romannikov, Vyacheslav N.; Salganik, Rudolph I.; Wlassoff, Wjatschesslaw A.; Khramtsov, Valeriy V.

    1997-08-01

    On the basis of experimental studies of the initial stages of glycine oligomerization in aqueous suspension of zeolite and kaolinite catalysts, a model is suggested for the prebiotic synthesis of oligopeptides from α-amino acids. The formation of linear dipeptides by hydrolysis of one amide bond in the cyclic piperazinedione intermediate (formed from glycine spontaneously) is found to be the critical stage of the reaction. This stage is base catalyzed and its rate increases when pH of the medium goes up. The linear glycyl-glycine yield rises under effect of hydroxyl anions generated from different sources including insoluble silicates and soluble sodium bicarbonate. During prebiotic evolution silicates capable of cation-exchange can serve as local sources of the hydroxyl anions which dramatically accelerate formation of linear dipeptides from cyclic ones. Oligopeptides of higher molecular weight are then easily formed from the linear dipeptides at neutral pH, even in the absence of catalysts or sources of energy (e.g. such as light). The described catalytic synthesis could occur in the proximity of submarine hydrothermal vents.

  1. Mapping sugar beet pectin acetylation pattern.

    PubMed

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

    2005-08-01

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

  2. Protein acetylation in archaea, bacteria, and eukaryotes.

    PubMed

    Soppa, Jörg

    2010-09-16

    Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  3. Quantifying contextuality.

    PubMed

    Grudka, A; Horodecki, K; Horodecki, M; Horodecki, P; Horodecki, R; Joshi, P; Kłobus, W; Wójcik, A

    2014-03-28

    Contextuality is central to both the foundations of quantum theory and to the novel information processing tasks. Despite some recent proposals, it still faces a fundamental problem: how to quantify its presence? In this work, we provide a universal framework for quantifying contextuality. We conduct two complementary approaches: (i) the bottom-up approach, where we introduce a communication game, which grasps the phenomenon of contextuality in a quantitative manner; (ii) the top-down approach, where we just postulate two measures, relative entropy of contextuality and contextuality cost, analogous to existent measures of nonlocality (a special case of contextuality). We then match the two approaches by showing that the measure emerging from the communication scenario turns out to be equal to the relative entropy of contextuality. Our framework allows for the quantitative, resource-type comparison of completely different games. We give analytical formulas for the proposed measures for some contextual systems, showing in particular that the Peres-Mermin game is by order of magnitude more contextual than that of Klyachko et al. Furthermore, we explore properties of these measures such as monotonicity or additivity. PMID:24724629

  4. Short one-pot chemo-enzymatic synthesis of L-lysine and L-alanine diblock co-oligopeptides.

    PubMed

    Fagerland, Jenny; Finne-Wistrand, Anna; Numata, Keiji

    2014-03-10

    Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using (1)H NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.

  5. Flow properties of acetylated chickpea protein dispersions.

    PubMed

    Liu, Li H; Hung, Tran V

    2010-06-01

    Chickpea protein concentrate was acetylated with acetic anhydride at 5 levels. Acetylated chickpea protein (ACP) dispersions at 3 levels (6%, 45%, and 49%) were chosen for this flow property study. Effects of protein concentration, temperature, concentrations of salt addition and particularly, degree of acetylation on these properties were examined. Compared with native chickpea proteins, the ACP dispersions exhibited a strong shear thinning behavior. Within measured temperature range (15 to 55 degrees C), the apparent viscosities of native chickpea protein dispersions were temperature independent; those of ACP dispersions were thermally affected. The flow index (n), consistency coefficient (m), apparent yield stress, and apparent viscosities of ACP dispersions increased progressively up to 45% acetylation but decreased at 49% acetylation level. Conformational studies by gel filtration suggested that chickpea proteins were associated or polymerized at up to 45% acetylation but the associated subunits gradually dissociated to smaller units at higher levels (49%) of acetylation.

  6. Chemical and enzymatic catalytic routes to polyesters and oligopeptides biobased materials

    NASA Astrophysics Data System (ADS)

    Zhu, Jianhui

    My Ph.D research focuses on the synthesis and property studies of different biobased materials, including polyesters, polyurethanes and oligopeptides. The first study describes the synthesis, crystal structure and physico-mechanical properties of a bio-based polyester prepared from 2,5-furandicarboxylic acid (FDCA) and 1,4-butanediol. Melt-polycondensation experiments were conducted by a two-stage polymerization using titanium tetraisopropoxide (Ti[OiPr] 4) as catalyst. Polymerization conditions (catalyst concentration, reaction time and 2nd stage reaction temperature) were varied to optimize poly(butylene furan dicarboxylate), PBF, molecular weight. A series of PBFs with different Mw were characterized by Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Dynamic Mechanical Thermal Analysis (DMTA), X-Ray diffraction and tensile testing. Influence of molecular weight and melting/crystallization enthalpy on PBF material tensile properties was explored. Cold-drawing tensile tests at room temperature for PBF with Mw 16K to 27K showed a brittle-to-ductile transition. When Mw reaches 38K, the Young's Modulus of PBF remains above 900 MPa, and the elongation at break increases to above 1000%. The mechanical properties, thermal properties and crystal structures of PBF were similar to petroleum derived poly(butylenes terephthalate), PBT. Fiber diagrams of uniaxially stretched PBF films were collected, indexed, and the unit cell was determined as triclinic (a=4.78(3) A, b=6.03(5) A, c=12.3(1) A, alpha=110.1(2)°, beta=121.1(3)°, gamma=100.6(2)°). A crystal structure was derived from this data and final atomic coordinates are reported. We concluded that there is a close similarity of the PBF structure to PBT alpha- and beta-forms. In the second study, a biobased long chain polyester polyol (PC14-OH) was synthesized from o-hydroxytetradecanoic acid (o-HOC14) and 1,4-butanediol. The first section about polyester polyurethanes describes the synthesis

  7. An Acute Acetyl Fentanyl Fatality: A Case Report With Postmortem Concentrations.

    PubMed

    McIntyre, Iain M; Trochta, Amber; Gary, Ray D; Malamatos, Mark; Lucas, Jonathan R

    2015-01-01

    In this case report, we present an evaluation of the distribution of postmortem concentrations of acetyl fentanyl in a fatality attributed to the drug. A young man who had a history of heroin abuse was found deceased at his parents' home. Toxicology testing, which initially screened positive for fentanyl by ELISA, subsequently confirmed acetyl fentanyl by gas chromatography-mass spectrometry specific ion monitoring (GC-MS SIM) analysis following liquid-liquid extraction. No other drugs or medications, including fentanyl, were detected. The acetyl fentanyl peripheral blood concentration was quantified at 260 ng/mL compared with the central blood concentration of 250 ng/mL. The liver concentration was 1,000 ng/kg, the vitreous was 240 ng/mL and the urine was 2,600 ng/mL. The cause of death was certified due to acute acetyl fentanyl intoxication, and the manner of death was certified as an accident.

  8. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated.

  9. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated. PMID:27423860

  10. Free Energy Landscapes of Alanine Oligopeptides in Rigid-Body and Hybrid Water Models.

    PubMed

    Nayar, Divya; Chakravarty, Charusita

    2015-08-27

    Replica exchange molecular dynamics is used to study the effect of different rigid-body (mTIP3P, TIP4P, SPC/E) and hybrid (H1.56, H3.00) water models on the conformational free energy landscape of the alanine oligopeptides (acAnme and acA5nme), in conjunction with the CHARMM22 force field. The free energy landscape is mapped out as a function of the Ramachandran angles. In addition, various secondary structure metrics, solvation shell properties, and the number of peptide-solvent hydrogen bonds are monitored. Alanine dipeptide is found to have similar free energy landscapes in different solvent models, an insensitivity which may be due to the absence of possibilities for forming i-(i + 4) or i-(i + 3) intrapeptide hydrogen bonds. The pentapeptide, acA5nme, where there are three intrapeptide backbone hydrogen bonds, shows a conformational free energy landscape with a much greater degree of sensitivity to the choice of solvent model, though the three rigid-body water models differ only quantitatively. The pentapeptide prefers nonhelical, non-native PPII and β-sheet populations as the solvent is changed from SPC/E to the less tetrahedral liquid (H1.56) to an LJ-like liquid (H3.00). The pentapeptide conformational order metrics indicate a preference for open, solvent-exposed, non-native structures in hybrid solvent models at all temperatures of study. The possible correlations between the properties of solvent models and secondary structure preferences of alanine oligopeptides are discussed, and the competition between intrapeptide, peptide-solvent, and solvent-solvent hydrogen bonding is shown to be crucial in the relative free energies of different conformers.

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

    PubMed

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas; Zechner, Rudolf; Choudhary, Chunaram

    2015-11-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3-targeted sites to chemical acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.

  12. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis.

  13. 2-Acetyl-pyridinium bromanilate.

    PubMed

    Thomas, Lynne H; Boyle, Bryan; Clive, Lesley A; Collins, Anna; Currie, Lynsey D; Gogol, Malgorzata; Hastings, Claire; Jones, Andrew O F; Kennedy, Jennifer L; Kerr, Graham B; Kidd, Alastair; Lawton, Lorreta M; Macintyre, Susan J; Maclean, Niall M; Martin, Alan R G; McGonagle, Kate; Melrose, Samantha; Rew, Gaius A; Robinson, Colin W; Schmidtmann, Marc; Turnbull, Felicity B; Williams, Lewis G; Wiseman, Alan Y; Wocial, Malgorzata H; Wilson, Chick C

    2009-01-01

    In the crystal of the title mol-ecular salt (systematic name: 2-acetyl-pyridinium 2,5-dibromo-4-hydr-oxy-3,6-dioxocyclo-hexa-1,4-dienolate), C(7)H(8)NO(+)·C(6)HBr(2)O(4) (-), centrosymmetric rings consisting of two cations and two anions are formed, with the components linked by alternating O-H⋯O and N-H⋯O hydrogen bonds. Short O⋯Br contacts [3.243 (2) and 3.359 (2) Å] may help to consolidate the packing. PMID:21583087

  14. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    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.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. 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 lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  15. Acetylation of woody lignocellulose: significance and regulation

    PubMed Central

    Pawar, Prashant Mohan-Anupama; Koutaniemi, Sanna; Tenkanen, Maija; Mellerowicz, Ewa J.

    2013-01-01

    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

  16. Acetylation regulates Jun protein turnover in Drosophila.

    PubMed

    Zhang, Daoyong; Suganuma, Tamaki; Workman, Jerry L

    2013-11-01

    C-Jun is a major transcription factor belonging to the activating protein 1 (AP-1) family. Phosphorylation has been shown to be critical for c-Jun activation and stability. Here, we report that Jra, the Drosophila Jun protein, is acetylated in vivo. We demonstrate that the acetylation of Jra leads to its rapid degradation in response to osmotic stress. Intriguingly, we also found that Jra phosphorylation antagonized its acetylation, indicating the opposite roles of acetylation and phosphorylation in Jra degradation process under osmotic stress. Our results provide new insights into how c-Jun proteins are precisely regulated by the interplay of different posttranslational modifications.

  17. [Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

    PubMed

    Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

    2012-01-01

    It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

  18. Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides.

    PubMed

    Kim, Sung-Woo; Kim, Jae-Bum; Lee, Weon Sup; Jung, Woo-Hyuk; Ryu, Ji-Myung; Jang, Hyung-Wook; Jo, Young-Bae; Jung, Joon-Ki; Kim, Jung-Hoe

    2007-09-01

    Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3.IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3.IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3.IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3.IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3.IL-2/HE molecule. The G3.IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16).

  19. Chemical and enzymatic catalytic routes to polyesters and oligopeptides biobased materials

    NASA Astrophysics Data System (ADS)

    Zhu, Jianhui

    My Ph.D research focuses on the synthesis and property studies of different biobased materials, including polyesters, polyurethanes and oligopeptides. The first study describes the synthesis, crystal structure and physico-mechanical properties of a bio-based polyester prepared from 2,5-furandicarboxylic acid (FDCA) and 1,4-butanediol. Melt-polycondensation experiments were conducted by a two-stage polymerization using titanium tetraisopropoxide (Ti[OiPr] 4) as catalyst. Polymerization conditions (catalyst concentration, reaction time and 2nd stage reaction temperature) were varied to optimize poly(butylene furan dicarboxylate), PBF, molecular weight. A series of PBFs with different Mw were characterized by Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Dynamic Mechanical Thermal Analysis (DMTA), X-Ray diffraction and tensile testing. Influence of molecular weight and melting/crystallization enthalpy on PBF material tensile properties was explored. Cold-drawing tensile tests at room temperature for PBF with Mw 16K to 27K showed a brittle-to-ductile transition. When Mw reaches 38K, the Young's Modulus of PBF remains above 900 MPa, and the elongation at break increases to above 1000%. The mechanical properties, thermal properties and crystal structures of PBF were similar to petroleum derived poly(butylenes terephthalate), PBT. Fiber diagrams of uniaxially stretched PBF films were collected, indexed, and the unit cell was determined as triclinic (a=4.78(3) A, b=6.03(5) A, c=12.3(1) A, alpha=110.1(2)°, beta=121.1(3)°, gamma=100.6(2)°). A crystal structure was derived from this data and final atomic coordinates are reported. We concluded that there is a close similarity of the PBF structure to PBT alpha- and beta-forms. In the second study, a biobased long chain polyester polyol (PC14-OH) was synthesized from o-hydroxytetradecanoic acid (o-HOC14) and 1,4-butanediol. The first section about polyester polyurethanes describes the synthesis

  20. Two-fold odd-even effect in self-assembled nanowires from oligopeptide-polymer-substituted perylene bisimides.

    PubMed

    Marty, Roman; Nigon, Robin; Leite, Deborah; Frauenrath, Holger

    2014-03-12

    Organic nanowires are important building blocks for nanoscopic organic electronic devices. In order to ensure efficient charge transport through such nanowires, it is important to understand in detail the molecular parameters that guide self-assembly of π-conjugated molecules into one-dimensional stacks with optimal constructive π-π overlap. Here, we investigated the subtle relationship between molecular structure and supramolecular arrangement of the chromophores in self-assembled nanowires prepared from perylene bisimides with oligopeptide-polymer side chains. We observed a "two-fold" odd-even effect in circular dichroism spectra of these derivatives, depending on both the number of l-alanine units in the oligopeptide segments and length of the alkylene spacer between chromophore and oligopeptide substituents. Our results indicate that there is a complex interplay between the translation of molecular chirality into supramolecular helicity and the molecules' inherent propensity for well-defined one-dimensional aggregation into β-sheet-like superstructures in the presence of a central chromophore. Strong excitonic coupling as expressed by the appearance of hypsochromically and bathochromically shifted UV-vis absorptions and strong CD signals was systematically observed for molecules with an odd number of l-alanines in the side chains. The latter derivatives gave rise to nanowires with a significantly higher electron mobility. Our results, hence, provide an important design rule for self-assembled organic nanowires.

  1. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGES

    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

    2014-01-01

    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

  2. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.828 Acetylated monoglycerides. The food additive acetylated... of catalytic agents that are not food additives or are authorized by regulation, followed by...

  3. Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotriene E4

    SciTech Connect

    Hagmann, W.; Denzlinger, C.; Rapp, S.; Weckbecker, G.; Keppler, D.

    1986-02-01

    Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats in vivo after endotoxin treatment. The mercapturate N-acetyl-leukotriene E4 (N-acetyl-LTE4) represented a major metabolite eliminated into bile after injection of (/sup 3/H)LTC4 as shown by cochromatography with synthetic N-acetyl-LTE4 in four different HPLC solvent systems. The identity of endogenous N-acetyl-LTE4 elicited by endotoxin in vivo was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE4 concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE4. The mercapturic acid pathway, leading from the glutathione conjugate LTC4 to N-acetyl-LTE4, thus plays an important role in the deactivation and elimination of these potent endogenous mediators.

  4. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    SciTech Connect

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang; and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  5. SPOTing Acetyl-Lysine Dependent Interactions.

    PubMed

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-08-17

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  6. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation. PMID:27600229

  7. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  8. Regulation of intracellular pH during H+-coupled oligopeptide absorption in enterocytes from guinea-pig ileum

    PubMed Central

    Hayashi, Hisayoshi; Suzuki, Yuichi

    1998-01-01

    The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3−-buffered solution was used. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 μM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3−, the pHi recovery was almost completely abolished by 0.3 mM amiloride. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3−-dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3− cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present

  9. [Inhibitory effect of methyl esters of arginine-containing oligopeptides on thrombin and trypsin].

    PubMed

    Poiarkova, S A; Kibirev, V K; Serebrianyĭ, S B

    1987-01-01

    Stereoisomeric oligopeptides were studied for their inhibitory effect on the hydrolysis of benzoyl-L-arginine methyl ester catalyzed by thrombin and trypsin, as well as on the thrombin-fibrinogen reaction. Comparison of the peptide structures, their conformational flexibility and inhibitory effects on thrombin and trypsin shows the availability of the essential differences in organization and functioning of the subsites S3, S2 and S'1 of these enzymes. In contrast to trypsin, thrombin is shown to be characterized by more pronounced secondary stereospecificity. This manifests in the more vigorous dropping of the catalytic constants of thrombin-catalyzed esterolysis than those of trypsin-catalyzed hydrolysis of the substrates, containing D-amino acids at the subsite P2. It is revealed that the peptide Tos-D-Val-D-Ala-D-Agr-D-Phe-OCH3 is the most powerful inhibitor among studied compounds. It is noteworthy that its antithrombin effect is almost an order of magnitude higher than its antitrypsin effect.

  10. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    PubMed

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  11. Crystal structure of a putative oligopeptide-binding periplasmic protein from a hyperthermophile.

    PubMed

    Yoon, Hye-Jin; Kim, Hee Jung; Mikami, Bunzo; Yu, Yeon Gyu; Lee, Hyung Ho

    2016-09-01

    Oligopeptide-binding proteins (Opps) are part of the ATP-binding cassette system, playing a crucial role in nutrient uptake and sensing the external environment in bacteria, including hyperthermophiles. Opps serve as a binding platform for diverse peptides; however, how these peptides are recognized by Opps is still largely unknown and few crystal structures of Opps from hyperthermophiles have been determined. To facilitate such an understanding, the crystal structure of a putative Opp, OppA from Thermotoga maritima (TmOppA), was solved at 2.6-Å resolution in the open conformation. TmOppA is composed of three domains. The N-terminal domain consists of twelve strands, nine helices, and four 310 helices, and the C-terminal domain consists of five strands, ten helices, and one 310 helix. These two domains are connected by the linker domain, which consists of two strands, three helices, and three 310 helices. Based on structural comparisons of TmOppA with other OppAs and binding studies, we suggest that TmOppA might be a periplasmic Opp. The most distinct feature of TmOppA is the insertion of two helices, which are lacking in other OppAs. A cavity volume between the N-terminal and C-terminal domains is suggested to be responsible for binding peptides of various lengths. PMID:27377296

  12. Thermodynamics, morphology, and kinetics of early- stage self-assembly of pi-conjugated oligopeptides

    NASA Astrophysics Data System (ADS)

    Thurston, Bryce; Tovar, John; Ferguson, Andrew

    Synthetic oligopeptides containing π-conjugated cores self-assemble novel materials with attractive electronic and photophysical properties. All-atom, explicit solvent molecular dynamics simulations of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp peptides were used to parameterize an implicit solvent model to simulate self-assembly. At low-pH conditions, peptides assemble into β-sheet-like stacks with strongly favorable monomer association free energies of ΔF ~ - 25kB T . Aggregation at high-pH produces disordered aggregates destabilized by Coulombic repulsion between negatively charged Asp termini. We model simulations of hundereds of monomers as a continuous-time Markov process. We infer transition rates between different aggregate sizes and microsecond relaxation times for early-stage assembly. Our data suggests a hierarchical model of assembly in which peptides coalesce into small clusters over tens of nanoseconds followed by structural ripening and diffusion limited aggregation on longer time scales. This work provides new molecular-level understanding of early-stage assembly, and a means to study the impact of peptide chemistry upon the thermodynamics, assembly kinetics, and morphology of the supramolecular aggregates. Supported by U.S. Department of Energy, Office of Science, Basic Energy Sciences, Award DE-SC0004857. Molecular simulations partially conducted on University of Illinois Computational Science and Engineering Program parallel computing resources.

  13. Polyamidoamine (PAMAM) dendrimers modified with short oligopeptides for early endosomal escape and enhanced gene delivery.

    PubMed

    Thuy, Le Thi; Mallick, Sudipta; Choi, Joon Sig

    2015-08-15

    Recently, non-viral vectors have become a popular research topic in the field of gene therapy. In this study, we conjugated short oligopeptides to polyamidoamine-generation 4 (PAMAM G4) to achieve higher transfection efficiency. Previous reports have shown that the PAMAM G4-histidine (H)-arginine (R) dendrimer enhances gene delivery by improving cell penetration and internalization mechanisms. Therefore, we synthesized PAMAM G4-H phenylalanine (F) R, PAMAM G4-FHR and PAMAM G4-FR derivatives to determine the best gene carrier with the lowest toxicity. Physicochemical studies were performed to determine mean diameters and surface charge of PAMAM derivatives/pDNA polyplexes. DNA condensation was confirmed using a gel retardation assay. Cytotoxicity and transfection efficiency were analyzed using human cervical carcinoma (HeLa) and human liver carcinoma (HepG2) cells. Similar levels of transfection were achieved in both cell lines by using gold standard transfection reagent PEI 25 kD. Therefore, our results show that these carriers are promising and may help achieve higher transfection with negligible cytotoxicity. PMID:26187169

  14. Protective Effects of Soy Oligopeptides in Ultraviolet B-Induced Acute Photodamage of Human Skin

    PubMed Central

    Ma, Li-wen; Liu, Juan; Zhang, Jia-an; Xu, Yang; Wu, Di; Permatasari, Felicia

    2016-01-01

    Aim. We explored the effects of soy oligopeptides (SOP) in ultraviolet B- (UVB-) induced acute photodamage of human skin in vivo and foreskin ex vivo. Methods. We irradiated the forearm with 1.5 minimal erythemal dose (MED) of UVB for 3 consecutive days, establishing acute photodamage of skin, and topically applied SOP. Erythema index (EI), melanin index, stratum corneum hydration, and transepidermal water loss were measured by using Multiprobe Adapter 9 device. We irradiated foreskin ex vivo with the same dose of UVB (180 mJ/cm2) for 3 consecutive days and topically applied SOP. Sunburn cells were detected by using hematoxylin and eosin staining. Apoptotic cells were detected by using terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Cyclobutane pyrimidine dimers (CPDs), p53 protein, Bax protein, and Bcl-2 protein were detected by using immunohistochemical staining. Results. Compared with UVB group, UVB-irradiated skin with topically applied SOP showed significantly decreased EI. Compared with UVB group, topical SOP significantly increased Bcl-2 protein expression and decreased CPDs-positive cells, sunburn cells, apoptotic cells, p53 protein expression, and Bax protein expressions in the epidermis of UVB-irradiated foreskin. Conclusion. Our study demonstrated that topical SOP can protect human skin against UVB-induced photodamage. PMID:27478534

  15. Protective Effects of Soy Oligopeptides in Ultraviolet B-Induced Acute Photodamage of Human Skin.

    PubMed

    Zhou, Bing-Rong; Ma, Li-Wen; Liu, Juan; Zhang, Jia-An; Xu, Yang; Wu, Di; Permatasari, Felicia; Luo, Dan

    2016-01-01

    Aim. We explored the effects of soy oligopeptides (SOP) in ultraviolet B- (UVB-) induced acute photodamage of human skin in vivo and foreskin ex vivo. Methods. We irradiated the forearm with 1.5 minimal erythemal dose (MED) of UVB for 3 consecutive days, establishing acute photodamage of skin, and topically applied SOP. Erythema index (EI), melanin index, stratum corneum hydration, and transepidermal water loss were measured by using Multiprobe Adapter 9 device. We irradiated foreskin ex vivo with the same dose of UVB (180 mJ/cm(2)) for 3 consecutive days and topically applied SOP. Sunburn cells were detected by using hematoxylin and eosin staining. Apoptotic cells were detected by using terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Cyclobutane pyrimidine dimers (CPDs), p53 protein, Bax protein, and Bcl-2 protein were detected by using immunohistochemical staining. Results. Compared with UVB group, UVB-irradiated skin with topically applied SOP showed significantly decreased EI. Compared with UVB group, topical SOP significantly increased Bcl-2 protein expression and decreased CPDs-positive cells, sunburn cells, apoptotic cells, p53 protein expression, and Bax protein expressions in the epidermis of UVB-irradiated foreskin. Conclusion. Our study demonstrated that topical SOP can protect human skin against UVB-induced photodamage. PMID:27478534

  16. Adsorption of a cell-adhesive oligopeptide on polymer surfaces irradiated by ion beams.

    PubMed

    Satriano, C; Manso, M; Gambino, G L; Rossi, F; Marletta, G

    2005-01-01

    The adsorption behavior of H-Arg-Gly-Asp-OH (RGD) oligopeptide on ion-irradiated polymer surfaces has been studied. The RGD-incubated surfaces of poly(ethylene terephtalate) (PET) and poly(hydroxymethylsiloxane) (PHMS) thin films, before and after irradiation with 50 keV Ar+ to 1x10(15) ions/cm2, were investigated by X-Ray Photoelectron Spectroscopy and Atomic Force Microscopy. It was found that no significant adsorption occurs on PET, while a measurable amount of RGD is preferentially adsorbed onto irradiated PHMS surfaces. The evaluated surface coverage was found to range between 5 and 12%. In situ adsorption measurements performed by using the Quartz Crystal Microbalance with Dissipation monitoring technique showed that the irradiation induced remarkable changes of mass uptake with respect to the unirradiated surfaces, mostly attributed to the change in the water adsorption capability of the irradiated surfaces. The adsorption results are discussed in terms of the ion-induced changes on the morphology, chemical structure and composition, surface free energy and surface charge. PMID:15623933

  17. Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai.

    PubMed

    Nguyen, Van-Tinh; Qian, Zhong-Ji; Ryu, Bomi; Kim, Kil-Nam; Kim, Daekyung; Kim, Young-Mog; Jeon, You-Jin; Park, Won Sun; Choi, Il-Whan; Kim, Geun Hyung; Je, Jae-Young; Jung, Won-Kyo

    2013-11-01

    Abalone (Haliotis discus hannai) is a marine gastropod, and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia and America. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purified abalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated from the digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQ column. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW=782.4 Da) with a de novo peptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitory effect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibrosarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibit MMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-κB)-mediated pathway. This data suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive potential for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases.

  18. Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability

    PubMed Central

    Jones, Megan M.

    2015-01-01

    Moraxella catarrhalis causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The five opp genes oppB, oppC, oppD, oppF, and oppA are in the same open reading frame. Sequence analysis predicted two promoters, one located upstream of oppB and one within the intergenic region between oppF and oppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream of oppA contributes to the transcription of oppA but is not influenced by the same environmental cues as the promoter upstream of oppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor in M. catarrhalis. PMID:26099587

  19. Acetylation modulates the STAT signaling code.

    PubMed

    Wieczorek, Martin; Ginter, Torsten; Brand, Peter; Heinzel, Thorsten; Krämer, Oliver H

    2012-12-01

    A fascinating question of modern biology is how a limited number of signaling pathways generate biological diversity and crosstalk phenomena in vivo. Well-defined posttranslational modification patterns dictate the functions and interactions of proteins. The signal transducers and activators of transcription (STATs) are physiologically important cytokine-induced transcription factors. They are targeted by a multitude of posttranslational modifications that control and modulate signaling responses and gene expression. Beyond phosphorylation of serine and tyrosine residues, lysine acetylation has recently emerged as a critical modification regulating STAT functions. Interestingly, acetylation can determine STAT signaling codes by various molecular mechanisms, including the modulation of other posttranslational modifications. Here, we provide an overview on the acetylation of STATs and how this protein modification shapes cellular cytokine responses. We summarize recent advances in understanding the impact of STAT acetylation on cell growth, apoptosis, innate immunity, inflammation, and tumorigenesis. Furthermore, we discuss how STAT acetylation can be targeted by small molecules and we consider the possibility that additional molecules controlling STAT signaling are regulated by acetylation. Our review also summarizes evolutionary aspects and we show similarities between the acetylation-dependent control of STATs and other important molecules. We propose the concept that, similar to the 'histone code', distinct posttranslational modifications and their crosstalk orchestrate the functions and interactions of STAT proteins. PMID:22795479

  20. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

    PubMed Central

    Snyder, Nathaniel W.; Wei, Shuanzeng; Venneti, Sriram; Worth, Andrew J.; Yuan, Zuo-Fei; Lim, Hee-Woong; Liu, Shichong; Jackson, Ellen; Aiello, Nicole M.; Haas, Naomi B.; Rebbeck, Timothy R.; Judkins, Alexander; Won, Kyoung-Jae; Chodosh, Lewis A.; Garcia, Benjamin A.; Stanger, Ben Z.; Feldman, Michael D.; Blair, Ian A.; Wellen, Kathryn E.

    2014-01-01

    SUMMARY Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. PMID:24998913

  1. Acetylator phenotypes in Papua New Guinea

    PubMed Central

    Penketh, R J A; Gibney, S F A; Nurse, G T; Hopkinson, D A

    1983-01-01

    Acetylator phenotypes have been determined in 139 unrelated subjects from the hitherto untested populations of Papua New Guinea, and their relevance to current antituberculous isoniazid chemotherapy is discussed. PMID:6842533

  2. Histone deacetylase 3 indirectly modulates tubulin acetylation.

    PubMed

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-12-15

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

  3. Levels of histone acetylation in thyroid tumors.

    PubMed

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  4. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.

    PubMed

    Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R; Hoppel, Charles

    2015-01-01

    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 β-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

  5. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer

    PubMed Central

    Miller, Kyle M.

    2016-01-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer. PMID:27631103

  6. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    PubMed

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  7. Histone acetylation and globin gene switching.

    PubMed Central

    Hebbes, T R; Thorne, A W; Clayton, A L; Crane-Robinson, C

    1992-01-01

    An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription. Images PMID:1549462

  8. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis. PMID:26503676

  9. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

    PubMed

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires Júnior, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

  10. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

    PubMed

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires Júnior, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. PMID:26661890

  11. Supramolecular assemblies of histidinylated β-cyclodextrin for enhanced oligopeptide delivery into osteoclast precursors.

    PubMed

    Liu, Wei; Zhang, Xuejin; Wang, Rui; Xu, Hong; Chi, Bo

    2016-01-01

    Much attention has been given to the problem of drug delivery through the cell membrane in order to treat and manage bone diseases recently. The aim of this study was to develop nanoparticles made of amino- and histidinyl-modified amphiphilic β-cyclodextrins (β-CDs) entrapping osteoclast inhibitor, a hydrophobic oligopeptides drug, across the membrane of bone marrow-derived macrophages (BMMs). Drug-loaded β-CDs nanoparticles (NPs) were prepared by the emulsion solvent evaporation technique and fully characterized for size, zeta potential, and entrapment efficiency. Spherical NPs displaying a hydrodynamic radius of about 295 nm which did not change upon storage as an aqueous dispersion, a positive zeta potential, and entrapment efficiency of drug very close to 98% were produced. Flow cytometry and spectrofluorimetry analysis indicated that the model drug itself was not taken up by the BMMs; however, NP systems underwent significant cellular uptake. In particular, histidinyl group-modified CD (β-CD-H) NPs were taken up more efficiently than amino group-modified (β-CD-A) ones. Cellular uptake mechanism study demonstrated that the permeability of drug-loaded NPs across the membrane of BMMs is probably due to macropinocytosis pathway. Cell viability studies showed that both β-CD-A and β-CD-H exhibited no significant cytotoxicity up to 1.0 mg/ml against the cells. These results highlight the developed β-CD-H NPs have great potential in safely and effectively delivering osteoclast inhibitors and other therapeutic agents toward bone disease. PMID:26907470

  12. Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella).

    PubMed

    Liu, Zhen; Zhou, Yi; Feng, Junchang; Lu, Shuangqing; Zhao, Qiong; Zhang, Jianshe

    2013-03-01

    The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.

  13. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium

    PubMed Central

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. PMID:26661890

  14. Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [REP-PSI (+)].

    PubMed

    Gasset-Rosa, Fátima; Giraldo, Rafael

    2015-01-01

    The yeast translation termination factor Sup35p, by aggregating as the [PSI (+)] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the Saccharomyces cerevisiae proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs), which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [REP-PSI (+)], (i) was functional in a stop codon read-through assay in S. cerevisiae; (ii) generates weak phenotypic variants upon both its expression or transformation into [psi (-)] cells; (iii) these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and (iv) according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of bona fide yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [PSI (+)]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.

  15. Effects of soya oligosaccharides and soya oligopeptides on lipid metabolism in hyperlipidaemic rats.

    PubMed

    Xie, Shali; Zhu, Jundong; Zhang, Yanqi; Shi, Kai; Shi, Yuangang; Ma, Xiao

    2012-08-01

    In the present study, we aimed to examine the effects of soya oligosaccharides (SOS) and soya oligopeptides (SOP) on blood lipid levels, release of vasoactive substances, antioxidant activity and faecal bile acid (FBA) excretion in rats fed a high-fat diet (HFD). Male Sprague-Dawley rats were evenly divided into five groups according to diets as follows: regular diet (control), HFD, HFD enriched with 2 % of SOS (SOS), HFD enriched with 3 % of SOP (SOP) and HFD enriched with 2 % SOS and 3 % SOP (SOSP). The results showed that SOS and SOP significantly reduced plasma total cholesterol, LDL-cholesterol and TAG, whereas HDL-cholesterol concentration was significantly increased. Furthermore, SOS and SOP reduced plasma apoB, apoE and the apoB:apoAI ratio, whereas apoAI was significantly increased. Moreover, SOS and SOP also reduced plasma thromboxane A₂ (TXA₂) and the TXA₂:prostacyclin (PGI₂) ratio, whereas plasma PGI₂ and nitric oxide were significantly increased. In addition, SOS and SOP significantly reduced serum and liver malondialdehyde concentrations and increased FBA excretion. However, we did not observe obvious influences of SOS and SOP on superoxide dismutase activities in the liver of HFD-fed rats. The combination of 2 % SOS and 3 % SOP showed a more marked effect than SOS or SOP alone in improving the lipid profile, release of vasoactive substances and increasing FBA excretion (P < 0.05). In summary, SOS and SOP might help prevent atherosclerosis through improving abnormal blood lipid levels, regulating vasoactive substances and protecting against oxidative stress.

  16. Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

    PubMed Central

    Sahu, Alexandria; Sorensen, Dylan; Minasov, George; Lima, Bruno P.; Scholle, Michael; Mrksich, Milan; Anderson, Wayne F.; Gibson, Bradford W.; Schilling, Birgit; Wolfe, Alan J.

    2014-01-01

    The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase. PMID:24756028

  17. The identification of oppA gene homologues as part of the oligopeptide transport system in mycoplasmas.

    PubMed

    Wium, Martha; Botes, Annelise; Bellstedt, Dirk U

    2015-03-01

    The lack of an annotated oppA gene as part of many oligopeptide permease (opp) operons has questioned the necessity of the oligopeptide-binding domain (OppA) as a part of the Opp transport system in mycoplasmas. This study investigated the occurrence of an oppA gene as part of the oppBCDF operon in 42 mycoplasma genomes. Except for hemoplasma, all mycoplasmas were found to possess one or more copies of the oppBCDF operon and with the help of similarity searches their oppA genes could be identified. Phylogenetic analysis of the combined OppABCDF amino acid sequences allowed them to be grouped into three types. Each type has a unique set of conserved motifs, which are likely to reflect substrate preference and adaption strategies. Our approach allowed the identification of oppA gene homologues for all mycoplasma opp operons and thereby provides a method for re-evaluating the current annotation of oppA genes in mycoplasma genomes. PMID:25528211

  18. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    PubMed

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.

  19. Fluorescent monitoring of copper-occupancy in His-ended catalytic oligo-peptides.

    PubMed

    Inokuchi, Reina; Kawano, Tomonori

    2016-01-01

    Controlled generation of reactive oxygen species (ROS) is widely beneficial to various medical, environmental, and agricultural studies. As inspired by the functional motifs in natural proteins, our group has been engaged in development of catalytically active oligo-peptides as minimum-sized metalloenzymes for generation of superoxide anion, an active member of ROS. In such candidate molecules, catalytically active metal-binding minimal motif was determined to be X-X-H, where X can be most amino acids followed by His. Based on above knowledge, we have designed a series of minimal copper-binding peptides designated as G n H series peptides, which are composed of oligo-glycyl chains ended with C-terminal His residue such as GGGGGH sequence (G5H). In order to further study the role of copper binding to the peptidic catalysts sharing the X-X-H motif such as G5H-conjugated peptides, we should be able to score the occupancy of the peptide population by copper ion in the reaction mixture. Here, model peptides with Cu-binding affinity which show intrinsic fluorescence due to tyrosyl residue (Y) in the UV region (excitation at ca. 230 and 280 nm, and emission at ca. 320 nm) were synthesized to score the effect of copper occupancy. Synthesized peptides include GFP-derived fluorophore sequence, TFSYGVQ (designated as Gfp), and Gfp sequence fused to C-terminal G5H (Gfp-G5H). In addition, two Y-containing tri-peptides derived from natural GFP fluorophores, namely, TYG and SYG were fused to the G5H (TYG-G5H and SYG-G5H). Conjugation of metal-binding G5H sequence to GFP-fluorophore peptide enhanced the action of Cu(2+) on quenching of intrinsic fluorescence due to Y residue. Two other Y-containing peptides, TYG-G5H and SYG-G5H, also showed intrinsic fluorescence which is sensitive to addition of Cu(2+). There was linear relationship between the loading of Cu(2+) and the quenching of fluorescence in these peptide, suggesting that Cu(2+)-dependent quenching of Y

  20. Fluorescent monitoring of copper-occupancy in His-ended catalytic oligo-peptides

    PubMed Central

    Inokuchi, Reina; Kawano, Tomonori

    2016-01-01

    ABSTRACT Controlled generation of reactive oxygen species (ROS) is widely beneficial to various medical, environmental, and agricultural studies. As inspired by the functional motifs in natural proteins, our group has been engaged in development of catalytically active oligo-peptides as minimum-sized metalloenzymes for generation of superoxide anion, an active member of ROS. In such candidate molecules, catalytically active metal-binding minimal motif was determined to be X-X-H, where X can be most amino acids followed by His. Based on above knowledge, we have designed a series of minimal copper-binding peptides designated as GnH series peptides, which are composed of oligo-glycyl chains ended with C-terminal His residue such as GGGGGH sequence (G5H). In order to further study the role of copper binding to the peptidic catalysts sharing the X-X-H motif such as G5H-conjugated peptides, we should be able to score the occupancy of the peptide population by copper ion in the reaction mixture. Here, model peptides with Cu-binding affinity which show intrinsic fluorescence due to tyrosyl residue (Y) in the UV region (excitation at ca. 230 and 280 nm, and emission at ca. 320 nm) were synthesized to score the effect of copper occupancy. Synthesized peptides include GFP-derived fluorophore sequence, TFSYGVQ (designated as Gfp), and Gfp sequence fused to C-terminal G5H (Gfp-G5H). In addition, two Y-containing tri-peptides derived from natural GFP fluorophores, namely, TYG and SYG were fused to the G5H (TYG-G5H and SYG-G5H). Conjugation of metal-binding G5H sequence to GFP-fluorophore peptide enhanced the action of Cu2+ on quenching of intrinsic fluorescence due to Y residue. Two other Y-containing peptides, TYG-G5H and SYG-G5H, also showed intrinsic fluorescence which is sensitive to addition of Cu2+. There was linear relationship between the loading of Cu2+ and the quenching of fluorescence in these peptide, suggesting that Cu2+-dependent quenching of Y

  1. Effect of condensation agents and minerals for oligopeptide formation under mild and hydrothermal conditions in related to chemical evolution of proteins

    NASA Astrophysics Data System (ADS)

    Kawamura, Kunio; Takeya, Hitoshi; Kushibe, Takao

    2009-07-01

    The role of condensation agents and minerals for oligopeptide formation was inspected to see whether minerals possess catalytic activity under mild and hydrothermal conditions. Under mild conditions, oligopeptide formation from negatively charged amino acids (Asp and Glu) using different minerals and the elongation of alanine oligopeptides ((Ala) 2-(Ala) 5) were attempted using apatite minerals. Oligo(Asp) up to 10 amino acid units from Asp were observed in the presence of 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC). Notable influence of minerals was not detected for the oligo(Asp) formation. Oligo(Asp) was gradually degraded by the further incubation in the presence of EDC in both the absence and presence of minerals. The formation of oligo(Glu) was less efficient in the presence of carbonyldiimidazole. The elongation from (Ala) 3, (Ala) 4, and (Ala) 5 and the formation of diketopiperazine from (Ala) 2 proceeded immediately in the presence of EDC in the meantime of the sample preparations. In addition, it was unexpected that the disappearance of the products and the reformation of the reactants occurred by the further incubation for 24 h; for instance, (Ala) 5 decreased but (Ala) 4 increased with increasing the reaction time in the reaction of (Ala) 4 with EDC. These facts suggest that the activation of the reactant amino acids or peptides immediately occurs. Under the simulated hydrothermal conditions, EDC did not enhance the formation of oligopeptides from Asp, Glu or Ala nor the spontaneous formation of (Ala) 5 from (Ala) 4.

  2. Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress

    PubMed Central

    Xu, Hong; Chen, Xuanyi; Xu, Xiaoli; Shi, Rongyi; Suo, Shasha; Cheng, Kaiying; Zheng, Zhiguo; Wang, Meixia; Wang, Liangyan; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2016-01-01

    Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions. PMID:27452117

  3. Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

    PubMed

    Xu, Hong; Chen, Xuanyi; Xu, Xiaoli; Shi, Rongyi; Suo, Shasha; Cheng, Kaiying; Zheng, Zhiguo; Wang, Meixia; Wang, Liangyan; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2016-01-01

    Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions. PMID:27452117

  4. Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection.

    PubMed

    Parker, Benjamin L; Shepherd, Nicholas E; Trefely, Sophie; Hoffman, Nolan J; White, Melanie Y; Engholm-Keller, Kasper; Hambly, Brett D; Larsen, Martin R; James, David E; Cordwell, Stuart J

    2014-09-12

    Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.

  5. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    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.

  6. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  7. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  8. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  9. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof 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.

  10. Histone deacetylase 3 indirectly modulates tubulin acetylation

    PubMed Central

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-01-01

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation. PMID:26450925

  11. H3K9 acetylation change patterns in rats after exposure to traffic-related air pollution.

    PubMed

    Ding, Rui; Jin, Yongtang; Liu, Xinneng; Zhu, Ziyi; Zhang, Yuan; Wang, Ting; Xu, Yinchun

    2016-03-01

    Traffic-related air pollution (TRAP) has been acknowledged as a potential risk factor for numerous respiratory disorders including lung cancer; however, the exact mechanisms involved are still unclear. Here we investigated the effects of TRAP exposure on the H3K9 acetylation in rats. The exposure was performed in both spring and autumn with identical study procedures. In each season, 48 healthy Wistar rats were exposed to different levels of TRAP for 4 h, 7 d, 14 d, and 28 d, respectively. H3K9 acetylation levels in both the peripheral blood mononuclear cells (PBMCs) and lung tissues were quantified. Multiple linear regression was applied to assess the influence of air pollutants on H3K9 acetylation levels. The levels of PM2.5, PM10, and NO2 in the tunnel and crossroad groups were significantly higher than in the control group. The H3K9 acetylation levels were not significantly different between spring and autumn. When spring and autumn data were analyzed together, no significant association between the TRAP and H3K9 acetylation was found in 4h exposure window. However, in the 7 d exposure window, PM2.5 and PM10 exposures were associated with changes in H3K9 acetylation ranging from 0.276 (0.053, 0.498) to 0.475 (0.103, 0.848) per 1 μg/m(3) increase in the pollutant concentration. In addition, prolonged exposure of the rats in the tunnel showed that both PM2.5 and PM10 concentrations were positively associated with H3k9 acetylation in both PBMCs and lung tissues. The findings showed that 7-d and prolonged TRAP exposure could effectively increase the H3K9 acetylation level in both PBMCs and lung tissues of rats.

  12. Property enhancement of optically transparent bionanofiber composites by acetylation

    NASA Astrophysics Data System (ADS)

    Nogi, Masaya; Abe, Kentaro; Handa, Keishin; Nakatsubo, Fumiaki; Ifuku, Shinsuke; Yano, Hiroyuki

    2006-12-01

    The authors studied acetylation of bacterial cellulose (BC) nanofibers to widen the applications of BC nanocomposites in optoelectronic devices. The slight acetylation of BC nanofibers significantly reduces the hygroscopicity of BC nanocomposites, while maintaining their high optical transparency and thermal stability. Furthermore, the degradation in optical transparency at elevated temperature (200°C) was significantly reduced by acetylation treatment. Therefore, the acetylation of bionanofibers has an extraordinary potential as treatment for property enhancement of bionanofiber composites.

  13. 40 CFR 721.10520 - Acetylated fatty acid glycerides (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Acetylated fatty acid glycerides... Specific Chemical Substances § 721.10520 Acetylated fatty acid glycerides (generic). (a) Chemical substance... acetylated fatty acid glycerides (PMN P-11-160) is subject to reporting under this section for...

  14. 40 CFR 721.10520 - Acetylated fatty acid glycerides (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Acetylated fatty acid glycerides... Specific Chemical Substances § 721.10520 Acetylated fatty acid glycerides (generic). (a) Chemical substance... acetylated fatty acid glycerides (PMN P-11-160) is subject to reporting under this section for...

  15. Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL.

    PubMed

    Jensen, Johanne M; Aduri, Nanda G; Prabhala, Bala K; Jahnsen, Rasmus; Franzyk, Henrik; Mirza, Osman

    2014-10-01

    Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguish between a dipeptide ligand and the corresponding dipeptide amide. The radically changed pH(Bulk) profiles of Lys117Gln and Lys117Arg mutants indicate an important role of Lys117 in facilitating protonation of the transporter; a notion that is supported by the close proximity of Lys117 to the conserved ExxERFxYY POT motif previously shown to be involved in proton translocation. These results point toward a novel dual role of Lys117 in direct or indirect interaction with both proton and peptide.

  16. Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL.

    PubMed

    Jensen, Johanne M; Aduri, Nanda G; Prabhala, Bala K; Jahnsen, Rasmus; Franzyk, Henrik; Mirza, Osman

    2014-10-01

    Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguish between a dipeptide ligand and the corresponding dipeptide amide. The radically changed pH(Bulk) profiles of Lys117Gln and Lys117Arg mutants indicate an important role of Lys117 in facilitating protonation of the transporter; a notion that is supported by the close proximity of Lys117 to the conserved ExxERFxYY POT motif previously shown to be involved in proton translocation. These results point toward a novel dual role of Lys117 in direct or indirect interaction with both proton and peptide. PMID:25261786

  17. Quantification of N-Acetyl Aspartyl Glutamate in Human Brain using Proton Magnetic Resonance Spectroscopy at 7 T

    NASA Astrophysics Data System (ADS)

    Elywa, M.

    2015-07-01

    The separation of N-acetyl aspartyl glutamate (NAAG) from N-acetyl aspartate (NAA) and other metabolites, such as glutamate, by in vivo proton magnetic resonance spectroscopy at 7 T is described. This method is based on the stimulated echo acquisition mode (STEAM), with short and long echo time (TE) and allows quantitative measurements of NAAG in the parietal and pregenual anterior cingulate cortex (pgACC) of human brain. Two basesets for the LCModel have been established using nuclear magnetic resonance simulator software (NMR-SIM). Six healthy volunteers (age 25-35 years) have been examined at 7 T. It has been established that NAAG can be separated and quantified in the parietal location and does not get quantified in the pgACC location when using a short echo time, TE = 20 ms. On the other hand, by using a long echo time, TE = 74 ms, NAAG can be quantified in pgACC structures.

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

    PubMed

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

    2000-01-01

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

  19. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    PubMed Central

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

  20. Fragrance material review on acetyl cedrene.

    PubMed

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

    2013-12-01

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

  1. Fragrance material review on acetyl carene.

    PubMed

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

    2013-12-01

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

  2. Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena

    PubMed Central

    1989-01-01

    In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly. PMID:2654136

  3. O-Acetylation of Plant Cell Wall Polysaccharides

    PubMed Central

    Gille, Sascha; Pauly, Markus

    2011-01-01

    Plant cell walls are composed of structurally diverse polymers, many of which are O-acetylated. How plants O-acetylate wall polymers and what its function is remained elusive until recently, when two protein families were identified in the model plant Arabidopsis that are involved in the O-acetylation of wall polysaccharides – the reduced wall acetylation (RWA) and the trichome birefringence-like (TBL) proteins. This review discusses the role of these two protein families in polysaccharide O-acetylation and outlines the differences and similarities of polymer acetylation mechanisms in plants, fungi, bacteria, and mammals. Members of the TBL protein family had been shown to impact pathogen resistance, freezing tolerance, and cellulose biosynthesis. The connection of TBLs to polysaccharide O-acetylation thus gives crucial leads into the biological function of wall polymer O-acetylation. From a biotechnological point understanding the O-acetylation mechanism is important as acetyl-substituents inhibit the enzymatic degradation of wall polymers and released acetate can be a potent inhibitor in microbial fermentations, thus impacting the economic viability of, e.g., lignocellulosic based biofuel production. PMID:22639638

  4. Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

    PubMed Central

    Yang, Haiquan; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Chen, Jian

    2013-01-01

    In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The kcat/Km was increased from 1.0 × 105 liters · mol−1 · s−1 for AmyK to 30.6 × and 23.2 × 105 liters · mol−1 · s−1 for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency. PMID:23956385

  5. Wireless quantified reflex device

    NASA Astrophysics Data System (ADS)

    Lemoyne, Robert Charles

    The deep tendon reflex is a fundamental aspect of a neurological examination. The two major parameters of the tendon reflex are response and latency, which are presently evaluated qualitatively during a neurological examination. The reflex loop is capable of providing insight for the status and therapy response of both upper and lower motor neuron syndromes. Attempts have been made to ascertain reflex response and latency, however these systems are relatively complex, resource intensive, with issues of consistent and reliable accuracy. The solution presented is a wireless quantified reflex device using tandem three dimensional wireless accelerometers to obtain response based on acceleration waveform amplitude and latency derived from temporal acceleration waveform disparity. Three specific aims have been established for the proposed wireless quantified reflex device: 1. Demonstrate the wireless quantified reflex device is reliably capable of ascertaining quantified reflex response and latency using a quantified input. 2. Evaluate the precision of the device using an artificial reflex system. 3.Conduct a longitudinal study respective of subjects with healthy patellar tendon reflexes, using the wireless quantified reflex evaluation device to obtain quantified reflex response and latency. Aim 1 has led to the steady evolution of the wireless quantified reflex device from a singular two dimensional wireless accelerometer capable of measuring reflex response to a tandem three dimensional wireless accelerometer capable of reliably measuring reflex response and latency. The hypothesis for aim 1 is that a reflex quantification device can be established for reliably measuring reflex response and latency for the patellar tendon reflex, comprised of an integrated system of wireless three dimensional MEMS accelerometers. Aim 2 further emphasized the reliability of the wireless quantified reflex device by evaluating an artificial reflex system. The hypothesis for aim 2 is that

  6. Preparation, physicochemical characterization and application of acetylated lotus rhizome starches.

    PubMed

    Sun, Suling; Zhang, Ganwei; Ma, Chaoyang

    2016-01-01

    Acetylated lotus rhizome starches were prepared, physicochemically characterized and used as food additives in puddings. The percentage content of the acetyl groups and degree of substitution increased linearly with the amount of acetic anhydride used. The introduction of acetyl groups was confirmed via Fourier transform infrared (FT-IR) spectroscopy. The values of the pasting parameters were lower for acetylated starch than for native starch. Acetylation was found to increase the light transmittance (%), the freeze-thaw stability, the swelling power and the solubility of the starch. Sensorial scores for puddings prepared using native and acetylated lotus rhizome starches as food additives indicated that puddings produced from the modified starches with superior properties over those prepared from native starch. PMID:26453845

  7. 2-Acetyl­pyridinium bromanilate

    PubMed Central

    Thomas, Lynne H.; Boyle, Bryan; Clive, Lesley A.; Collins, Anna; Currie, Lynsey D.; Gogol, Malgorzata; Hastings, Claire; Jones, Andrew O. F.; Kennedy, Jennifer L.; Kerr, Graham B.; Kidd, Alastair; Lawton, Lorreta M.; Macintyre, Susan J.; MacLean, Niall M.; Martin, Alan R. G.; McGonagle, Kate; Melrose, Samantha; Rew, Gaius A.; Robinson, Colin W.; Schmidtmann, Marc; Turnbull, Felicity B.; Williams, Lewis G.; Wiseman, Alan Y.; Wocial, Malgorzata H.; Wilson, Chick C.

    2009-01-01

    In the crystal of the title mol­ecular salt (systematic name: 2-acetyl­pyridinium 2,5-dibromo-4-hydr­oxy-3,6-dioxocyclo­hexa-1,4-dienolate), C7H8NO+·C6HBr2O4 −, centrosymmetric rings consisting of two cations and two anions are formed, with the components linked by alternating O—H⋯O and N—H⋯O hydrogen bonds. Short O⋯Br contacts [3.243 (2) and 3.359 (2) Å] may help to consolidate the packing. PMID:21583087

  8. Survey of the human acetylator polymorphism in spontaneous disorders.

    PubMed Central

    Evans, D A

    1984-01-01

    There is ample evidence that the human acetylator phenotypes are associated with drug induced phenomena. It is principally the slow acetylators who exhibit toxic adverse effects because of their relative inability to detoxify the original drug compounds. In rare instances, however, it is the rapid acetylators who are at a disadvantage. In the matter of association of spontaneous disease with either acetylator phenotype, there are two groups of disorders to consider. First, disorders in which carcinogenic amines are known to be an aetiological factor. This is because these amines are substrates for the polymorphic N-acetyltransferase activity and hence there is a possible rational basis for searching for an association. Secondly, other disorders where searches for associations are based more on hunches. In the first group there is a definite statistical association between cancer of the bladder and the slow acetylator phenotype. In prevalence studies the slow phenotype is 39% more associated with bladder cancer than is the rapid phenotype. On the basis of the evidence now available it is not possible to say whether this association is because slow acetylators develop the disease more frequently or whether they survive longer. In the second group the relevant studies show (1) a greatly increased prevalence of slow acetylators in Gilbert's disease; (2) a confirmed association between the rapid acetylator phenotype and diabetes; (3) a possible association between the rapid acetylator phenotype and breast cancer; (4) a possible association between the slow acetylator phenotype and leprosy in Chinese patients; (5) an earlier age of onset of thyrotoxicosis (Graves' disease) in slow acetylators than in rapid acetylators; (6) no evidence of an association between either phenotype and spontaneous systemic lupus erythematosus. PMID:6387123

  9. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  10. Determination of Acetylation of the Gli Transcription Factors.

    PubMed

    Coni, Sonia; Di Magno, Laura; Canettieri, Gianluca

    2015-01-01

    The Gli transcription factors (Gli1, Gli2, and Gli3) are the final effectors of the Hedgehog (Hh) signaling and play a key role in development and cancer. The activity of the Gli proteins is finely regulated by covalent modifications, such as phosphorylation, ubiquitination, and acetylation. Both Gli1 and Gli2 are acetylated at a conserved lysine, and this modification causes the inhibition of their transcriptional activity. Thus, the acetylation status of these proteins represents a useful marker to monitor Hh activation in pathophysiological conditions. Herein we describe the techniques utilized to detect in vitro and intracellular acetylation of the Gli transcription factors. PMID:26179046

  11. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  12. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+) T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+) T cell immunity.

  13. Multi-responsive Hydrogels Derived from the Self-assembly of Tethered Allyl-functionalized Racemic Oligopeptides

    PubMed Central

    He, Xun; Fan, Jingwei; Zhang, Fuwu; Li, Richen; Pollack, Kevin A.; Raymond, Jeffery E.; Zou, Jiong; Wooley, Karen L.

    2014-01-01

    A multi-responsive triblock hydrogelator oligo(dl-allylglycine)-block-poly(ethylene glycol)-block-oligo(dl-allylglycine) (ODLAG-b-PEG-b-ODLAG) was synthesized facilely by ring-opening polymerization (ROP) of DLAG N-carboxyanhydride (NCA) with a diamino-terminated PEG as the macroinitiator. This system exhibited heat-induced sol-to-gel transitions and either sonication- or enzyme-induced gel-to-sol transitions. The β-sheeting of the oligopeptide segments was confirmed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and wide-angle X-ray scattering (WAXS). The β-sheets further displayed tertiary ordering into fibrillar structures that, in turn generated a porous and interconnected hydrogel matrix, as observed via transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The reversible macroscopic sol-to-gel transitions triggered by heat and gel-to-sol transitions triggered by sonication were correlated with the transformation of nanostructural morphologies, with fibrillar structures observed in gel and spherical aggregates in sol, respectively. The enzymatic breakdown of the hydrogels was also investigated. This allyl-functionalized hydrogelator can serve as a platform for the design of smart hydrogels, appropriate for expansion into biological systems as bio-functional and bio-responsive materials. PMID:25485113

  14. Isolation and purification of oligopeptides from Ruditapes philippinarum and its inhibition on the growth of DU‑145 cells in vitro.

    PubMed

    Yang, Zuisu; Zhao, Yuqin; Yan, Haiqiang; Xu, Lv; Ding, Guofang; Yu, Di; Sun, Yu

    2015-02-01

    Ruditapes philippinarum is a member of the Veneridae family of marine bivalve molluscs. RPOI‑1 (Ruditapes philippinarum oligopeptide) is a tetrapeptide that can be extracted from Ruditapes philippinarum by means of enzymolysis. This study showed that RPOI‑1 strongly inhibits proliferation and induces apoptosis in DU‑145 human prostate cancer cells. When cells were treated with varying concentrations of RPOI‑1, significant inhibition of proliferation was detected by an MTT assay, and sub‑G1 and G2/M phase cell cycle arrest was observed using flow cytometric (FCM) analysis. Furthermore, morphological changes characteristic of apoptosis and an increase in the proportion of apoptotic cells were observed using double sequential acridine orange/ethidium bromide staining, FCM analysis and transmission election microscopy. FCM studies showed that exposing DU‑145 cells to 10, 20 and 30 mg/ml RPOI‑1 for 24 h increased the percentage of cells in the early‑stages of apoptotis in a dose‑dependent manner, with the numbers rising from 3.01% in the control group to 13.40% in the group treated with the highest dose.

  15. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    PubMed

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-01-01

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  16. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    PubMed

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-01-01

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data. PMID:27092480

  17. Sea cucumber (Codonopsis pilosula) oligopeptides: immunomodulatory effects based on stimulating Th cells, cytokine secretion and antibody production.

    PubMed

    He, Li-Xia; Zhang, Zhao-Feng; Sun, Bin; Chen, Qi-He; Liu, Rui; Ren, Jin-Wei; Wang, Jun-Bo; Li, Yong

    2016-02-01

    This study aimed to investigate the immunomodulating activity of small molecule oligopeptides from sea cucumber (Codonopsis pilosula) (SOP) in mice. Seven assays were performed to determine the immunomodulatory effects, including splenic lymphocyte proliferation and delayed-type hypersensitivity assays (cell-mediated immunity), IgM antibody response of spleen to sheep red blood cells (SRBC) and serum hemolysin level assays (humoral immunity), the carbon clearance assay and the phagocytic capacity of peritoneal cavity phagocytes assay (macrophage phagocytosis), and the NK cell activity assay. Spleen T lymphocyte subpopulations, multiplex sandwich immunoassays of serum cytokine and immunoglobulin levels and enzyme-linked immunosorbent assays for small intestinal secretory immunoglobulin were performed to study the mechanism by which SOP affects the immune system. We found that SOP could improve immune functions in mice, which may be due to the enhancement of the functions of cell-mediated immunity, humoral immunity, macrophage phagocytosis and NK cell activity. From the cellular and molecular assays, we postulated that the immunomodulatory effects are most likely attributed to the stimulation of Th cells, cytokine secretion and antibody production. PMID:26838796

  18. Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production.

    PubMed

    Podbielski, A; Pohl, B; Woischnik, M; Körner, C; Schmidt, K H; Rozdzinski, E; Leonard, B A

    1996-09-01

    Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.

  19. The Oligopeptide Permease Opp Mediates Illicit Transport of the Bacterial P-site Decoding Inhibitor GE81112 †

    PubMed Central

    Maio, Alessandro; Brandi, Letizia; Donadio, Stefano; Gualerzi, Claudio O.

    2016-01-01

    GE81112 is a tetrapeptide antibiotic that binds to the 30S ribosomal subunit and specifically inhibits P-site decoding of the mRNA initiation codon by the fMet-tRNA anticodon. GE81112 displays excellent microbiological activity against some Gram-positive and Gram-negative bacteria in both minimal and complete, chemically defined, broth, but is essentially inactive in complete complex media. This is due to the presence of peptides that compete with the antibiotic for the oligopeptide permease system (Opp) responsible for its illicit transport into the bacterial cells as demonstrated in the cases of Escherichia coli and Bacillus subtilis. Mutations that inactivate the Opp system and confer GE81112 resistance arise spontaneously with a frequency of ca. 1 × 10−6, similar to that of the mutants resistant to tri-l-ornithine, a known Opp substrate. On the contrary, cells expressing extrachromosomal copies of the opp genes are extremely sensitive to GE81112 in rich medium and GE81112-resistant mutations affecting the molecular target of the antibiotic were not detected upon examining >109 cells of this type. However, some mutations introduced in the 16S rRNA to confer kasugamycin resistance were found to reduce the sensitivity of the cells to GE81112. PMID:27231947

  20. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  1. Lysine Acetylation Activates Mitochondrial Aconitase in the Heart

    PubMed Central

    Fernandes, Jolyn; Weddle, Alexis; Kinter, Caroline S.; Humphries, Kenneth M.; Mather, Timothy; Szweda, Luke I.; Kinter, Michael

    2015-01-01

    High throughput proteomics studies have identified several thousand acetylation sites on over one thousand proteins. Mitochondrial aconitase, the Krebs cycle enzyme that converts citrate to isocitrate, has been identified in many of these reports. Acetylated mitochondrial aconitase has also been identified as a target for sirtuin 3 (SIRT3) catalyzed deacetylation. However, the functional significance of mitochondrial aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of obesity, we found a significant acetylation-dependent activation of aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either acetic anhydride or acetyl-CoA resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and found significant increases with both in vitro treatments. A high fat diet (60% kcal from fat) was used as an in vivo model and also showed significantly increased mitochondrial aconitase activity without changes in protein level. The high fat diet also produced increased aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with SIRT3 abolished the high fat diet-induced activation of aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the enzyme. The results of this study reveal a novel activation of mitochondrial aconitase by acetylation. PMID:26061789

  2. Catalysis: Quantifying charge transfer

    NASA Astrophysics Data System (ADS)

    James, Trevor E.; Campbell, Charles T.

    2016-02-01

    Improving the design of catalytic materials for clean energy production requires a better understanding of their electronic properties, which remains experimentally challenging. Researchers now quantify the number of electrons transferred from metal nanoparticles to an oxide support as a function of particle size.

  3. Quantifying Faculty Workloads.

    ERIC Educational Resources Information Center

    Archer, J. Andrew

    Teaching load depends on many variables, however most colleges define it strictly in terms of contact or credit hours. The failure to give weight to variables such as number of preparations, number of students served, committee and other noninstructional assignments is usually due to the lack of a formula that will quantify the effects of these…

  4. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation

    PubMed Central

    Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies. PMID:27606599

  5. Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

    PubMed

    Tahir, I M; Iqbal, T; Saleem, S; Mehboob, H; Akhter, N; Riaz, M

    2016-03-01

    The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n=19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P<0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P<0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P<0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies.

  6. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

    PubMed

    Kim, Sun-Yee; Sim, Choon Kiat; Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.

  7. Global analysis of lysine acetylation in strawberry leaves.

    PubMed

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants. PMID:26442052

  8. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L... section. The minimum amount of the additive to achieve the desired effect must be used, and the...

  9. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

    PubMed

    Kim, Sun-Yee; Sim, Choon Kiat; Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies. PMID:27606599

  10. A facile and practical synthesis of N-acetyl enamides.

    PubMed

    Tang, Wenjun; Capacci, Andrew; Sarvestani, Max; Wei, Xudong; Yee, Nathan K; Senanayake, Chris H

    2009-12-18

    A facile and practical method for the synthesis of N-acetyl alpha-arylenamides has been developed from corresponding ketoximes as the starting materials with ferrous acetate as the reducing reagent. This methodology offers mild reaction conditions, simple purification procedures, and high yields for a variety of N-acetyl enamides. PMID:19921804

  11. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  12. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  13. Study on Dendrobium officinale O-acetyl-glucomannan (Dendronan®): part II. Fine structures of O-acetylated residues.

    PubMed

    Xing, Xiaohui; Cui, Steve W; Nie, Shaoping; Phillips, Glyn O; Goff, H Douglas; Wang, Qi

    2015-03-01

    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-β-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: β-D-mannopyranose; G: β-D-glucopyranose; a: O-acetyl group.

  14. Cell biology (Communication arising): Tubulin acetylation and cell motility

    NASA Astrophysics Data System (ADS)

    Palazzo, Alexander; Ackerman, Brian; Gundersen, Gregg G.

    2003-01-01

    Although the protein tubulin is known to undergo several post-translational modifications that accumulate in stable but not dynamic microtubules inside cells, the function of these modifications is unknown. Hubbert et al. have shown that the enzyme HDAC6 (for histone deacetylase 6) reverses the post-translational acetylation of tubulin, and provide evidence that reducing tubulin acetylation enhances cell motility. They also suggest that decreasing tubulin acetylation reduces microtubule stability. However, we find that microtubule stabilization is not promoted by tubulin acetylation. We conclude that the alteration in cell motility observed by Hubbert et al. in cells overexpressing HDAC6 results not from changes in the formation of stable microtubules, but from alterations in the degree of tubulin acetylation.

  15. Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

    PubMed

    Nie, Zuoming; Zhu, Honglin; Zhou, Yong; Wu, Chengcheng; Liu, Yue; Sheng, Qing; Lv, Zhengbing; Zhang, Wenping; Yu, Wei; Jiang, Caiying; Xie, Longfei; Zhang, Yaozhou; Yao, Juming

    2015-09-01

    Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.

  16. Quantifying Ubiquitin Signaling

    PubMed Central

    Ordureau, Alban; Münch, Christian; Harper, J. Wade

    2015-01-01

    Ubiquitin (UB)-driven signaling systems permeate biology, and are often integrated with other types of post-translational modifications (PTMs), most notably phosphorylation. Flux through such pathways is typically dictated by the fractional stoichiometry of distinct regulatory modifications and protein assemblies as well as the spatial organization of pathway components. Yet, we rarely understand the dynamics and stoichiometry of rate-limiting intermediates along a reaction trajectory. Here, we review how quantitative proteomic tools and enrichment strategies are being used to quantify UB-dependent signaling systems, and to integrate UB signaling with regulatory phosphorylation events. A key regulatory feature of ubiquitylation is that the identity of UB chain linkage types can control downstream processes. We also describe how proteomic and enzymological tools can be used to identify and quantify UB chain synthesis and linkage preferences. The emergence of sophisticated quantitative proteomic approaches will set a new standard for elucidating biochemical mechanisms of UB-driven signaling systems. PMID:26000850

  17. Specific Oligopeptides in Fermented Soybean Extract Inhibit NF-κB-Dependent iNOS and Cytokine Induction by Toll-Like Receptor Ligands

    PubMed Central

    Lee, Woo Hyung; Wu, Hong Min; Lee, Chan Gyu; Sung, Dae Il; Song, Hye Jung; Matsui, Toshiro

    2014-01-01

    Abstract The ethanol extract of fermented soybean from Glycine max (chungkookjang, CHU) has been claimed to have chemopreventive and cytoprotective effects. In the present study, we examined the inhibitory effect of CHU on inducible nitric oxide synthase (iNOS) and cytokine induction by toll-like receptor (TLR) ligands treatment and attempted to identify the responsible active components. Nitric oxide (NO) content and iNOS levels in the media or RAW264.7 cells were measured using the Griess reagent and real-time polymerase chain reaction assays. CHU treatment inhibited NO production and iNOS induction elicited by lipopolysaccharide (LPS, TLR4L) in a concentration-dependent manner. Tumor necrosis factor-α and interleukin-6 productions were also diminished. Peptidoglycans (TLR2/6L) and CpG-oligodeoxynucleotides (TLR9L) from CHU inhibited iNOS induction, but not poly I:C (TLR3L) or loxoribine (TLF7L). The anti-inflammatory effect resulted from the inhibition of nuclear factor-kappa B (NF-κB) through the inhibition of inhibitory-κB degradation. Of the representative components in CHU, specific oligopeptides (AFPG and GVAWWMY) had the ability to inhibit iNOS induction by LPS, whereas others failed to do so. Daidzein, an isoflavone used for comparative purposes, was active at a relatively higher concentration. In an animal model, oral administration of CHU to rats significantly diminished carrageenan-induced paw edema and iNOS induction. Our results demonstrate that CHU has anti-inflammatory effects against TLR ligands by inhibiting NF-κB activation, which may result from specific oligopeptide components in CHU. Since CHU is orally effective, dietary applications of CHU and/or the identified oligopeptides may be of use in the prevention of inflammatory diseases. PMID:25184943

  18. Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

    PubMed Central

    Jones, Megan M.; Johnson, Antoinette; Koszelak-Rosenblum, Mary; Kirkham, Charmaine; Brauer, Aimee L.; Malkowski, Michael G.

    2014-01-01

    Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract. PMID:25156736

  19. Proton-coupled oligopeptide transporter (POT) family expression in human nasal epithelium and their drug transport potential.

    PubMed

    Agu, Remigius; Cowley, Elizabeth; Shao, Di; Macdonald, Christopher; Kirkpatrick, David; Renton, Ken; Massoud, Emad

    2011-06-01

    The molecular and functional expression of peptide transporters (PEPT1 and PEPT2, PHT1, PHT2) in human nasal epithelium was investigated. Quantitative/reverse transcriptase polymerase chain reaction (qPCR/RT-PCR), Western blotting and indirect immuno-histochemistry were used to investigate the functional gene and protein expression for the transporters. Uptake and transport studies were performed using metabolically stable peptides [β-alanyl-L-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) and β-alanyl-L-histidine (carnosine)]. The effects of concentration, temperature, polarity, competing peptides, and inhibitors on peptide uptake and transport were investigated. PCR products corresponding to PEPT1 (150 bp), PEPT2 (127 bp), PHT1 (110 bp) and PHT2 (198 bp) were detected. Immunohistochemistry and Western blotting confirmed the functional expression of PEPT1 and PEPT2 genes. The uptake of β-Ala-Lys-AMCA was concentration-dependent and saturable (Vmax =4.1 ( 0.07 μmol/min/mg protein, Km = 0.6 ( 0.07 μM). The optimal pH for intracellular accumulation of β-Ala-Lys-AMCA was 6.5. Whereas dipeptides and carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited peptide uptake and transport, L-Phe had no effect on peptide transport. The permeation of β-alanyl-L-histidine was concentration-, direction-, and temperature-dependent. The uptake, permeation, qPCR/RT-PCR and protein expression data showed that the human nasal epithelium functionally expresses proton-coupled oligopeptide transporters.

  20. Interaction of the Hydrophobic Tip of an Atomic Force Microscope with Oligopeptides Immobilized Using Short and Long Tethers.

    PubMed

    Ma, C Derek; Acevedo-Vélez, Claribel; Wang, Chenxuan; Gellman, Samuel H; Abbott, Nicholas L

    2016-03-29

    We report an investigation of the adhesive force generated between the hydrophobic tip of an atomic force microscope (AFM) and surfaces presenting oligopeptides immobilized using either short (∼1 nm) or long (∼60 nm) tethers. Specifically, we used either sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) or 10 kDa polyethylene glycol (PEG) end-functionalized with maleimide and N-hydroxysuccinimide groups to immobilize helical oligomers of β-amino acids (β-peptides) to mixed monolayers presenting tetraethylene glycol (EG4) and amine-terminated EG4 (EG4N) groups. When SSMCC was used to immobilize the β-peptides, we measured the adhesive interaction between the AFM tip and surface to rupture through a single event with magnitude consistent with the interaction of a single β-peptide with the AFM tip. Surprisingly, this occurred even when, on average, multiple β-peptides were located within the interaction area between the AFM tip and surface. In contrast, when using the long 10 kDa PEG tether, we observed the magnitude of the adhesive interaction as well as the dynamics of the rupture events to unmask the presence of the multiple β-peptides within the interaction area. To provide insight into these observations, we formulated a simple mechanical model of the interaction of the AFM tip with the immobilized β-peptides and used the model to demonstrate that adhesion measurements performed using short tethers (but not long tethers) are dominated by the interaction of single β-peptides because (i) the mechanical properties of the short tether are highly nonlinear, thus causing one β-peptide to dominate the adhesion force at the point of rupture, and (ii) the AFM cantilever is mechanically unstable following the rupture of the adhesive interaction with a single β-peptide. Overall, our study reveals that short tethers offer the basis of an approach that facilitates measurement of adhesive interactions with single molecules presented at

  1. Postmortem tissue distribution of acetyl fentanyl, fentanyl and their respective nor-metabolites analyzed by ultrahigh performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Poklis, Justin; Poklis, Alphonse; Wolf, Carl; Mainland, Mary; Hair, Laura; Devers, Kelly; Chrostowski, Leszek; Arbefeville, Elise; Merves, Michele; Pearson, Julia

    2015-12-01

    In the last two years, an epidemic of fatal narcotic overdose cases has occurred in the Tampa area of Florida. Fourteen of these deaths involved fentanyl and/or the new designer drug, acetyl fentanyl. Victim demographics, case histories, toxicology findings and causes and manners of death, as well as, disposition of fentanyl derivatives and their nor-metabolites in postmortem heart blood, peripheral blood, bile, brain, liver, urine and vitreous humor are presented. In the cases involving only acetyl fentanyl (without fentanyl, n=4), the average peripheral blood acetyl fentanyl concentration was 0.467 mg/L (range 0.31 to 0.60 mg/L) and average acetyl norfentanyl concentration was 0.053 mg/L (range 0.002 to 0.086 mg/L). In the cases involving fentanyl (without acetyl fentanyl, n=7), the average peripheral blood fentanyl concentration was 0.012 mg/L (range 0.004 to 0.027 mg/L) and average norfentanyl blood concentration was 0.001 mg/L (range 0.0002 to 0.003 mg/L). In the cases involving both acetyl fentanyl and fentanyl (n=3), the average peripheral blood acetyl fentanyl concentration was 0.008 mg/L (range 0.006 to 0.012 mg/L), the average peripheral blood acetyl norfentanyl concentration was 0.001 mg/L (range 0.001 to 0.002 mg/L), the average peripheral blood fentanyl concentration was 0.018 mg/L (range 0.015 to 0.021mg/L) and the average peripheral blood norfentanyl concentration was 0.002 mg/L (range 0.001 mg/L to 0.003 mg/L). Based on the toxicology results, it is evident that when fentanyl and/or acetyl fentanyl were present, they contributed to the cause of death. A novel ultrahigh performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) method to identify and quantify acetyl fentanyl, acetyl norfentanyl, fentanyl and norfentanyl in postmortem fluids and tissues is also presented.

  2. Microbial acetyl conjugation of T-2 toxin and its derivatives.

    PubMed Central

    Yoshizawa, T; Onomoto, C; Morooka, N

    1980-01-01

    The acetyl conjugation of T-2 toxin and its derivatives, the 12,13-epoxytrichothecene mycotoxins, was studied by using mycelia of trichothecene-producing strains of Fusarium graminearum, F. nivale, Calonectria nivalis, and F. sporotrichoides, T-2 toxin was efficiently converted into acetyl T-2 toxin by all strains except a T-2 toxin-producing strain of F. sporotrichoides, which hydrolyzed the substrate to HT-2-toxin and neosolaniol. HT-2 toxin was conjugated to 3-acetyl HT-2 toxin as an only product by mycelia of F. graminearum and C. nivalis, but was also resistant to conjugation by both F. nivale and F. sporotrichoides. Neosolaniol was also biotransformed selectively into 3-acetyl neosolaniol by F. graminearum. However, 3-acetyl HT-2 toxin was not acetylated by any of the strains under the conditions employed, but was hydrolyzed to HT-2 toxin by F. graminearum and F. nivale. This is the first report on the biological 3 alpha-O-acetyl conjugation of T-2 toxin and its derivatives. PMID:7396487

  3. Chitosan Molecular Structure as a Function of N-Acetylation

    SciTech Connect

    Franca, Eduardo F.; Freitas, Luiz C.; Lins, Roberto D.

    2011-07-01

    Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with f and w values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3- HO3(n) ... O5(n+ 1) hydrogen bond, which in turn controls particle aggregation.

  4. Quantifying light pollution

    NASA Astrophysics Data System (ADS)

    Cinzano, P.; Falchi, F.

    2014-05-01

    In this paper we review new available indicators useful to quantify and monitor light pollution, defined as the alteration of the natural quantity of light in the night environment due to introduction of manmade light. With the introduction of recent radiative transfer methods for the computation of light pollution propagation, several new indicators become available. These indicators represent a primary step in light pollution quantification, beyond the bare evaluation of the night sky brightness, which is an observational effect integrated along the line of sight and thus lacking the three-dimensional information.

  5. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

    PubMed

    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

    2015-01-01

    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

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

    PubMed

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

    2016-10-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. PMID:27402865

  7. Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

    PubMed Central

    Dupont, C; Clarke, A J

    1991-01-01

    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

  8. Sulfonate group-modified FePtCu nanoparticles as a selective probe for LDI-MS analysis of oligopeptides from a peptide mixture and human serum proteins.

    PubMed

    Kawasaki, Hideya; Akira, Tarui; Watanabe, Takehiro; Nozaki, Kazuyoshi; Yonezawa, Tetsu; Arakawa, Ryuichi

    2009-11-01

    Bare FePtCu nanoparticles (NPs) are first prepared for laser desorption/ionization mass spectroscopy (LDI-MS) analysis as affinity probes to selectively trap oppositely charged analytes from a sample solution. Our present results demonstrate bare FePtCu NPs to be a potentially useful matrix for surface-assisted laser desorption/ionization mass spectroscopy (SALDI-MS), for the analysis of small proteins and peptides. The upper detectable mass range of peptides was approximately 5 kDa, and the detection limit for peptides approximately 5 fmol. Sulfonate group-modified FePtCu nanoparticles (FePtCu-SO(3)(-) NPs), with ionization being independent of the solution pH, can interact with a positively charged analyte, and the analyte-bound NPs can be separated from the reaction supernatant by centrifugation or an external magnetic field. An oligopeptide, Gly-Gly-Tyr-Arg (GGYR) from an oligopeptide mixture containing Asp-Asp-Asp-Asp (DDDD), Gly-Gly-Gly-Gly (GGGG) and GGYR, was detected using SALDI-MS with FePtCu-SO(3)(-) NPs employing electrostatic interaction. Furthermore, FePtCu-SO(3)(-) NPs can detect lysozyme (Lyz) in human serum through the electrostatic attraction between positively charged Lyz and FePtCu-SO(3)(-) NPs at pH 8, while detection of negatively charged albumin in human serum is not possible.

  9. Staphylococcus aureus NorD, a Putative Efflux Pump Coregulated with the Opp1 Oligopeptide Permease, Contributes Selectively to Fitness In Vivo

    PubMed Central

    Ding, Yanpeng; Fu, Yingmei; Lee, Jean C.

    2012-01-01

    Staphylococcus aureus readily infects humans, causing infections from mild superficial skin infections to lethal bacteremia and endocarditis. Transporters produced by S. aureus allow the pathogen to adapt to a variety of settings, including survival at sites of infection and in the presence of antibiotics. The native functions of many transporters are unknown, but their potential dual contribution to fitness and antimicrobial resistance highlights their importance in staphylococcal infections. Here, we show that S. aureus NorD, a newly recognized efflux pump of the major facilitator superfamily, contributes to fitness in a murine subcutaneous abscess model. In community-associated methicillin-resistant S. aureus (CA-MRSA) strain MW2, norD was selectively upregulated 36-fold at the infection site relative to growth in vitro, and the norD mutant demonstrated significant fitness impairment in abscesses, with fitness 20- to 40-fold lower than that of the parent MW2 strain. Plasmid-encoded NorD could complement the fitness defect of the MW2 norD mutant. Chromosomal norD expression is polycistronic with the upstream oligopeptide permease genes (opp1ABCDF), which encode an ABC oligopeptide transporter. Both norD and opp1 were upregulated in abscesses and iron-restricted culture medium and negatively regulated by Fur, but only NorD contributed to fitness in the murine abscess model. PMID:23042988

  10. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    EPA Science Inventory

    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 ...

  11. Acetylation of C/EBPα inhibits its granulopoietic function

    PubMed Central

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S.; Numata, Akihiko; Sárosi, Menyhárt B.; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K.; Gunaratne, Jayantha; Tenen, Daniel G.

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  12. Acetylation of banana fibre to improve oil absorbency.

    PubMed

    Teli, M D; Valia, Sanket P

    2013-01-30

    Oil spill leaves detrimental effects on the environment, living organisms and economy. In the present work, an attempt is made to provide an efficient, easily deployable method of cleaning up oil spills and recovering of the oil. The work reports the use of banana fibres which were acetylated for oil spill recovery. The product so formed was characterized by FT-IR, TG, SEM and its degree of acetylation was also evaluated. The extent of acetylation was measured by weight percent gain. The oil sorption capacity of the acetylated fibre was higher than that of the commercial synthetic oil sorbents such as polypropylene fibres as well as un-modified fibre. Therefore, these oil sorption-active materials which are also biodegradable can be used to substitute non-biodegradable synthetic materials in oil spill cleanup. PMID:23218302

  13. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

    Aslan, Joseph E.; David, Larry L.; McCarty, Owen J.T.

    2015-01-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification – mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  14. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  15. On quantifying insect movements

    SciTech Connect

    Wiens, J.A.; Crist, T.O. ); Milne, B.T. )

    1993-08-01

    We elaborate on methods described by Turchin, Odendaal Rausher for quantifying insect movement pathways. We note the need to scale measurement resolution to the study insects and the questions being asked, and we discuss the use of surveying instrumentation for recording sequential positions of individuals on pathways. We itemize several measures that may be used to characterize movement pathways and illustrate these by comparisons among several Eleodes beetles occurring in shortgrass steppe. The fractal dimension of pathways may provide insights not available from absolute measures of pathway configuration. Finally, we describe a renormalization procedure that may be used to remove sequential interdependence among locations of moving individuals while preserving the basic attributes of the pathway.

  16. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    PubMed Central

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  17. Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin.

    PubMed

    Ralet, Marie-Christine; Crépeau, Marie-Jeanne; Bonnin, Estelle

    2008-06-01

    Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.

  18. A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.

    PubMed

    Petrovas, C J; Vlachoyiannopoulos, P G; Tzioufas, A G; Alexopoulos, C; Tsikaris, V; Sakarellos-Daitsiotis, M; Sakarellos, C; Moutsopoulos, H M

    1998-11-01

    A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for

  19. Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation*

    PubMed Central

    Wang, Yun; Kavran, Jennifer M.; Chen, Zan; Karukurichi, Kannan R.; Leahy, Daniel J.; Cole, Philip A.

    2014-01-01

    S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. PMID:25248746

  20. Quantitative determination of sulfisoxazole and its three N-acetylated metabolites using HPLC-MS/MS, and the saturable pharmacokinetics of sulfisoxazole in mice.

    PubMed

    Oh, Kyungsoo; Baek, Moon-Chang; Kang, Wonku

    2016-09-10

    Sulfisoxazole (SFX) is still used in combination with trimethoprim in cattle despite adverse drug reactions (e.g., urolithiasis). Recently, SFX is known to be a promising repositioned drug candidate for pulmonary hypertension and cancer. We developed a simultaneous determination method of SFX and its N-acetylated metabolites (N(1)-acetyl SFX, N1AS; N(4)-acetyl SFX, N4AS; diacetyl SFX, DAS) using HPLC-MS/MS for the first time, and examined the pharmacokinetics of SFX in mice. N1AS and DAS were converted rapidly to SFX and N4AS, respectively, in mouse plasma. The time courses of plasma SFX and N4AS concentrations were well-characterised following the oral administration of SFX to mice. The absorption, metabolism, and/or excretion of SFX given at >700mg/kg may be saturable, and in contrast to humans and rats, the extent of systemic exposure of mice to N4AS was much greater than that of SFX. Interestingly, the acetyl groups at both N1- and N4-positions were degraded during the ionisation required to generate precursor ions. In additional experiments the carboxyl group of N-acetyl-5-aminosalicylic acid (NA5AS) was lost instead of the acetyl group during the ionisation, and acetaminophen (AAP) appeared. As the acetyl and carboxyl groups of some substances can be degraded during ionisation in the mass spectrometer, caution is appropriate when it is sought to simultaneously quantify similar structures containing these moieties; chromatographic separation is essential. PMID:27454084

  1. Hexavalent chromium-induced differential disruption of cortical microtubules in some Fabaceae species is correlated with acetylation of α-tubulin.

    PubMed

    Eleftheriou, Eleftherios P; Adamakis, Ioannis-Dimosthenis S; Michalopoulou, Vasiliki A

    2016-03-01

    The effects of hexavalent chromium [Cr(VI)] on the cortical microtubules (MTs) of five species of the Fabaceae family (Vicia faba, Pisum sativum, Vigna sinensis, Vigna angularis, and Medicago sativa) were investigated by confocal laser scanning microscopy after immunolocalization of total tubulin with conventional immunofluorescence techniques and of acetylated α-tubulin with the specific 6-11B-1 monoclonal antibody. Moreover, total α-tubulin and acetylated α-tubulin were quantified by Western immunoblotting and scanning densitometry. Results showed the universality of Cr(VI) detrimental effects to cortical MTs, which proved to be a sensitive and reliable subcellular marker for monitoring Cr(VI) toxicity in plant cells. However, a species-specific response was recorded, and a correlation of MT disturbance with the acetylation status of α-tubulin was demonstrated. In V. faba, MTs were depolymerized at the gain of cytoplasmic tubulin background and displayed low α-tubulin acetylation, while in P. sativum, V. sinensis, V. angularis, and M. sativa, MTs became bundled and changed orientation from perpendicular to oblique or longitudinal. Bundled MTs were highly acetylated as determined by both immunofluorescence and Western immunoblotting. Tubulin acetylation in P. sativum and M. sativa preceded MT bundling; in V. sinensis it followed MT derangement, while in V. angularis the two phenomena coincided. Total α-tubulin remained constant in all treatments. Should acetylation be an indicator of MT stabilization, it is deduced that bundled MTs became stabilized, lost their dynamic properties, and were rendered inactive. Results of this report allow the conclusion that Cr(VI) toxicity disrupts MTs and deranges the MT-mediated functions either by depolymerizing or stabilizing them.

  2. Quantifier Comprehension in Corticobasal Degeneration

    ERIC Educational Resources Information Center

    McMillan, Corey T.; Clark, Robin; Moore, Peachie; Grossman, Murray

    2006-01-01

    In this study, we investigated patients with focal neurodegenerative diseases to examine a formal linguistic distinction between classes of generalized quantifiers, like "some X" and "less than half of X." Our model of quantifier comprehension proposes that number knowledge is required to understand both first-order and higher-order quantifiers.…

  3. Quantifying the Adaptive Cycle.

    PubMed

    Angeler, David G; Allen, Craig R; Garmestani, Ahjond S; Gunderson, Lance H; Hjerne, Olle; Winder, Monika

    2015-01-01

    The adaptive cycle was proposed as a conceptual model to portray patterns of change in complex systems. Despite the model having potential for elucidating change across systems, it has been used mainly as a metaphor, describing system dynamics qualitatively. We use a quantitative approach for testing premises (reorganisation, conservatism, adaptation) in the adaptive cycle, using Baltic Sea phytoplankton communities as an example of such complex system dynamics. Phytoplankton organizes in recurring spring and summer blooms, a well-established paradigm in planktology and succession theory, with characteristic temporal trajectories during blooms that may be consistent with adaptive cycle phases. We used long-term (1994-2011) data and multivariate analysis of community structure to assess key components of the adaptive cycle. Specifically, we tested predictions about: reorganisation: spring and summer blooms comprise distinct community states; conservatism: community trajectories during individual adaptive cycles are conservative; and adaptation: phytoplankton species during blooms change in the long term. All predictions were supported by our analyses. Results suggest that traditional ecological paradigms such as phytoplankton successional models have potential for moving the adaptive cycle from a metaphor to a framework that can improve our understanding how complex systems organize and reorganize following collapse. Quantifying reorganization, conservatism and adaptation provides opportunities to cope with the intricacies and uncertainties associated with fast ecological change, driven by shifting system controls. Ultimately, combining traditional ecological paradigms with heuristics of complex system dynamics using quantitative approaches may help refine ecological theory and improve our understanding of the resilience of ecosystems. PMID:26716453

  4. Uncertainty quantified trait predictions

    NASA Astrophysics Data System (ADS)

    Fazayeli, Farideh; Kattge, Jens; Banerjee, Arindam; Schrodt, Franziska; Reich, Peter

    2015-04-01

    Functional traits of organisms are key to understanding and predicting biodiversity and ecological change, which motivates continuous collection of traits and their integration into global databases. Such composite trait matrices are inherently sparse, severely limiting their usefulness for further analyses. On the other hand, traits are characterized by the phylogenetic trait signal, trait-trait correlations and environmental constraints, all of which provide information that could be used to statistically fill gaps. We propose the application of probabilistic models which, for the first time, utilize all three characteristics to fill gaps in trait databases and predict trait values at larger spatial scales. For this purpose we introduce BHPMF, a hierarchical Bayesian extension of Probabilistic Matrix Factorization (PMF). PMF is a machine learning technique which exploits the correlation structure of sparse matrices to impute missing entries. BHPMF additionally utilizes the taxonomic hierarchy for trait prediction. Implemented in the context of a Gibbs Sampler MCMC approach BHPMF provides uncertainty estimates for each trait prediction. We present comprehensive experimental results on the problem of plant trait prediction using the largest database of plant traits, where BHPMF shows strong empirical performance in uncertainty quantified trait prediction, outperforming the state-of-the-art based on point estimates. Further, we show that BHPMF is more accurate when it is confident, whereas the error is high when the uncertainty is high.

  5. Quantifying the adaptive cycle

    USGS Publications Warehouse

    Angeler, David G.; Allen, Craig R.; Garmestani, Ahjond S.; Gunderson, Lance H.; Hjerne, Olle; Winder, Monika

    2015-01-01

    The adaptive cycle was proposed as a conceptual model to portray patterns of change in complex systems. Despite the model having potential for elucidating change across systems, it has been used mainly as a metaphor, describing system dynamics qualitatively. We use a quantitative approach for testing premises (reorganisation, conservatism, adaptation) in the adaptive cycle, using Baltic Sea phytoplankton communities as an example of such complex system dynamics. Phytoplankton organizes in recurring spring and summer blooms, a well-established paradigm in planktology and succession theory, with characteristic temporal trajectories during blooms that may be consistent with adaptive cycle phases. We used long-term (1994–2011) data and multivariate analysis of community structure to assess key components of the adaptive cycle. Specifically, we tested predictions about: reorganisation: spring and summer blooms comprise distinct community states; conservatism: community trajectories during individual adaptive cycles are conservative; and adaptation: phytoplankton species during blooms change in the long term. All predictions were supported by our analyses. Results suggest that traditional ecological paradigms such as phytoplankton successional models have potential for moving the adaptive cycle from a metaphor to a framework that can improve our understanding how complex systems organize and reorganize following collapse. Quantifying reorganization, conservatism and adaptation provides opportunities to cope with the intricacies and uncertainties associated with fast ecological change, driven by shifting system controls. Ultimately, combining traditional ecological paradigms with heuristics of complex system dynamics using quantitative approaches may help refine ecological theory and improve our understanding of the resilience of ecosystems.

  6. Quantifying solvated electrons' delocalization.

    PubMed

    Janesko, Benjamin G; Scalmani, Giovanni; Frisch, Michael J

    2015-07-28

    Delocalized, solvated electrons are a topic of much recent interest. We apply the electron delocalization range EDR(r;u) (J. Chem. Phys., 2014, 141, 144104) to quantify the extent to which a solvated electron at point r in a calculated wavefunction delocalizes over distance u. Calculations on electrons in one-dimensional model cavities illustrate fundamental properties of the EDR. Mean-field calculations on hydrated electrons (H2O)n(-) show that the density-matrix-based EDR reproduces existing molecular-orbital-based measures of delocalization. Correlated calculations on hydrated electrons and electrons in lithium-ammonia clusters illustrates how electron correlation tends to move surface- and cavity-bound electrons onto the cluster or cavity surface. Applications to multiple solvated electrons in lithium-ammonia clusters provide a novel perspective on the interplay of delocalization and strong correlation central to lithium-ammonia solutions' concentration-dependent insulator-to-metal transition. The results motivate continued application of the EDR to simulations of delocalized electrons.

  7. Quantifying the Adaptive Cycle

    PubMed Central

    Angeler, David G.; Allen, Craig R.; Garmestani, Ahjond S.; Gunderson, Lance H.; Hjerne, Olle; Winder, Monika

    2015-01-01

    The adaptive cycle was proposed as a conceptual model to portray patterns of change in complex systems. Despite the model having potential for elucidating change across systems, it has been used mainly as a metaphor, describing system dynamics qualitatively. We use a quantitative approach for testing premises (reorganisation, conservatism, adaptation) in the adaptive cycle, using Baltic Sea phytoplankton communities as an example of such complex system dynamics. Phytoplankton organizes in recurring spring and summer blooms, a well-established paradigm in planktology and succession theory, with characteristic temporal trajectories during blooms that may be consistent with adaptive cycle phases. We used long-term (1994–2011) data and multivariate analysis of community structure to assess key components of the adaptive cycle. Specifically, we tested predictions about: reorganisation: spring and summer blooms comprise distinct community states; conservatism: community trajectories during individual adaptive cycles are conservative; and adaptation: phytoplankton species during blooms change in the long term. All predictions were supported by our analyses. Results suggest that traditional ecological paradigms such as phytoplankton successional models have potential for moving the adaptive cycle from a metaphor to a framework that can improve our understanding how complex systems organize and reorganize following collapse. Quantifying reorganization, conservatism and adaptation provides opportunities to cope with the intricacies and uncertainties associated with fast ecological change, driven by shifting system controls. Ultimately, combining traditional ecological paradigms with heuristics of complex system dynamics using quantitative approaches may help refine ecological theory and improve our understanding of the resilience of ecosystems. PMID:26716453

  8. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle.

  9. Quantifying Anderson's fault types

    USGS Publications Warehouse

    Simpson, R.W.

    1997-01-01

    Anderson [1905] explained three basic types of faulting (normal, strike-slip, and reverse) in terms of the shape of the causative stress tensor and its orientation relative to the Earth's surface. Quantitative parameters can be defined which contain information about both shape and orientation [Ce??le??rier, 1995], thereby offering a way to distinguish fault-type domains on plots of regional stress fields and to quantify, for example, the degree of normal-faulting tendencies within strike-slip domains. This paper offers a geometrically motivated generalization of Angelier's [1979, 1984, 1990] shape parameters ?? and ?? to new quantities named A?? and A??. In their simple forms, A?? varies from 0 to 1 for normal, 1 to 2 for strike-slip, and 2 to 3 for reverse faulting, and A?? ranges from 0?? to 60??, 60?? to 120??, and 120?? to 180??, respectively. After scaling, A?? and A?? agree to within 2% (or 1??), a difference of little practical significance, although A?? has smoother analytical properties. A formulation distinguishing horizontal axes as well as the vertical axis is also possible, yielding an A?? ranging from -3 to +3 and A?? from -180?? to +180??. The geometrically motivated derivation in three-dimensional stress space presented here may aid intuition and offers a natural link with traditional ways of plotting yield and failure criteria. Examples are given, based on models of Bird [1996] and Bird and Kong [1994], of the use of Anderson fault parameters A?? and A?? for visualizing tectonic regimes defined by regional stress fields. Copyright 1997 by the American Geophysical Union.

  10. Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

    PubMed Central

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D. Ramesh; Alfonso, Lloyd F.; Marimuthu, Srinivasan; Bhat, G. Jayarama

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  11. Relationship of histone acetylation to DNA topology and transcription.

    PubMed

    Krajewski, W A; Luchnik, A N

    1991-12-01

    An autonomously replicating plasmid constructed from bovine papiloma virus (BPV) and pBR322 was stably maintained as a nuclear episome in a mouse cell culture. Addition to a cell culture of sodium butyrate (5 mM) induced an increase in plasmid DNA supercoiling of 3-5 turns, an increase in acetylation of cellular histones, and a decrease in plasmid transcription by 2- to 4-fold. After withdrawal of butyrate, DNA supercoiling began to fluctuate in a wave-like manner with an amplitude of up to 3 turns and a period of 3-4 h. These waves gradually faded by 24 h. The transcription of the plasmid and acetylation of cellular histones also oscillated with the same period. The wave-like alterations were not correlated with the cell cycle, for there was no resumption of DNA replication after butyrate withdrawal for at least 24 h. In vitro chemical acetylation of histones with acetyl adenylate also led to an increase in the superhelical density of plasmid DNA. The parallel changes in transcription, histone acetylation, and DNA supercoiling in vivo may indicate a functional innerconnection. Also, the observed in vivo variation in the level of DNA supercoiling directly indicates the possibility of its natural regulation in eukaryotic cells.

  12. Cytotoxicity of chitosans with different acetylation degrees and molecular weights on bladder carcinoma cells.

    PubMed

    Younes, Islem; Frachet, Véronique; Rinaudo, Marguerite; Jellouli, Kemel; Nasri, Moncef

    2016-03-01

    The purpose of this research was to evaluate the cytotoxicity of chitosans with different degrees of acetylation (DA) and molecular weights (MW), as well as the effect of their positive ionic charges controlled by pH on bladder carcinoma cells (RT112 and RT112cp) using the tetrazolium salt colorimetric (MTT) assay. Our data showed that all chitosan samples were cytotoxic on RT112 and RT112cp cells with a higher cytotoxicity obtained at lower pH. Further, it was found that the toxicity increased with increasing DA. However, no significant difference in cytotoxicity between chitosans with different molecular weights was observed. Annexin V-FITC staining test was then used to study and quantify the induction of apoptosis. Data shows that chitosans induce apoptosis of RT112 and RT112cp cells with the same dependence with DA.

  13. On the use of an explicit chemical mechanism to dissect peroxy acetyl nitrate formation.

    PubMed

    Xue, Likun; Wang, Tao; Wang, Xinfeng; Blake, Donald R; Gao, Jian; Nie, Wei; Gao, Rui; Gao, Xiaomei; Xu, Zheng; Ding, Aijun; Huang, Yu; Lee, Shuncheng; Chen, Yizhen; Wang, Shulan; Chai, Fahe; Zhang, Qingzhu; Wang, Wenxing

    2014-12-01

    Peroxy acetyl nitrate (PAN) is a key component of photochemical smog and plays an important role in atmospheric chemistry. Though it has been known that PAN is produced via reactions of nitrogen oxides (NOx) with some volatile organic compounds (VOCs), it is difficult to quantify the contributions of individual precursor species. Here we use an explicit photochemical model--Master Chemical Mechanism (MCM) model--to dissect PAN formation and identify principal precursors, by analyzing measurements made in Beijing in summer 2008. PAN production was sensitive to both NOx and VOCs. Isoprene was the predominant VOC precursor at suburb with biogenic impact, whilst anthropogenic hydrocarbons dominated at downtown. PAN production was attributable to a relatively small class of compounds including NOx, xylenes, trimethylbenzenes, trans/cis-2-butenes, toluene, and propene. MCM can advance understanding of PAN photochemistry to a species level, and provide more relevant recommendations for mitigating photochemical pollution in large cities. PMID:25194270

  14. Peptide binding in OppA, the crystal structures of the periplasmic oligopeptide binding protein in the unliganded form and in complex with lysyllysine.

    PubMed

    Sleigh, S H; Tame, J R; Dodson, E J; Wilkinson, A J

    1997-08-12

    The periplasmic oligopeptide binding protein, OppA, acts as the initial receptor for the uptake of peptides by the oligopeptide permease (Opp) in Gram-negative bacteria. Opp will handle peptides between two and five amino acid residues regardless of their sequence. The crystal structures of a series of OppA-peptide complexes have revealed an enclosed but versatile peptide binding pocket and have illustrated how tri- and tetrapeptide ligands are accommodated. Here, the crystal structures of (i) OppA complexed with a dipeptide (lysyllysine) and (ii) unliganded OppA have been solved using X-ray data extending to 1.8 and 2.4 A spacing, respectively. In the dipeptide complex, the alpha-amino group of the ligand is anchored through an ion pair interaction with Asp419, as observed in complexes with longer peptides. However, its alpha-carboxylate group forms water-mediated interactions with the guanidinium groups of Arg404 and Arg413 rather than the direct salt bridges to Arg413 and His371 observed in the tripeptide and tetrapeptide complexes, respectively. Isothermal titration calorimetric measurements of the binding of lysine-containing peptides of different lengths to OppA show that the dipeptide, KK, is bound with approximately 60-fold lower affinity than related tri- and tetrapeptides (KKK and KKKA, respectively). These data are discussed with reference to the calculated enthalpic and entropic contributions to ligand binding and the structures of the OppA peptide complexes. In the unliganded molecule, domain III has rotated as a rigid body through 26 degrees away from domains I and II, exposing the ligand binding site. The water structure in the binding cleft shows similarities to that in the various OppA-peptide complexes.

  15. Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

    PubMed Central

    Lorenzana, Luis M.; Pérez-Redondo, Rosario; Santamarta, Irene; Martín, Juan F.; Liras, Paloma

    2004-01-01

    orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2. The mutant S. clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1. Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1::acc and S. clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins. PMID:15150229

  16. Dynamic changes in histone acetylation regulate origins of DNA replication

    PubMed Central

    Unnikrishnan, Ashwin; Gafken, Philip R.; Tsukiyama, Toshio

    2011-01-01

    While histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here, we describe a very efficient, single-step method to specifically purify histones located around an origin of replication from S. cerevisiae. Using high-resolution mass spectrometry, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that histone H3 and H4 acetylation is dynamically regulated around an origin of replication, at the level of multiply-acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S-phase. PMID:20228802

  17. Synthetic biology for engineering acetyl coenzyme A metabolism in yeast.

    PubMed

    Nielsen, Jens

    2014-01-01

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

  18. An acetylation rheostat for the control of muscle energy homeostasis

    PubMed Central

    Menzies, Keir; Auwerx, Johan

    2013-01-01

    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 governs 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, have helped biologists 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

  19. An acetylation rheostat for the control of muscle energy homeostasis.

    PubMed

    Menzies, Keir; Auwerx, Johan

    2013-12-01

    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

  20. Fate and effects of acetyl cedrene in sediments inhabited by different densities of the deposit feeder, Capitella teleta.

    PubMed

    Dai, Lina; Selck, Henriette; Salvito, Daniel; Forbes, Valery E

    2012-11-01

    Fragrance materials, such as acetyl cedrene (AC), are of environmental concern because they are continuously released to aquatic systems down the drain. In the present study, Capitella teleta (formerly Capitella capitata species I) was exposed to AC-amended sediment at two population densities corresponding to 44,000 and 88,000 worms/m(2). The fate of AC in systems with worms was compared to that in identical systems without worms. We examined the toxicity of AC on worm survival, growth, and feeding rate, and quantified the fate of AC in exposure systems by mass balance. Worm survival was close to 100% in all treatments. Acetyl cedrene had some positive effects on worm growth, but not feeding, whereas density had negative effects on both growth and feeding rates. After 14 d, the sediment concentration of AC was reduced by 88 to 99% in the presence of worms, whereas sediment AC concentration was reduced by 13 to 31% or less in the absence of worms. Acetyl cedrene was detected in fecal pellets, at low concentrations compared to the initial concentration in the sediment, but not in worm tissue, suggesting that ingested AC is bioavailable to Capitella teleta and that worms can biotransform sediment-associated AC effectively.

  1. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  2. Complex N-Acetylation of TriethylenetetramineS⃞

    PubMed Central

    Cerrada-Gimenez, Marc; Weisell, Janne; Hyvönen, Mervi T.; Hee Park, Myung; Alhonen, Leena; Vepsäläinen, Jouko

    2011-01-01

    Triethylenetetramine (TETA) is an efficient copper chelator that has versatile clinical potential. We have recently shown that spermidine/spermine-N1-acetyltransferase (SSAT1), the key polyamine catabolic enzyme, acetylates TETA in vitro. Here, we studied the metabolism of TETA in three different mouse lines: syngenic, SSAT1-overexpressing, and SSAT1-deficient (SSAT1-KO) mice. The mice were sacrificed at 1, 2, or 4 h after TETA injection (300 mg/kg i.p.). We found only N1-acetyltriethylenetetramine (N1AcTETA) and/or TETA in the liver, kidney, and plasma samples. As expected, SSAT1-overexpressing mice acetylated TETA at an accelerated rate compared with syngenic and SSAT1-KO mice. It is noteworthy that SSAT1-KO mice metabolized TETA as syngenic mice did, probably by thialysine acetyltransferase, which had a Km value of 2.5 ± 0.3 mM and a kcat value of 1.3 s−1 for TETA when tested in vitro with the human recombinant enzyme. Thus, the present results suggest that there are at least two N-acetylases potentially metabolizing TETA. However, their physiological significance for TETA acetylation requires further studies. Furthermore, we detected chemical intramolecular N-acetyl migration from the N1 to N3 position of N1AcTETA and N1,N8-diacetyltriethylenetetramine in an acidified high-performance liquid chromatography sample matrix. The complex metabolism of TETA together with the intramolecular N-acetyl migration may explain the huge individual variations in the acetylation rate of TETA reported earlier. PMID:21878558

  3. Acetylated histone H4 is reduced in human gastric adenomas and carcinomas.

    PubMed

    Ono, S; Oue, N; Kuniyasu, H; Suzuki, T; Ito, R; Matsusaki, K; Ishikawa, T; Tahara, E; Yasui, W

    2002-09-01

    Acetylation of core histones is closely linked to transcriptional activation of various genes. The acetylation levels of nucleosomal histones can be modified through a balance of histone acetyltransferases and deacetylases. To elucidate the role of histone acetylation in human gastric carcinogenesis, we studied the status of histone H4 acetylation in gastric carcinoma tissues and corresponding non-neoplastic mucosa. The status of histone acetylation was assessed by examining the expression of acetylated histone H4 through Western blotting and immunohistochemistry using an anti-acetylated histone H4 antibody. The levels of acetylated histone H4 expression were obviously reduced in 72% (13/18) of gastric carcinomas in comparison with non-neoplastic mucosa by Western blotting. In immunohistochemistry, acetylated histone H4 was clearly detected in the nuclei of both non-neoplastic epithelial and stromal cells, whereas the levels of acetylated histone H4 were heterogeneous or reduced in 66% (38/57) of gastric carcinomas and 46% (6/13) of gastric adenomas. Reduced expression of acetylated histone H4 was also observed in some areas of intestinal metaplasia adjacent to carcinomas. Reduction in the expression of acetylated histone H4 was significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. These results suggest that low levels of histone acetylation may be closely associated with the development and progression of gastric carcinomas, possibly through alteration of gene expression.

  4. 2H2O incorporation into hepatic acetyl-CoA and de novo lipogenesis as measured by Krebs cycle-mediated 2H-enrichment of glutamate and glutamine.

    PubMed

    Silva, Ana Maria; Martins, Fatima; Jones, John G; Carvalho, Rui

    2011-12-01

    Deuterated water is widely used for measuring de novo lipogenesis on the basis of quantifying lipid (2)H-enrichment relative to that of body water. However, incorporation of (2)H-enrichment from body water into newly synthesized lipid molecules is incomplete therefore the true lipid precursor enrichment differs from that of body water. We describe a novel measurement of de novo lipogenesis that is based on a true precursor-product analysis of hepatic acetyl-CoA and triglyceride methyl enrichments from deuterated water. After deuterated water administration to seven in situ and seven perfused livers, acetyl-CoA methyl enrichment was inferred from (2)H nuclear magnetic resonance analysis of hepatic glutamate/glutamine (Glx) enrichment and triglyceride methyl enrichment was directly determined by (2)H nuclear magnetic resonance of triglycerides. Acetyl-CoA (2) H-enrichment was 71% ± 1% that of body water for in situ livers and 53% ± 2% of perfusate water for perfused livers. From the ratio of triglyceride-methyl/acetyl-CoA enrichments, fractional de novo lipogenesis rates of 0.97% ± 0.09%/2 hr and 7.92% ± 1.47%/48 hr were obtained for perfused and in situ liver triglycerides, respectively. Our method reveals that acetyl-CoA enrichment is significantly less than body water both for in situ and perfused livers. Furthermore, the difference between acetyl-CoA and body water enrichments is sensitive to the experimental setting.

  5. Genetic Control of Differential Acetylation in Diabetic Rats

    PubMed Central

    Kaisaki, Pamela J.; Otto, Georg W.; McGouran, Joanna F.; Toubal, Amine; Argoud, Karène; Waller-Evans, Helen; Finlay, Clare; Caldérari, Sophie; Bihoreau, Marie-Thérèse; Kessler, Benedikt M.; Gauguier, Dominique; Mott, Richard

    2014-01-01

    Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression. PMID:24743600

  6. SCANDIUM TRIFLATE CATALYZED ACETYLATION OF STARCH UNDER MILD CONDITIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scandium (III) trifluoromethan sulfonate (Sc(OTf)3) was investigated as a catalyst for the acetylation of starch in order to determine the potential for preparing new types of starch esters under mild conditions. At room temperature, dry granular corn starch reacts with acetic anhydride in the pres...

  7. Mass spectrometry-based detection of protein acetylation

    PubMed Central

    Li, Yu; Silva, Jeffrey C.; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    Summary Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful characterizing changes in the acetylome in response to biological interventions (1). PMID:24014401

  8. Tubulin acetylation: responsible enzymes, biological functions and human diseases.

    PubMed

    Li, Lin; Yang, Xiang-Jiao

    2015-11-01

    Microtubules have important functions ranging from maintenance of cell morphology to subcellular transport, cellular signaling, cell migration, and formation of cell polarity. At the organismal level, microtubules are crucial for various biological processes, such as viral entry, inflammation, immunity, learning and memory in mammals. Microtubules are subject to various covalent modifications. One such modification is tubulin acetylation, which is associated with stable microtubules and conserved from protists to humans. In the past three decades, this reversible modification has been studied extensively. In mammals, its level is mainly governed by opposing actions of α-tubulin acetyltransferase 1 (ATAT1) and histone deacetylase 6 (HDAC6). Knockout studies of the mouse enzymes have yielded new insights into biological functions of tubulin acetylation. Abnormal levels of this modification are linked to neurological disorders, cancer, heart diseases and other pathological conditions, thereby yielding important therapeutic implications. This review summarizes related studies and concludes that tubulin acetylation is important for regulating microtubule architecture and maintaining microtubule integrity. Together with detyrosination, glutamylation and other modifications, tubulin acetylation may form a unique 'language' to regulate microtubule structure and function.

  9. Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome.

    PubMed

    Kim, Jongseong; Lee, Jaehyoun; Lee, Tae-Hee

    2015-12-01

    The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit.

  10. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    PubMed Central

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  11. Acetylation mediates Cx43 reduction caused by electrical stimulation

    PubMed Central

    Meraviglia, Viviana; Azzimato, Valerio; Colussi, Claudia; Florio, Maria Cristina; Binda, Anna; Panariti, Alice; Qanud, Khaled; Suffredini, Silvia; Gennaccaro, Laura; Miragoli, Michele; Barbuti, Andrea; Lampe, Paul D.; Gaetano, Carlo; Pramstaller, Peter P.; Capogrossi, Maurizio C.; Recchia, Fabio A.; Pompilio, Giulio; Rivolta, Ilaria; Rossini, Alessandra

    2015-01-01

    Communication between cardiomyocytes depends upon Gap Junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylases (HAT) and deacetylases (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 hours significantly reduced Connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43. PMID:26264759

  12. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  13. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  15. Children's interpretations of general quantifiers, specific quantifiers, and generics

    PubMed Central

    Gelman, Susan A.; Leslie, Sarah-Jane; Was, Alexandra M.; Koch, Christina M.

    2014-01-01

    Recently, several scholars have hypothesized that generics are a default mode of generalization, and thus that young children may at first treat quantifiers as if they were generic in meaning. To address this issue, the present experiment provides the first in-depth, controlled examination of the interpretation of generics compared to both general quantifiers ("all Xs", "some Xs") and specific quantifiers ("all of these Xs", "some of these Xs"). We provided children (3 and 5 years) and adults with explicit frequency information regarding properties of novel categories, to chart when "some", "all", and generics are deemed appropriate. The data reveal three main findings. First, even 3-year-olds distinguish generics from quantifiers. Second, when children make errors, they tend to be in the direction of treating quantifiers like generics. Third, children were more accurate when interpreting specific versus general quantifiers. We interpret these data as providing evidence for the position that generics are a default mode of generalization, especially when reasoning about kinds. PMID:25893205

  16. Glucose-6-phosphate dehydrogenase deficiency and sulfadimidin acetylation phenotypes in Egyptian oases.

    PubMed

    Hussein, L; Yamamah, G; Saleh, A

    1992-04-01

    Screening of 1315 males from two Egyptian oases for glucose-6-phosphate dehydrogenase deficiency (G-6PD) found an incidence of 5.9%. The rate of acetylation of sulfadimidin was also studied, and a bimodal distribution was found with 73% rapid acetylators. There is a correlation between high frequency of G-6PD deficiency and high frequency of slow acetylation rate.

  17. Human acetylator polymorphism: estimate of allele frequency in Libya and details of global distribution.

    PubMed Central

    Karim, A K; Elfellah, M S; Evans, D A

    1981-01-01

    Acetylator phenotyping by means of a sulphadimidine tests revealed 65% of Libyan Arabs to be slow acetylators. Hence the frequency of the allele controlling slow acetylation (As) is estimated as q = 0.81 +/- 0.05. This estimate is similar to those previously recorded in European and adjacent Middle Eastern populations. PMID:7328611

  18. Use of lignin extracted from different plant sources as standards in the spectrophotometric acetyl bromide lignin method.

    PubMed

    Fukushima, Romualdo S; Kerley, Monty S

    2011-04-27

    A nongravimetric acetyl bromide lignin (ABL) method was evaluated to quantify lignin concentration in a variety of plant materials. The traditional approach to lignin quantification required extraction of lignin with acidic dioxane and its isolation from each plant sample to construct a standard curve via spectrophotometric analysis. Lignin concentration was then measured in pre-extracted plant cell walls. However, this presented a methodological complexity because extraction and isolation procedures are lengthy and tedious, particularly if there are many samples involved. This work was targeted to simplify lignin quantification. Our hypothesis was that any lignin, regardless of its botanical origin, could be used to construct a standard curve for the purpose of determining lignin concentration in a variety of plants. To test our hypothesis, lignins were isolated from a range of diverse plants and, along with three commercial lignins, standard curves were built and compared among them. Slopes and intercepts derived from these standard curves were close enough to allow utilization of a mean extinction coefficient in the regression equation to estimate lignin concentration in any plant, independent of its botanical origin. Lignin quantification by use of a common regression equation obviates the steps of lignin extraction, isolation, and standard curve construction, which substantially expedites the ABL method. Acetyl bromide lignin method is a fast, convenient analytical procedure that may routinely be used to quantify lignin.

  19. Treating Colon Cancer Cells with FK228 Reveals a Link between Histone Lysine Acetylation and Extensive Changes in the Cellular Proteome.

    PubMed

    Wang, Tian-yun; Jia, Yan-long; Zhang, Xi; Sun, Qiu-li; Li, Yi-chun; Zhang, Jun-he; Zhao, Chun-peng; Wang, Xiao-yin; Wang, Li

    2015-12-17

    The therapeutic value of FK228 as a cancer treatment option is well known, and various types of cancer have been shown to respond to this drug. However, the complete mechanism of FK228 and the affect it has on histone lysine acetylation and the colon cancer cell proteome are largely unknown. In the present study, we used stable isotope labeling by amino acids in cell culture (SILAC) and affinity enrichment followed by high-resolution liquid chromatograph-mass spectrometer (LC-MS)/MS analysis to quantitate the changes in the lysine acetylome in HCT-8 cells after FK228 treatment. A total of 1,194 lysine acetylation sites in 751 proteins were quantified, with 115 of the sites in 85 proteins being significantly upregulated and 38 of the sites in 32 proteins being significantly downregulated in response to FK228 treatment. Interestingly, 47 histone lysine acetylation sites were identified in the core histone proteins. We also found a novel lysine acetylation site on H2BK121. These significantly altered proteins are involved in multiple biological functions as well as a myriad of metabolic and enzyme-regulated pathways. Taken together, the link between FK228 function and the downstream changes in the HCT-8 cell proteome observed in response to FK228 treatment is established.

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

    PubMed Central

    Davies, Michael N.; Kjalarsdottir, Lilja; Thompson, J. Will; Dubois, Laura G.; Stevens, Robert D.; Ilkayeva, Olga R.; Brosnan, M. Julia; Rolph, Timothy P.; Grimsrud, Paul A.; Muoio, Deborah M.

    2016-01-01

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  1. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    PubMed

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

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

    PubMed

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

    2012-07-01

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

  3. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    PubMed Central

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  4. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation.

    PubMed

    Bailey, Zachary S; Grinter, Michael B; VandeVord, Pamela J

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  5. Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts

    PubMed Central

    Li, Qingling; Deng, Shuang; Ibarra, Rafael A.; Anderson, Vernon E.; Brunengraber, Henri; Zhang, Guo-Fang

    2015-01-01

    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 [13C2,2H3]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. [13C6]glucose or [1,2-13C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-13C]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

  6. αTAT1 controls longitudinal spreading of acetylation marks from open microtubules extremities

    PubMed Central

    Ly, Nathalie; Elkhatib, Nadia; Bresteau, Enzo; Piétrement, Olivier; Khaled, Mehdi; Magiera, Maria M.; Janke, Carsten; Le Cam, Eric; Rutenberg, Andrew D.; Montagnac, Guillaume

    2016-01-01

    Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules. PMID:27752143

  7. Quantifying errors without random sampling

    PubMed Central

    Phillips, Carl V; LaPole, Luwanna M

    2003-01-01

    Background All quantifications of mortality, morbidity, and other health measures involve numerous sources of error. The routine quantification of random sampling error makes it easy to forget that other sources of error can and should be quantified. When a quantification does not involve sampling, error is almost never quantified and results are often reported in ways that dramatically overstate their precision. Discussion We argue that the precision implicit in typical reporting is problematic and sketch methods for quantifying the various sources of error, building up from simple examples that can be solved analytically to more complex cases. There are straightforward ways to partially quantify the uncertainty surrounding a parameter that is not characterized by random sampling, such as limiting reported significant figures. We present simple methods for doing such quantifications, and for incorporating them into calculations. More complicated methods become necessary when multiple sources of uncertainty must be combined. We demonstrate that Monte Carlo simulation, using available software, can estimate the uncertainty resulting from complicated calculations with many sources of uncertainty. We apply the method to the current estimate of the annual incidence of foodborne illness in the United States. Summary Quantifying uncertainty from systematic errors is practical. Reporting this uncertainty would more honestly represent study results, help show the probability that estimated values fall within some critical range, and facilitate better targeting of further research. PMID:12892568

  8. [Effect of acetylation and oxidation on some properties of breadfruit (Artocarpus altilis) seed starch].

    PubMed

    Rincón, Alicia Mariela; Bou Rached, Lizet; Aragoza, Luis E; Padilla, Fanny

    2007-09-01

    Starch extracted from seeds of Artocarpus altilis (Breadfruit) was chemically modified by acetylation and oxidation, and its functional properties were evaluated and compared with these of native starch. Analysis of the chemical composition showed that moisture content was higher for modified starches. Ash, protein, crude fiber and amylose contents were reduced by the modifications, but did not alter the native starch granules' irregularity, oval shape and smooth surface. Acetylation produced changes in water absorption, swelling power and soluble solids, these values were higher for acetylated starch, while values for native and oxidized starches were similar. Both modifications reduced pasting temperature; oxidation reduced maximum peak viscosity but it was increased by acetylation. Hot paste viscosity was reduced by both modifications, whereas cold paste viscosity was lower in the oxidized starch and higher in the acetylated starch. Breakdown was increased by acetylation and reduced with oxidation. Setback value was reduced after acetylation, indicating it could minimize retrogradation of the starch.

  9. N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

    SciTech Connect

    Scott, Daniel C.; Monda, Julie K.; Bennett, Eric J.; Harper, J. Wade; Schulman, Brenda A.

    2012-10-25

    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.

  10. Histone acetylation dependent energy landscapes in tri-nucleosome revealed by residue-resolved molecular simulations

    PubMed Central

    Chang, Le; Takada, Shoji

    2016-01-01

    Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366

  11. Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

    PubMed Central

    Wang, Yu-Gang; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wen-Ying; Wang, Na; Shi, Min

    2015-01-01

    AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation. METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclinD1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy. RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediated through p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase (acetyl K68) and nuclear factor-κB p65 (acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models. CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis. PMID:26217084

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

    SciTech Connect

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

    1991-04-23

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  15. Acetylation modification regulates GRP78 secretion in colon cancer cells.

    PubMed

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  16. Carbon isotope fractionation and the acetyl-CoA pathway

    NASA Astrophysics Data System (ADS)

    Blaser, Martin; Conrad, Ralf

    2010-05-01

    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.

  17. Acetylation modification regulates GRP78 secretion in colon cancer cells

    PubMed Central

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  18. Investigation of acetylated chitosan microspheres as potential chemoembolic agents.

    PubMed

    Zhou, Xuan; Kong, Ming; Cheng, Xiaojie; Li, Jingjing; Li, Jing; Chen, Xiguang

    2014-11-01

    The aim was to investigate the potential of chitosan microspheres (CMs) with different acetylation using as a chemoembolic agent. Chitosan microspheres (CMs) were prepared via water-in-oil (W/O) emulsification cross-linking method, and acetylated chitosan microspheres (ACMs) were obtained by acetylation of CMs. Next, we characterized the morphology, size, composition and degrees of deacetylation using scanning electron microscopy (TEM), dynamic laser light scattering (DLS), and Fourier transform infrared spectrometer (FTIR). All microspheres had smooth surfaces and good mechanical flexibility, and all could pass through a 5F catheter. The swelling rate (SR) of CMs decreased significantly with the increase of pH (4.0-10.0) but ACMs did not change under the same conditions. Protein absorption assays suggested that albumin was more greatly adsorbed on CMs than on ACMs. Furthermore, CMs caused more blood clots than ACMs. ACMs caused hemolysis less than CMs (<5% of the time). Data indicated that ACMs had more hemocompatibility. Cytotoxicity tests indicated that ACMs initially had less cell attached proliferation but increased with incubation. In contrast, the relative growth rate of mouse embryo fibroblasts (MEFs) on CMs decreased gradually. The results suggested that ACMs could stimulate the growth of MEFs, and CMs were not cytotoxic to MEFs. Thus, ACMs were more biocompatible with greater potential to be used as chemoembolic material.

  19. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides

    PubMed Central

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  20. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides.

    PubMed

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-09-23

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water.

  1. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides

    NASA Astrophysics Data System (ADS)

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-09-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water.

  2. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides.

    PubMed

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  3. Visualizing and quantifying Fusarium oxysporum in the plant host.

    PubMed

    Diener, Andrew

    2012-12-01

    Host-specific forms of Fusarium oxysporum infect the roots of numerous plant species. I present a novel application of familiar methodology to visualize and quantify F. oxysporum in roots. Infection in the roots of Arabidopsis thaliana, tomato, and cotton was detected with colorimetric reagents that are substrates for Fusarium spp.-derived arabinofuranosidase and N-acetyl-glucosaminidase activities and without the need for genetic modification of either plant host or fungal pathogen. Similar patterns of blue precipitation were produced by treatment with 5-bromo-4-chloro-3-indoxyl-α-l-arabinofuranoside and 5-bromo-4-chloro-3-indoxyl-2-acetamido-2-deoxy-β-d-glucopyranoside, and these patterns were consistent with prior histological descriptions of F. oxysporum in roots. Infection was quantified in roots of wild-type and mutant Arabidopsis using 4-nitrophenyl-α-l-arabinofuranoside. In keeping with an expectation that disease severity above ground is correlated with F. oxysporum infection below ground, elevated levels of arabinofuranosidase activity were measured in the roots of susceptible agb1 and rfo1 while a reduced level was detected in the resistant eir1. In contrast, disease severity and F. oxysporum infection were uncoupled in tir3. The distribution of staining patterns in roots suggests that AGB1 and RFO1 restrict colonization of the vascular cylinder by F. oxysporum whereas EIR1 promotes colonization of root apices.

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

    SciTech Connect

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

    2003-01-31

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

  5. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340 nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme.

  6. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite

    PubMed Central

    Cobbold, Simon A.; Santos, Joana M.; Ochoa, Alejandro; Perlman, David H.; Llinás, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite ‘acetylome’, characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

  7. Cell differentiation along multiple pathways accompanied by changes in histone acetylation status.

    PubMed

    Legartová, Soňa; Kozubek, Stanislav; Franek, Michal; Zdráhal, Zbyněk; Lochmanová, Gabriela; Martinet, Nadine; Bártová, Eva

    2014-04-01

    Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.

  8. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  9. Quantifying Poststroke Apathy With Actimeters.

    PubMed

    Goldfine, Andrew M; Dehbandi, Behdad; Kennedy, Juliana M; Sabot, Briana; Semper, Cory; Putrino, David

    2016-01-01

    The authors tested the hypothesis that wrist-worn actimeters can quantify the severity of poststroke apathy. The authors studied 57 patients admitted to an acute rehabilitation unit for ischemic or hemorrhagic stroke. After accounting for motor deficit of the affected arm and accounting for age, each increment of the Apathy Inventory score correlated with 5.6 fewer minutes of moving per hour. The overall statistical model had an R(2) of only 0.34, suggesting unexplained factors for total movement time. Wrist-worn actimeters may serve as an objective, quantifiable measure of poststroke apathy in patients with an intact upper extremity but cannot be used alone to diagnose apathy. PMID:26900735

  10. Quantifying and measuring cyber resiliency

    NASA Astrophysics Data System (ADS)

    Cybenko, George

    2016-05-01

    Cyber resliency has become an increasingly attractive research and operational concept in cyber security. While several metrics have been proposed for quantifying cyber resiliency, a considerable gap remains between those metrics and operationally measurable and meaningful concepts that can be empirically determined in a scientific manner. This paper describes a concrete notion of cyber resiliency that can be tailored to meet specific needs of organizations that seek to introduce resiliency into their assessment of their cyber security posture.

  11. O-acetylated oligosaccharides from pectins of potato tuber cell walls.

    PubMed Central

    Ishii, T

    1997-01-01

    Acetylated trigalacturonides and rhamnogalacturonan I (RG-I)-derived oligosaccharides were isolated from a Driselase digest of potato tuber cell walls by ion-exchange and size-exclusion chromatography. The oligosaccharides were structurally characterized by fast atom bombardment-mass spectroscopy, nuclear magnetic resonance spectroscopy, and glycosyl-linkage composition analysis. One trigalacturonide contained a single acetyl group at O-3 of the reducing galacturonic acid residue. A second trigalacturonide contained two acetyl substituents, which were located on O-3 or O-4 of the nonreducing galacturonic acid residue and O-3 of the reducing galacturonic acid residue. RG-I backbone-derived oligomers had acetyl groups at O-2 of the galacturonic acid residues. Some of these galacturonic acid residues were O-acetylated at both O-2 and O-3 positions. Rhamnosyl residues of RG-I oligomers were not acetylated. PMID:9112775

  12. Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci

    PubMed Central

    Litt, Michael D.; Simpson, Melanie; Recillas-Targa, Félix; Prioleau, Marie-Noëlle; Felsenfeld, Gary

    2001-01-01

    We have studied developmentally regulated patterns of histone acetylation at high resolution across ∼54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken β-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5′ insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases. PMID:11331588

  13. Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity

    PubMed Central

    Sartor, Gregory C.; Powell, Samuel K.; Brothers, Shaun P.

    2015-01-01

    Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic “reader” proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction. SIGNIFICANCE STATEMENT Proteins involved in the “readout” of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and

  14. Kinetic analysis of histone acetylation turnover and Trichostatin A induced hyper- and hypoacetylation in alfalfa.

    PubMed

    Waterborg, Jakob H; Kapros, Tamás

    2002-01-01

    Dynamic histone acetylation is a characteristic of chromatin transcription. The first estimates for the rate of acetylation turnover of plants are reported, measured in alfalfa cells by pulse, pulse-chase, and steady-state acetylation labeling. Acetylation turnover half-lives of about 0.5 h were observed by all methods used for histones H3, H4, and H2B. This is consistent with the rate at which changes in gene expression occur in plants. Treatment with histone deacetylase inhibitor Trichostatin A (TSA) induced hyperacetylation at a similar rate. Replacement histone variant H3.2, preferentially localized in highly acetylated chromatin, displayed faster acetyl turnover. Histone H2A with a low level of acetylation was not subject to rapid turnover or hyperacetylation. Patterns of acetate labeling revealed fundamental differences between histone H3 versus histones H4 and H2B. In H3, acetylation of all molecules, limited by lysine methylation, had similar rates, independent of the level of lysine acetylation. Acetylation of histones H4 and H2B was seen in only a fraction of all molecules and involved multiacetylation. Acetylation turnover rates increased from mono- to penta- and hexaacetylated forms, respectively. TSA was an effective inhibitor of alfalfa histone deacetylases in vivo and caused a doubling in steady-state acetylation levels by 4-6 h after addition. However, hyperacetylation was transient due to loss of TSA inhibition. TSA-induced overexpression of cellular deacetylase activity produced hypoacetylation by 18 h treatment with enhanced acetate turnover labeling of alfalfa histones. Thus, application of TSA to change gene expression in vivo in plants may have unexpected consequences. PMID:12123281

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

    SciTech Connect

    Saare, Mario; Rebane, Ana; Rajashekar, Balaji; Vilo, Jaak; Peterson, Paert

    2012-08-15

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

  16. Fusion of an oligopeptide to the N terminus of an alkaline α-amylase from Alkalimonas amylolytica simultaneously improves the enzyme's catalytic efficiency, thermal stability, and resistance to oxidation.

    PubMed

    Yang, Haiquan; Lu, Xinyao; Liu, Long; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

    2013-05-01

    In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: kcat/Km increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.

  17. Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

    PubMed Central

    Yang, Haiquan; Lu, Xinyao; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R.; Du, Guocheng

    2013-01-01

    In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: kcat/Km increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes. PMID:23455344

  18. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis1[C][W][OPEN

    PubMed Central

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha; Harholt, Jesper; Chong, Sun-Li; Pawar, Prashant Mohan-Anupama; Mellerowicz, Ewa J.; Tenkanen, Maija; Cheng, Kun; Pauly, Markus; Scheller, Henrik Vibe

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls. PMID:24019426

  19. Glutathione Utilization by Candida albicans Requires a Functional Glutathione Degradation (DUG) Pathway and OPT7, an Unusual Member of the Oligopeptide Transporter Family

    PubMed Central

    Desai, Prashant Ramesh; Thakur, Anil; Ganguli, Dwaipayan; Paul, Sanjoy; Morschhäuser, Joachim; Bachhawat, Anand K.

    2011-01-01

    Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1–OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a Km of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence. PMID:21994941

  20. Teleost fish models in membrane transport research: the PEPT1(SLC15A1) H+–oligopeptide transporter as a case study

    PubMed Central

    Romano, Alessandro; Barca, Amilcare; Storelli, Carlo; Verri, Tiziano

    2014-01-01

    Human genes for passive, ion-coupled transporters and exchangers are included in the so-called solute carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC-like form in 50 families and at least 338 genes after systematic GenBank database mining (December 2010–March 2011). When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary ‘molecular diversity’ with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+–oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g. the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g. the adaptation of icefish PEPT1 to sub-zero temperatures) and/or substrate specificity (e.g. the species-specificity of PEPT1 for the uptake of l-lysine-containing peptides). The revelation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g. molecular structure–function analyses) and applied research (e.g. optimizing diets to enhance growth of commercially valuable fish). PMID:23981715

  1. The multicopy sRNA LhrC controls expression of the oligopeptide-binding protein OppA in Listeria monocytogenes

    PubMed Central

    Sievers, Susanne; Lund, Anja; Menendez-Gil, Pilar; Nielsen, Aaraby; Storm Mollerup, Maria; Lambert Nielsen, Stine; Buch Larsson, Pernille; Borch-Jensen, Jonas; Johansson, Jörgen; Kallipolitis, Birgitte Haahr

    2015-01-01

    Listeria monocytogenes is the causative agent of the foodborne disease listeriosis. During infection, L. monocytogenes produces an array of non-coding RNAs, including the multicopy sRNA LhrC. These five, nearly identical sRNAs are highly induced in response to cell envelope stress and target the virulence adhesin lapB at the post-transcriptional level. Here, we demonstrate that LhrC controls expression of additional genes encoding cell envelope-associated proteins with virulence function. Using transcriptomics and proteomics, we identified a set of genes affected by LhrC in response to cell envelope stress. Three targets were significantly down-regulated by LhrC at both the RNA and protein level: lmo2349, tcsA and oppA. All three genes encode membrane-associated proteins: A putative substrate binding protein of an amino acid ABC transporter (Lmo2349); the CD4+ T cell-stimulating antigen TcsA, and the oligopeptide binding protein OppA, of which the latter 2 are required for full virulence of L. monocytogenes. For OppA, we show that LhrC acts by direct base paring to the ribosome binding site of the oppA mRNA, leading to an impediment of its translation and a decreased mRNA level. The sRNA-mRNA interaction depends on 2 of 3 CU-rich regions in LhrC allowing binding of 2 oppA mRNAs to a single LhrC molecule. Finally, we found that LhrC contributes to infection in macrophage-like cells. These findings demonstrate a central role for LhrC in controlling the level of OppA and other virulence-associated cell envelope proteins in response to cell envelope stress. PMID:26176322

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

    PubMed

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

    2015-12-01

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

  3. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    SciTech Connect

    Lee, Juhyung; Yun, Nuri; Kim, Chiho; Song, Min-Young; Park, Kang-Sik; Oh, Young J.

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  4. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    PubMed

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  5. Aspirin-mediated acetylation induces structural alteration and aggregation of bovine pancreatic insulin.

    PubMed

    Yousefi, Reza; Taheri, Behnaz; Alavi, Parnian; Shahsavani, Mohammad Bagher; Asadi, Zahra; Ghahramani, Maryam; Niazi, Ali; Alavianmehr, Mohammad Mehdi; Moosavi-Movahedi, Ali Akbar

    2016-01-01

    The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37°C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation.

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

    PubMed Central

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

    2015-01-01

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

  7. Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea

    PubMed Central

    Lv, Binna; Yang, Qianqian; Li, Delong; Liang, Wenxing; Song, Limin

    2016-01-01

    Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in diverse cellular processes. Botrytis cinerea is the most thoroughly studied necrotrophic species due to its broad host range and huge economic impact. However, to date, little is known about the functions of lysine acetylation in this plant pathogen. In this study, we determined the lysine acetylome of B. cinerea through the combination of affinity enrichment and high-resolution LC-MS/MS analysis. Overall, 1582 lysine acetylation sites in 954 proteins were identified. Bioinformatics analysis shows that the acetylated proteins are involved in diverse biological functions and show multiple cellular localizations. Several particular amino acids preferred near acetylation sites, including KacY, KacH, Kac***R, KacF, FKac and Kac***K, were identified in this organism. Protein interaction network analysis demonstrates that a variety of interactions are modulated by protein acetylation. Interestingly, 6 proteins involved in virulence of B. cinerea, including 3 key components of the high-osmolarity glycerol pathway, were found to be acetylated, suggesting that lysine acetylation plays regulatory roles in pathogenesis. These data provides the first comprehensive view of the acetylome of B. cinerea and serves as a rich resource for functional analysis of lysine acetylation in this plant pathogen. PMID:27381557

  8. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells

    PubMed Central

    Barjaktarovic, Zarko; Kempf, Stefan J.; Sriharshan, Arundhathi; Merl-Pham, Juliane; Atkinson, Michael J.; Tapio, Soile

    2015-01-01

    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

  9. First Comprehensive Proteome Analyses of Lysine Acetylation and Succinylation in Seedling Leaves of Brachypodium distachyon L.

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Wang, Jian; Zhu, Gengrui; Cao, Hui; Yuan, Linlin; Yan, Yueming

    2016-01-01

    Protein acetylation and succinylation are the most crucial protein post-translational modifications (PTMs) involved in the regulation of plant growth and development. In this study, we present the first lysine-acetylation and lysine-succinylation proteome analysis of seedling leaves in Brachypodium distachyon L (Bd). Using high accuracy nano LC-MS/MS combined with affinity purification, we identified a total of 636 lysine-acetylated sites in 353 proteins and 605 lysine-succinylated sites in 262 proteins. These proteins participated in many biology processes, with various molecular functions. In particular, 119 proteins and 115 sites were found to be both acetylated and succinylated, simultaneously. Among the 353 acetylated proteins, 148 had acetylation orthologs in Oryza sativa L., Arabidopsis thaliana, Synechocystis sp. PCC 6803, and Glycine max L. Among the 262 succinylated proteins, 170 of them were found to have homologous proteins in Oryza sativa L., Escherichia coli, Sacchayromyces cerevisiae, or Homo sapiens. Motif-X analysis of the acetylated and succinylated sites identified two new acetylated motifs (K---K and K-I-K) and twelve significantly enriched succinylated motifs for the first time, which could serve as possible binding loci for future studies in plants. Our comprehensive dataset provides a promising starting point for further functional analysis of acetylation and succinylation in Bd and other plant species. PMID:27515067

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

    SciTech Connect

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

    1991-04-15

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

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

    PubMed Central

    Schmeitzl, Clemens; Warth, Benedikt; Fruhmann, Philipp; Michlmayr, Herbert; Malachová, Alexandra; Berthiller, Franz; Schuhmacher, Rainer; Krska, Rudolf; Adam, Gerhard

    2015-01-01

    Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives. PMID:26274975

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

    PubMed

    Schmeitzl, Clemens; Warth, Benedikt; Fruhmann, Philipp; Michlmayr, Herbert; Malachová, Alexandra; Berthiller, Franz; Schuhmacher, Rainer; Krska, Rudolf; Adam, Gerhard

    2015-08-12

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

  13. The dynamic organization of fungal acetyl-CoA carboxylase

    PubMed Central

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

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  14. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    SciTech Connect

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

    2008-05-23

    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.

  15. The dynamic organization of fungal acetyl-CoA carboxylase

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  16. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

    PubMed Central

    Shieh, J; Whitman, W B

    1988-01-01

    To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation. PMID:3133359

  17. Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.

    PubMed

    Hinnebusch, Brian F; Henderson, J Welles; Siddique, Aleem; Malo, Madhu S; Zhang, Wenying; Abedrapo, Mario A; Hodin, Richard A

    2003-02-01

    Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between -378 and -303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP

  18. Quantifying torso deformity in scoliosis

    NASA Astrophysics Data System (ADS)

    Ajemba, Peter O.; Kumar, Anish; Durdle, Nelson G.; Raso, V. James

    2006-03-01

    Scoliosis affects the alignment of the spine and the shape of the torso. Most scoliosis patients and their families are more concerned about the effect of scoliosis on the torso than its effect on the spine. There is a need to develop robust techniques for quantifying torso deformity based on full torso scans. In this paper, deformation indices obtained from orthogonal maps of full torso scans are used to quantify torso deformity in scoliosis. 'Orthogonal maps' are obtained by applying orthogonal transforms to 3D surface maps. (An 'orthogonal transform' maps a cylindrical coordinate system to a Cartesian coordinate system.) The technique was tested on 361 deformed computer models of the human torso and on 22 scans of volunteers (8 normal and 14 scoliosis). Deformation indices from the orthogonal maps correctly classified up to 95% of the volunteers with a specificity of 1.00 and a sensitivity of 0.91. In addition to classifying scoliosis, the system gives a visual representation of the entire torso in one view and is viable for use in a clinical environment for managing scoliosis.

  19. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    PubMed

    Tsuchiya, Yugo; Pham, Uyen; Hu, Wanzhou; Ohnuma, Shin-Ichi; Gout, Ivan

    2014-01-01

    Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis. PMID:24831956

  20. Insights into K-Ras 4B regulation by post-translational lysine acetylation.

    PubMed

    Knyphausen, Philipp; Lang, Franziska; Baldus, Linda; Extra, Antje; Lammers, Michael

    2016-10-01

    Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

  1. Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

    PubMed Central

    2015-01-01

    The human lens contains three major protein families: α-, β-, and γ-crystallin. Among the several variants of γ-crystallin in the human lens, γD-crystallin is a major form. γD-Crystallin is primarily present in the nuclear region of the lens and contains a single lysine residue at the second position (K2). In this study, we investigated the acetylation of K2 in γD-crystallin in aging and cataractous human lenses. Our results indicated that K2 is acetylated at an early age and that the amount of K2-acetylated γD-crystallin increased with age. Mass spectrometric analysis revealed that in addition to K2, glycine 1 (G1) was acetylated in γD-crystallin from human lenses and in γD-crystallin acetylated in vitro. The chaperone ability of α-crystallin for acetylated γD-crystallin was lower than that for the nonacetylated protein. The tertiary structure and the microenvironment of the cysteine residues were significantly altered by acetylation. The acetylated protein exhibited higher surface hydrophobicity, was unstable against thermal and chemical denaturation, and exhibited a higher propensity to aggregate at 80 °C in comparison to the nonacetylated protein. Acetylation enhanced the GdnHCl-induced unfolding and slowed the subsequent refolding of γD-crystallin. Theoretical analysis indicated that the acetylation of K2 and G1 reduced the structural stability of the protein and brought the distal cysteine residues (C18 and C78) into close proximity. Collectively, these results indicate that the acetylation of G1 and K2 residues in γD-crystallin likely induced a molten globule-like structure, predisposing it to aggregation, which may account for the high content of aggregated proteins in the nucleus of aged and cataractous human lenses. PMID:25393041

  2. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.

    PubMed Central

    Lowe, D M; Tubbs, P K

    1985-01-01

    Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA. PMID:2860896

  3. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE PAGES

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng -Hua; Zhang, Jin -Song

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  4. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

    PubMed

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-04-01

    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

  5. Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)*

    PubMed Central

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-01-01

    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

  6. Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

    PubMed Central

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng-Hua

    2016-01-01

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. PMID:26745802

  7. In vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis.

    PubMed Central

    Dupont, C; Clarke, A J

    1991-01-01

    The synthesis and O acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl-3H]N-acetyl-D-glucosamine or [glucosamine-1,6-3H]N-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-6-3H]N-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-14C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of O-linked [14C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and [14C]acetate transfer. These results support the hypothesis that the base-labile O-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N----O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate. PMID:1856164

  8. Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

    PubMed Central

    Ryder, Daniel J.; Judge, Sarah M.; Beharry, Adam W.; Farnsworth, Charles L.; Silva, Jeffrey C.; Judge, Andrew R.

    2015-01-01

    Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the

  9. Topical effects of N-acetyl-L-hydroxyproline on ceramide synthesis and alleviation of pruritus

    PubMed Central

    Hashizume, Erika; Nakano, Tetsuo; Kamimura, Ayako; Morishita, Koji

    2013-01-01

    Purpose N-acetyl-l-hydroxyproline (AHYP) is an acetylated form of l-hydroxyproline that is used to treat skin ulcers and porphyria cutanea tarda. Its other biological and physiological effects on the skin have not been elucidated. We investigated the effects of AHYP on the skin-barrier function, focusing on ceramide synthesis and the effects of topical AHYP on atopic dermatitis. Materials and methods AHYP was applied to a three-dimensional cultured skin model. Ceramides were quantified by high-performance thin-layer chromatography. Serine palmitoyltransferase (SPT) is the rate-limiting enzyme in de novo ceramide synthesis, and the mRNA of its long-chain base subunit 1 (SPTLC1) was evaluated by quantitative reverse-transcription polymerase chain reaction. A clinical trial in the form of an intraindividual, comparative, double-blind, randomized, vehicle-controlled test involving 15 female subjects suffering from slight atopic dermatitis was performed. Subjects applied 1% (w/w) AHYP cream to one forearm and a control cream to the other forearm twice daily for 4 weeks. Skin condition was evaluated by measuring transepidermal water loss (TEWL). Dermatological observations were made by a dermatologist, and subjects evaluated their own pruritus intensity before beginning treatment and 4 weeks after the start of treatment. Results SPTLC1 expression and ceramide synthesis were significantly increased in an AHYP-treated skin model (P < 0.05). In the clinical trial, no adverse effects were observed in any subjects. TEWL was increased in the control-treated region of the forearm (P < 0.05) after 4 weeks’ application, whereas there was no change in the AHYP-treated region of the forearm. Pruritus intensity declined in the AHYP-treated forearms between 0 and 4 weeks (P < 0.05), but there was no change in the control-treated forearms. Conclusion AHYP increased ceramide synthesis by upregulating SPTLC1 in a three-dimensional cultured skin model, and it prevented TEWL increase

  10. Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2010-05-25

    Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

  11. A redox-sensitive, oligopeptide-guided, self-assembling, and efficiency-enhanced (ROSE) system for functional delivery of microRNA therapeutics for treatment of hepatocellular carcinoma.

    PubMed

    Hu, Qida; Wang, Kai; Sun, Xu; Li, Yang; Fu, Qihan; Liang, Tingbo; Tang, Guping

    2016-10-01

    Lack of efficient adjuvant therapy contributes to a high incidence of recurrence and metastasis of hepatocellular carcinoma (HCC). A novel therapeutic is required for adjuvant treatment of HCC. We developed a polymer-based nanosystem (ROSE) for functional gene therapy by synthesizing a supramolecular complex self-assembled from polycations and functional adamantyl modules. The ROSE system condensing tumor suppressor microRNA-34a (miR-34a) therapeutics becomes ROSE/miR-34a nanoparticles that could facilitate gene transfection in HCC cells with satisfied stability and efficiency, possibly due to proton sponge effect by polycations, PEGlyation protection, and controlled release by breakdown of disulfide bonds. Meanwhile, modification with a targeting oligopeptide SP94 in ROSE/miR-34a enables approximately higher affinity for LM3 HCC cells than hepatocytes in vitro and greater HCC specificity in vivo. Furthermore, ROSE/miR-34a nanoparticles significantly inhibits HCC cell proliferation and in vivo tumor growth, representing a notable effect improvement over conventional gene delivery strategies. ROSE/miR-34a, featuring redox-responsiveness, oligopeptide-guided specificity, self-assembly, and enhanced transfection, is therefore a potential therapeutic agent in future adjuvant therapy for HCC treatment. PMID:27459325

  12. A redox-sensitive, oligopeptide-guided, self-assembling, and efficiency-enhanced (ROSE) system for functional delivery of microRNA therapeutics for treatment of hepatocellular carcinoma.

    PubMed

    Hu, Qida; Wang, Kai; Sun, Xu; Li, Yang; Fu, Qihan; Liang, Tingbo; Tang, Guping

    2016-10-01

    Lack of efficient adjuvant therapy contributes to a high incidence of recurrence and metastasis of hepatocellular carcinoma (HCC). A novel therapeutic is required for adjuvant treatment of HCC. We developed a polymer-based nanosystem (ROSE) for functional gene therapy by synthesizing a supramolecular complex self-assembled from polycations and functional adamantyl modules. The ROSE system condensing tumor suppressor microRNA-34a (miR-34a) therapeutics becomes ROSE/miR-34a nanoparticles that could facilitate gene transfection in HCC cells with satisfied stability and efficiency, possibly due to proton sponge effect by polycations, PEGlyation protection, and controlled release by breakdown of disulfide bonds. Meanwhile, modification with a targeting oligopeptide SP94 in ROSE/miR-34a enables approximately higher affinity for LM3 HCC cells than hepatocytes in vitro and greater HCC specificity in vivo. Furthermore, ROSE/miR-34a nanoparticles significantly inhibits HCC cell proliferation and in vivo tumor growth, representing a notable effect improvement over conventional gene delivery strategies. ROSE/miR-34a, featuring redox-responsiveness, oligopeptide-guided specificity, self-assembly, and enhanced transfection, is therefore a potential therapeutic agent in future adjuvant therapy for HCC treatment.

  13. N-Ace: using solvent accessibility and physicochemical properties to identify protein N-acetylation sites.

    PubMed

    Lee, Tzong-Yi; Hsu, Justin Bo-Kai; Lin, Feng-Mao; Chang, Wen-Chi; Hsu, Po-Chiang; Huang, Hsien-Da

    2010-11-30

    Protein acetylation, which is catalyzed by acetyltransferases, is a type of post-translational modification and crucial to numerous essential biological processes, including transcriptional regulation, apoptosis, and cytokine signaling. As the experimental identification of protein acetylation sites is time consuming and laboratory intensive, several computational approaches have been developed for identifying the candidates of experimental validation. In this work, solvent accessibility and the physicochemical properties of proteins are utilized to identify acetylated alanine, glycine, lysine, methionine, serine, and threonine. A two-stage support vector machine was applied to learn the computational models with combinations of amino acid sequences, and the accessible surface area and physicochemical properties of proteins. The predictive accuracy thus achieved is 5% to 14% higher than that of models trained using only amino acid sequences. Additionally, the substrate specificity of the acetylated site was investigated in detail with reference to the subcellular colocalization of acetyltransferases and acetylated proteins. The proposed method, N-Ace, is evaluated using independent test sets in various acetylated residues and predictive accuracies of 90% were achieved, indicating that the performance of N-Ace is comparable with that of other acetylation prediction methods. N-Ace not only provides a user-friendly input/output interface but also is a creative method for predicting protein acetylation sites. This novel analytical resource is now freely available at http://N-Ace.mbc.NCTU.edu.tw/. PMID:20839302

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

    ERIC Educational Resources Information Center

    Pueschel, Siegfried M.

    2006-01-01

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

  15. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

    PubMed Central

    van Rossum, Harmen M.; Kozak, Barbara U.; Niemeijer, Matthijs S.; Dykstra, James C.; Luttik, Marijke A. H.; van Maris, Antonius J. A.

    2016-01-01

    ABSTRACT In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. PMID:27143389

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    ERIC Educational Resources Information Center

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

    2009-01-01

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

  18. Acetylation of Starch with Vinyl Acetate in Imidazolium Ionic Liquids and Characterization of Acetate Distribution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch was acetylated with vinyl acetate in different 1-butyl-3-methylimidazolium (BMIM) salts as solvent in effort to produce starches with different acetylation patterns. Overall degree of substitution was much higher for basic anions such as acetate and dicyanimide (dca) than for neutral anions ...

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

    PubMed Central

    Moafian, Zeinab; Khoshaman, Kazem; Oryan, Ahmad; Kurganov, Boris I.; Yousefi, Reza

    2016-01-01

    Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL). Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of α-crystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins. PMID:27706231

  20. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  1. Microbial conversion of daunorubicin into N-acetyl-13(S)-dihydrodaunomycin and bisanhydro-13-dihydrodaunomycinone.

    PubMed

    Dornberger, K; Hübener, R; Ihn, W; Thrum, H; Radics, L

    1985-09-01

    By using a strain of Streptomyces willmorii, daunorubicin (daunomycin) was stereoselectively converted into N-acetyl-13(S)-dihydrodaunomycin and bisanhydro-13-dihydrodaunomycinone. The absolute stereochemistry of the new chiral center in N-acetyl-13(S)-dihydrodaunomycin was established by means of nuclear Overhauser effect measured in the 9,13-O-isopropylidene derivative.

  2. A dysregulated acetyl/SUMO switch of FXR promotes hepatic inflammation in obesity

    PubMed Central

    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

    2015-01-01

    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-κB but blocked that with RXRα, so that SUMO2-modified FXR was selectively recruited to and trans-repressed inflammatory genes without affecting FXR/RXRα 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

  3. Location of the O-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan.

    PubMed

    York, W S; Oates, J E; van Halbeek, H; Darvill, A G; Albersheim, P; Tiller, P R; Dell, A

    1988-02-15

    The locations of the O-acetyl substituents on the major nonasaccharide repeating unit of the xyloglucan isolated from sycamore extracellular polysaccharides were determined by a combination of analytical methods, including f.a.b.-m.s. and 1H-n.m.r. spectroscopy. The O-2-linked-beta-D-galactosyl residue of the nonasaccharide was found to be the dominant site of O-acetyl substitution. Both mono-O-acetylated and di-O-acetylated beta-D-galactosyl residues were detected. The degree of O-acetylation of the beta-D-galactosyl residue, was estimated by 1H-n.m.r. spectroscopy to be 55-60% at O-6, 15-20% at O-4, and 20-25% at O-3. 1H-n.m.r. spectroscopy also indicated that approximately 50% of the beta-D-galactosyl residues are mono-O-acetylated, 25-30% are di-O-acetylated, and 20% are not acetylated.

  4. Roles of Arabidopsis TBL34 and TBL35 in xylan acetylation and plant growth.

    PubMed

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Ye, Zheng-Hua

    2016-02-01

    Xylan is one of the major polymers in lignocellulosic biomass and about 60% of its xylosyl residues are acetylated at O-2 and/or O-3. Because acetylation of cell wall polymers contributes to biomass recalcitrance for biofuel production, it is important to investigate the biochemical mechanism underlying xylan acetylation, the knowledge of which could be applied to custom-design biomass composition tailored for biofuel production. In this report, we investigated the functions of Arabidopsis TRICHOME BIREFRINGENCE-LIKE 34 (TBL34) and TBL35, two DUF231-containing proteins, in xylan acetylation. The TBL34 gene was found to be specifically expressed in xylem cells in stems and root-hypocotyls, and both TBL34 and TBL35 were shown to be localized in the Golgi, where xylan biosynthesis occurs. Chemical analysis revealed that simultaneous mutations of TBL34 and TBL35 caused a mild decrease in xylan acetyl content and a specific reduction in xylan 3-O-monoacetylation and 2,3-di-O-acetylation. Furthermore, simultaneous mutations of TBL34, TBL35 and ESKIMO1 (ESK1) resulted in severely collapsed xylem vessels with altered secondary wall structure, and an extremely retarded plant growth. These findings indicate that TBL34 and TBL35 are putative acetyltransferases required for xylan 3-O-monoacetylation and 2,3-di-O-acetylation and that xylan acetylation is essential for normal secondary wall deposition and plant growth. PMID:26795157

  5. Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori.

    PubMed

    Zhou, Yong; Wu, Chengcheng; Sheng, Qing; Jiang, Caiying; Chen, Qin; Lv, Zhengbing; Yao, Juming; Nie, Zuoming

    2016-01-01

    Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated-particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm. PMID:27374983

  6. Effects of acetylation on the emulsifying properties of Artemisia sphaerocephala Krasch. polysaccharide.

    PubMed

    Li, Junjun; Hu, Xinzhong; Li, Xiaoping; Ma, Zhen

    2016-06-25

    In the present study, polysaccharides extracted from Artemisia sphaerocephala Krasch. seeds (ASKP) were acetylated to improve the emulsifying properties of the macromolecules. Several methods were applied for the acetylation purpose, among which the acetic anhydride-pyridine method with formamide as solvent was found to be the most effective one. Acetylated ASKPs with various degree of substitution (DS) were successfully produced and structurally characterized using HPSEC-MALS, FTIR and (1)H NMR techniques in this study. Results showed that acetylation treatment could cause the degradation of ASKP. Moreover, with the increase of DS, both the molecular weight and radius of gyration increased, as well as the molecular conformation trended to be more compact. Low DS (DS: 0.04 and 0.13) conferred acetylated ASKP a lower viscosity than that of ASKP. With the increase of DS, the viscosity of acetylated ASKPs increased and exceeded that of ASKP. Compared with ASKP, acetylated ASKPs could reduce the surface tension to a greater extent and demonstrated a much smaller droplet size (ZD) in an oil/water emulsion system. Acetylated ASKPs were capable of stabilizing the oil/water emulsion for 3 days at 60°C, whose performance was as good as that of gum acacia. In conclusion, such a hydrophobic modification on ASKP conferred it better emulsifying properties. PMID:27083845

  7. Quantifying Aggressive Behavior in Zebrafish.

    PubMed

    Teles, Magda C; Oliveira, Rui F

    2016-01-01

    Aggression is a complex behavior that influences social relationships and can be seen as adaptive or maladaptive depending on the context and intensity of expression. A model organism suitable for genetic dissection of the underlying neural mechanisms of aggressive behavior is still needed. Zebrafish has already proven to be a powerful vertebrate model organism for the study of normal and pathological brain function. Despite the fact that zebrafish is a gregarious species that forms shoals, when allowed to interact in pairs, both males and females express aggressive behavior and establish dominance hierarchies. Here, we describe two protocols that can be used to quantify aggressive behavior in zebrafish, using two different paradigms: (1) staged fights between real opponents and (2) mirror-elicited fights. We also discuss the methodology for the behavior analysis, the expected results for both paradigms, and the advantages and disadvantages of each paradigm in face of the specific goals of the study. PMID:27464816

  8. Loss-of-Function Mutation of REDUCED WALL ACETYLATION2 in Arabidopsis Leads to Reduced Cell Wall Acetylation and Increased Resistance to Botrytis cinerea1[W][OA

    PubMed Central

    Manabe, Yuzuki; Nafisi, Majse; Verhertbruggen, Yves; Orfila, Caroline; Gille, Sascha; Rautengarten, Carsten; Cherk, Candice; Marcus, Susan E.; Somerville, Shauna; Pauly, Markus; Knox, J. Paul; Sakuragi, Yumiko; Scheller, Henrik Vibe

    2011-01-01

    Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea. PMID:21212300

  9. Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

    PubMed

    Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul; Wallace, John C; Jitrapakdee, Sarawut; Attwood, Paul V

    2015-07-22

    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

  10. Improving the O/W emusifying properties of rapeseed lecithin ethanol insoluble fraction by acetylation.

    PubMed

    Sosada, Marian; Pasker, Beata; Bogocz, Marzena

    2003-01-01

    The effect of acetylation of rapeseed lecithin ethanol insoluble fraction (LEIF) containing 25% phosphatidylethanolamine (PE) on the O/W emulsifying properties was reported. In the study, acetic anhydride (50-150 mmol/100 g) and pyridine (0-30 mmole/100 g) were used. The PE conversion to N-acetyl-PE in LEIF determined by the HPLC method was varied from 18.2 to 84.7% and depended essentially on the acetylating agent amount and pyridine quantity used in acetylation. Emulsions of the O/W systems containing lecithin emulsifiers with different PE conversion degree were prepared and evaluated for its stability. It was found that the acetylation of LEIF improves its emulsifying properties and in the formation of emulsions containing soya oil, provided a decrease in oil droplet size and polydispersity index.

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

    PubMed Central

    Cui, Junhe; Yu, Zechuan; Lau, Denvid

    2016-01-01

    Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered. PMID:26742033

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

    PubMed

    Cui, Junhe; Yu, Zechuan; Lau, Denvid

    2016-01-05

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

  13. Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells

    PubMed Central

    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

    2014-01-01

    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

  14. Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

    PubMed Central

    1987-01-01

    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

  15. Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae

    PubMed Central

    Li, Delong; Lv, Binna; Tan, Lingling; Yang, Qianqian; Liang, Wenxing

    2016-01-01

    Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens. PMID:27412925

  16. Alamethicin biosynthesis: acetylation of the amino terminus and attachment of phenylalaninol.

    PubMed

    Mohr, H; Kleinkauf, H

    1978-10-12

    Alamethicin synthetase was extracted from the fungus Trichoderma viride at the end of its exponential growth phase. It is multienzyme complex with a molecular weight of approx. 480 000. The biosynthesis of alamethicin is initiated on the synthetase by acetylation of thiolester-bound aminoisobutyric acid, which remains enzyme bound. Acetyl-CoA serves as the acetate donor. Of the alamethicin constituents, glycine, alanine and valine are also acetylated when incubated alone. This acetylation is prevented by added aminoisobutyric acid, which indicates that the site on alamethicin synthetase catalyzing the acetylation has a preference for aminoisobutyric acid. Alamethicin formation on the synthetase is terminated by linkage of phenylalaninol to the carboxyl terminus of the peptide. It is unlikely that the amino alcohol is a degradation product of alamethicin or that it had been split off from the synthetase complex. Thus it is probably the reaction product of a separate enzyme system. PMID:568941

  17. Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies

    PubMed Central

    Saturnino, Carmela; Sinicropi, Maria Stefania; Puoci, Francesco

    2014-01-01

    Hyaluronic acid (HA), a macropolysaccharidic component of the extracellular matrix, is common to most species and it is found in many sites of the human body, including skin and soft tissue. Not only does HA play a variety of roles in physiologic and in pathologic events, but it also has been extensively employed in cosmetic and skin-care products as drug delivery agent or for several biomedical applications. The most important limitations of HA are due to its short half-life and quick degradation in vivo and its consequently poor bioavailability. In the aim to overcome these difficulties, HA is generally subjected to several chemical changes. In this paper we obtained an acetylated form of HA with increased bioavailability with respect to the HA free form. Furthermore, an improved radical scavenging and anti-inflammatory activity has been evidenced, respectively, on ABTS radical cation and murine monocyte/macrophage cell lines (J774.A1). PMID:25114930

  18. Writers and Readers of Histone Acetylation: Structure, Mechanism, and Inhibition

    PubMed Central

    Marmorstein, Ronen; Zhou, Ming-Ming

    2014-01-01

    Histone acetylation marks are written by histone acetyltransferases (HATs) and read by bromodomains (BrDs), and less commonly by other protein modules. These proteins regulate many transcription-mediated biological processes, and their aberrant activities are correlated with several human diseases. Consequently, small molecule HAT and BrD inhibitors with therapeutic potential have been developed. Structural and biochemical studies of HATs and BrDs have revealed that HATs fall into distinct subfamilies containing a structurally related core for cofactor binding, but divergent flanking regions for substrate-specific binding, catalysis, and autoregulation. BrDs adopt a conserved left-handed four-helix bundle to recognize acetyllysine; divergent loop residues contribute to substrate-specific acetyllysine recognition. PMID:24984779

  19. Acetylation of barnyardgrass starch with acetic anhydride under iodine catalysis.

    PubMed

    Bartz, Josiane; Goebel, Jorge Tiago; Giovanaz, Marcos Antônio; Zavareze, Elessandra da Rosa; Schirmer, Manoel Artigas; Dias, Alvaro Renato Guerra

    2015-07-01

    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.

  20. Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

    PubMed Central

    Pietrocola, Federico; Mariño, Guillermo; Lissa, Delphine; Vacchelli, Erika; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Zamzami, Naoufal; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2012-01-01

    Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components. PMID:23070521

  1. Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient

    NASA Technical Reports Server (NTRS)

    Thomas, B. R.; Chernov, A. A.

    2000-01-01

    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 or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity 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 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. 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 impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

  2. Acetylation of the response regulator RcsB controls transcription from a small RNA promoter.

    PubMed

    Hu, Linda I; Chi, Bui Khanh; Kuhn, Misty L; Filippova, Ekaterina V; Walker-Peddakotla, Arti J; Bäsell, Katrin; Becher, Dörte; Anderson, Wayne F; Antelmann, Haike; Wolfe, Alan J

    2013-09-01

    Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154. PMID:23852870

  3. Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter

    PubMed Central

    Hu, Linda I.; Chi, Bui Khanh; Kuhn, Misty L.; Filippova, Ekaterina V.; Walker-Peddakotla, Arti J.; Bäsell, Katrin; Becher, Dörte; Anderson, Wayne F.; Antelmann, Haike

    2013-01-01

    Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154. PMID:23852870

  4. Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Abbanat, D R; Ferry, J G

    1990-01-01

    The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide. PMID:2123865

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

    SciTech Connect

    Bame, K.J.

    1986-01-01

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

  6. Quantifying Evaporation in a Permeable Pavement System

    EPA Science Inventory

    Studies quantifying evaporation from permeable pavement systems are limited to a few laboratory studies and one field application. This research quantifies evaporation for a larger-scale field application by measuring the water balance from lined permeable pavement sections. Th...

  7. Unifying mechanism for bacterial cell signalers (4,5-dihydroxy-2,3-pentanedione, lactones and oligopeptides): electron transfer and reactive oxygen species. Practical medical features.

    PubMed

    Kovacic, Peter

    2007-01-01

    Cell signaling has attracted much attention involving higher organisms, and more recently is of considerable interest concerning involvement in the bacterial realm. Many aspects can apply, including quorum sensing. Of the participating molecules, designated autoinducers, 4,5-dihydroxy-2,3-pentanedione (DPD) is one of the most important. It is in equilibrium with a furanone and a furanosyl-borate diester (AI-2). A prior hypothesis for cell signaling in higher organisms invoked a key role for electron transfer (ET) and reactive oxygen species (ROS), as well as conduits, relays and electrical effects. The principal ET functionalities are quinones, metal complexes, ArNO(2), and iminium species. A lesser known type is the alpha-dicarbonyl class. Diacetyl, a member, as well as its imine derivatives, can serve as a model for DPD, since the parent possesses a reduction potential amenable to ET in the biological domain. Hence, it is conceivable that DPD and its imine derivatives may be involved in ET-ROS processes. Presence of hydroxy groups should facilitate ET by DPD vs. diacetyl. Extensive prior literature supports participation of ET functionalities in action of therapeutic drugs, toxins and various illnesses. This biochemical behavior also applies to the alpha-dicarbonyl parent models. A second important bacterial autoinducer is the lactone category. Although ET functionality is lacking, the presence of the 1,3-dicarbonyl structure can provide a site for avid chelation with redox metal, e.g., iron or copper, followed by ET-ROS. Findings with added iron furnish support for the proposal. Oligopeptides comprise the third principal type of bacterial signaling agent. A prior review incorporates these within the theoretical framework based on ET by redox amino acids and redox enzymes. In recent years there has been a rapid increase in resistance to antibiotics by pathogenic bacteria. Infectious diseases are the leading cause of death worldwide and the third leading cause in

  8. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array*

    PubMed Central

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-01-01

    Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our

  9. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.

    PubMed

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-04-01

    Calpains are intracellular Ca(2+)-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10' of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. Thekcat/Kms for 119 sites ranged from 12.5-1,710 M(-1)s(-1) Although most sites were cleaved by both calpain-1 and -2 with a similarkcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5'. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P'-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achievedkcat/Kmprediction withr= 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3', and P4' sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model

  10. ABC and SLC transporter expression and proton oligopeptide transporter (POT) mediated permeation across the human blood--brain barrier cell line, hCMEC/D3 [corrected].

    PubMed

    Carl, Stephen M; Lindley, David J; Das, Debanjan; Couraud, Pierre O; Weksler, Babette B; Romero, Ignacio; Mowery, Stephanie A; Knipp, Gregory T

    2010-08-01

    Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential

  11. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    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.

  12. Detecting, visualising, and quantifying mucins.

    PubMed

    Harrop, Ceri A; Thornton, David J; McGuckin, Michael A

    2012-01-01

    The extreme size, extensive glycosylation, and gel-forming nature of mucins make them a challenge to work with, and methodologies for the detection of mucins must take into consideration these features to ensure that one obtains both accurate and meaningful results. In understanding and appreciating the nature of mucins, this affords the researcher a valuable toolkit which can be used to full advantage in detecting, quantifying, and visualising mucins. The employment of a combinatorial approach to mucin detection, using antibody, chemical, and lectin detection methods, allows important information to be gleaned regarding the size, extent of glycosylation, specific mucin species, and distribution of mucins within a given sample. In this chapter, the researcher is guided through considerations into the structure of mucins and how this both affects the detection of mucins and can be used to full advantage. Techniques including ELISA, dot/slot blotting, and Western blotting, use of lectins and antibodies in mucin detection on membranes as well as immunohistochemistry and immunofluorescence on both tissues and cells grown on Transwell™ inserts are described. Notes along with each section advice the researcher on best practice and describe any associated limitations of a particular technique from which the researcher can further develop a particular protocol.

  13. Quantifying the vitamin D economy.

    PubMed

    Heaney, Robert P; Armas, Laura A G

    2015-01-01

    Vitamin D enters the body through multiple routes and in a variety of chemical forms. Utilization varies with input, demand, and genetics. Vitamin D and its metabolites are carried in the blood on a Gc protein that has three principal alleles with differing binding affinities and ethnic prevalences. Three major metabolites are produced, which act via two routes, endocrine and autocrine/paracrine, and in two compartments, extracellular and intracellular. Metabolic consumption is influenced by physiological controls, noxious stimuli, and tissue demand. When administered as a supplement, varying dosing schedules produce major differences in serum metabolite profiles. To understand vitamin D's role in human physiology, it is necessary both to identify the foregoing entities, mechanisms, and pathways and, specifically, to quantify them. This review was performed to delineate the principal entities and transitions involved in the vitamin D economy, summarize the status of present knowledge of the applicable rates and masses, draw inferences about functions that are implicit in these quantifications, and point out implications for the determination of adequacy. PMID:26024057

  14. Quantifying the vitamin D economy.

    PubMed

    Heaney, Robert P; Armas, Laura A G

    2015-01-01

    Vitamin D enters the body through multiple routes and in a variety of chemical forms. Utilization varies with input, demand, and genetics. Vitamin D and its metabolites are carried in the blood on a Gc protein that has three principal alleles with differing binding affinities and ethnic prevalences. Three major metabolites are produced, which act via two routes, endocrine and autocrine/paracrine, and in two compartments, extracellular and intracellular. Metabolic consumption is influenced by physiological controls, noxious stimuli, and tissue demand. When administered as a supplement, varying dosing schedules produce major differences in serum metabolite profiles. To understand vitamin D's role in human physiology, it is necessary both to identify the foregoing entities, mechanisms, and pathways and, specifically, to quantify them. This review was performed to delineate the principal entities and transitions involved in the vitamin D economy, summarize the status of present knowledge of the applicable rates and masses, draw inferences about functions that are implicit in these quantifications, and point out implications for the determination of adequacy.

  15. Quantifying Uncertainty in Epidemiological Models

    SciTech Connect

    Ramanathan, Arvind; Jha, Sumit Kumar

    2012-01-01

    Modern epidemiology has made use of a number of mathematical models, including ordinary differential equation (ODE) based models and agent based models (ABMs) to describe the dynamics of how a disease may spread within a population and enable the rational design of strategies for intervention that effectively contain the spread of the disease. Although such predictions are of fundamental importance in preventing the next global pandemic, there is a significant gap in trusting the outcomes/predictions solely based on such models. Hence, there is a need to develop approaches such that mathematical models can be calibrated against historical data. In addition, there is a need to develop rigorous uncertainty quantification approaches that can provide insights into when a model will fail and characterize the confidence in the (possibly multiple) model outcomes/predictions, when such retrospective analysis cannot be performed. In this paper, we outline an approach to develop uncertainty quantification approaches for epidemiological models using formal methods and model checking. By specifying the outcomes expected from a model in a suitable spatio-temporal logic, we use probabilistic model checking methods to quantify the probability with which the epidemiological model satisfies the specification. We argue that statistical model checking methods can solve the uncertainty quantification problem for complex epidemiological models.

  16. Quantifying macromolecular conformational transition pathways

    NASA Astrophysics Data System (ADS)

    Seyler, Sean; Kumar, Avishek; Thorpe, Michael; Beckstein, Oliver

    2015-03-01

    Diverse classes of proteins function through large-scale conformational changes that are challenging for computer simulations. A range of fast path-sampling techniques have been used to generate transitions, but it has been difficult to compare paths from (and assess the relative strengths of) different methods. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Fréchet metrics (known from computational geometry) are used to quantify the degree of similarity between polygonal curves in configuration space. A strength of PSA is its use of the full information available from the 3 N-dimensional configuration space trajectory without requiring additional specific knowledge about the system. We compare a sample of eleven different methods for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK) and also apply PSA to an ensemble of 400 AdK trajectories produced by dynamic importance sampling MD and the Geometrical Pathways algorithm. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and help guide future research toward deeper physical insights into conformational transitions.

  17. Quantifying periodicity in omics data.

    PubMed

    Amariei, Cornelia; Tomita, Masaru; Murray, Douglas B

    2014-01-01

    Oscillations play a significant role in biological systems, with many examples in the fast, ultradian, circadian, circalunar, and yearly time domains. However, determining periodicity in such data can be problematic. There are a number of computational methods to identify the periodic components in large datasets, such as signal-to-noise based Fourier decomposition, Fisher's g-test and autocorrelation. However, the available methods assume a sinusoidal model and do not attempt to quantify the waveform shape and the presence of multiple periodicities, which provide vital clues in determining the underlying dynamics. Here, we developed a Fourier based measure that generates a de-noised waveform from multiple significant frequencies. This waveform is then correlated with the raw data from the respiratory oscillation found in yeast, to provide oscillation statistics including waveform metrics and multi-periods. The method is compared and contrasted to commonly used statistics. Moreover, we show the utility of the program in the analysis of noisy datasets and other high-throughput analyses, such as metabolomics and flow cytometry, respectively.

  18. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development

    PubMed Central

    Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J.; Cui, Liwang

    2013-01-01

    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

  19. Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Alfonso, Lloyd F; Marimuthu, Srinivasan; Bhat, G Jayarama

    2016-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT‑29 colorectal cancer cells, in order to compare aspirin‑mediated acetylation of G6PD and its activity between HCT 116 and HT‑29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT‑29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin‑acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  20. Mitochondrial protein acetylation as a cell-intrinsic, evolutionary driver of fat storage: chemical and metabolic logic of acetyl-lysine modifications.

    PubMed

    Ghanta, Sirisha; Grossmann, Ruth E; Brenner, Charles

    2013-01-01

    Hormone systems evolved over 500 million years of animal natural history to motivate feeding behavior and convert excess calories to fat. These systems produced vertebrates, including humans, who are famine-resistant but sensitive to obesity in environments of persistent overnutrition. We looked for cell-intrinsic metabolic features, which might have been subject to an evolutionary drive favoring lipogenesis. Mitochondrial protein acetylation appears to be such a system. Because mitochondrial acetyl-coA is the central mediator of fuel oxidation and is saturable, this metabolite is postulated to be the fundamental indicator of energy excess, which imprints a memory of nutritional imbalances by covalent modification. Fungal and invertebrate mitochondria have highly acetylated mitochondrial proteomes without an apparent mitochondrially targeted protein lysine acetyltransferase. Thus, mitochondrial acetylation is hypothesized to have evolved as a nonenzymatic phenomenon. Because the pKa of a nonperturbed Lys is 10.4 and linkage of a carbonyl carbon to an ε amino group cannot be formed with a protonated Lys, we hypothesize that acetylation occurs on residues with depressed pKa values, accounting for the propensity of acetylation to hit active sites and suggesting that regulatory Lys residues may have been under selective pressure to avoid or attract acetylation throughout animal evolution. In addition, a shortage of mitochondrial oxaloacetate under ketotic conditions can explain why macronutrient insufficiency also produces mitochondrial hyperacetylation. Reduced mitochondrial activity during times of overnutrition and undernutrition would improve fitness by virtue of resource conservation. Micronutrient insufficiency is predicted to exacerbate mitochondrial hyperacetylation. Nicotinamide riboside and Sirt3 activity are predicted to relieve mitochondrial inhibition. PMID:24050258

  1. Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Alfonso, Lloyd F; Marimuthu, Srinivasan; Bhat, G Jayarama

    2016-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT‑29 colorectal cancer cells, in order to compare aspirin‑mediated acetylation of G6PD and its activity between HCT 116 and HT‑29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT‑29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin‑acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

  2. FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

    PubMed Central

    Xie, Jenny; Peng, Min; Guillemette, Shawna; Quan, Steven; Maniatis, Stephanie; Wu, Yuliang; Venkatesh, Aditya; Shaffer, Scott A.; Brosh, Robert M.; Cantor, Sharon B.

    2012-01-01

    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

  3. Monitoring the effect of belinostat in solid tumors by H4 acetylation

    PubMed Central

    MARQUARD, LENA; PETERSEN, KAMILLE DUMONG; PERSSON, MORTEN; HOFF, KIRSTEN DAMGAARD; JENSEN, PETER BUHL; SEHESTED, MAXWELL

    2008-01-01

    Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors. PMID:18452428

  4. Preparation and investigation of acetyl salicylic acid-caffeine complex for rectal administration.

    PubMed

    Fouad, Ehab A; El-Badry, Mahmoud; Alanazi, Fars K; Arafah, Maha M; Al-Ashban, Riyadh; Alsarra, Ibrahim A

    2010-06-01

    An acetyl salicylic acid-caffeine complex was prepared and evaluated for the potential use in rectal administration. The results revealed the formation of a complex between acetyl salicylic acid and caffeine in a 1:1 molar ratio by a charge transfer mechanism. The effects of acetyl salicylic acid and complex on the rectal tissues showed destruction in the mucosal epithelium in case of acetyl salicylic acid; however, no change in the rectal tissues was noticed upon the administration of the complex. The effect of suppository bases on the release of the complex was studied using Witepsol H15 as fatty base and polyethylene glycols (PEG) 1000 and 4000 as a water soluble suppository base. The release profiles of acetyl salicylic acid and the complex were faster from PEG than from that of Witepsol H15. The percent release for the complex and acetyl salicylic acid from PEG base were 45.8, and 34.9%, respectively. However, it was 8.7 and 7.8%, respectively, from Witepsol H15 fatty base. The release kinetic was found to follow the non-Fickian diffusion model for complex from the suppository bases. It was concluded that acetyl salicylic acid caffeine complex can be used safely for rectal administration.

  5. Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF.

    PubMed

    Chatterjee, Nilanjana; North, Justin A; Dechassa, Mekonnen Lemma; Manohar, Mridula; Prasad, Rashmi; Luger, Karolin; Ottesen, Jennifer J; Poirier, Michael G; Bartholomew, Blaine

    2015-12-01

    Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as "readers," which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers.

  6. A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.

    PubMed

    Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

    2014-12-01

    Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

  7. Effect of acetyl esterification on physicochemical properties of chick pea (Cicer arietinum L.) starch.

    PubMed

    Yadav, Dev Kumar; Patki, Prakash Eknatharao

    2015-07-01

    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 ΔH of acetylated starch than native starch which resulted in reduced retrogradation by 56 %.

  8. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set.

    PubMed

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  9. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set

    PubMed Central

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  10. An Smc3 Acetylation Cycle Is Essential for Establishment of Sister Chromatid Cohesion

    PubMed Central

    Beckouët, Frederic; Hu, Bin; Roig, Maurici B.; Sutani, Takashi; Komata, Makiko; Uluocak, Pelin; Katis, Vittorio L.; Shirahige, Katsuhiko; Nasmyth, Kim

    2015-01-01

    SUMMARY Sister chromatid cohesion is thought to involve entrapment of sister DNAs by a tripartite ring composed of the cohesin subunits Smc1, Smc3, and Scc1. Establishment of cohesion during S phase depends on acetylation of Smc3’s nucleotide-binding domain (NBD) by the Eco1 acetyl transferase. It is destroyed at the onset of anaphase due to Scc1 cleavage by separase. In yeast, Smc3 acetylation is reversed at anaphase by the Hos1 deacetylase as a consequence of Scc1 cleavage. Smc3 molecules that remain acetylated after mitosis due to Hos1 inactivation cannot generate cohesion during the subsequent S phase, implying that cohesion establishment depends on de novo acetylation during DNA replication. By inducing Smc3 deacetylation in postreplicative cells due to Hos1 overexpression, we provide evidence that Smc3 acetylation contributes to the maintenance of sister chromatid cohesion. A cycle of Smc3 NBD acetylation is therefore an essential aspect of the chromosome cycle in eukaryotic cells. PMID:20832721

  11. Proteomic Investigations of Lysine Acetylation Identify Diverse Substrates of Mitochondrial Deacetylase Sirt3

    PubMed Central

    Weinert, Brian T.; Kumar, Amit; Kim, Hyun-Seok; Deng, Chu-Xia; Choudhary, Chunaram

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases. PMID:23236377

  12. Quantifying uncertainty from material inhomogeneity.

    SciTech Connect

    Battaile, Corbett Chandler; Emery, John M.; Brewer, Luke N.; Boyce, Brad Lee

    2009-09-01

    Most engineering materials are inherently inhomogeneous in their processing, internal structure, properties, and performance. Their properties are therefore statistical rather than deterministic. These inhomogeneities manifest across multiple length and time scales, leading to variabilities, i.e. statistical distributions, that are necessary to accurately describe each stage in the process-structure-properties hierarchy, and are ultimately the primary source of uncertainty in performance of the material and component. When localized events are responsible for component failure, or when component dimensions are on the order of microstructural features, this uncertainty is particularly important. For ultra-high reliability applications, the uncertainty is compounded by a lack of data describing the extremely rare events. Hands-on testing alone cannot supply sufficient data for this purpose. To date, there is no robust or coherent method to quantify this uncertainty so that it can be used in a predictive manner at the component length scale. The research presented in this report begins to address this lack of capability through a systematic study of the effects of microstructure on the strain concentration at a hole. To achieve the strain concentration, small circular holes (approximately 100 {micro}m in diameter) were machined into brass tensile specimens using a femto-second laser. The brass was annealed at 450 C, 600 C, and 800 C to produce three hole-to-grain size ratios of approximately 7, 1, and 1/7. Electron backscatter diffraction experiments were used to guide the construction of digital microstructures for finite element simulations of uniaxial tension. Digital image correlation experiments were used to qualitatively validate the numerical simulations. The simulations were performed iteratively to generate statistics describing the distribution of plastic strain at the hole in varying microstructural environments. In both the experiments and simulations, the

  13. Quantifying magma segregation in dykes

    NASA Astrophysics Data System (ADS)

    Yamato, P.; Duretz, T.; May, D. A.; Tartèse, R.

    2015-10-01

    The dynamics of magma flow is highly affected by the presence of a crystalline load. During magma ascent, it has been demonstrated that crystal-melt segregation constitutes a viable mechanism for magmatic differentiation. Moreover, crystal-melt segregation during magma transport has important implications not only in terms of magma rheology, but also in terms of differentiation of the continental crust. However, the influences of the crystal volume percentage (φ), of their geometry, their size and their density on crystal-melt segregation are still not well constrained. To address these issues, we performed a parametric study using 2D direct numerical simulations, which model the ascension of a crystal-bearing magma in a vertical dyke. Using these models, we have characterised the amount of segregation as a function of different physical properties including φ, the density contrast between crystals and the melt phase (Δρ), the size of the crystals (Ac) and their aspect ratio (R). Results show that small values of R do not affect the segregation. In this case, the amount of segregation depends upon four parameters. Segregation is highest when Δρ and Ac are large, and lowest for large pressure gradient (Pd) and/or large values of dyke width (Wd). These four parameters can be combined into a single one, the Snumber, which can be used to quantify the amount of segregation occurring during magma ascent. Based on systematic numerical modelling and dimensional analysis, we provide a first order scaling law which allows quantification of the segregation for an arbitrary Snumber and φ, encompassing a wide range of typical parameters encountered in terrestrial magmatic systems. Although developed in a simplified system, this study has strong implications regarding our understanding of crystal segregation processes during magma transport. Our first order scaling law allows to immediately determine the amount of crystal-melt segregation occurring in any given magmatic

  14. 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.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

  15. Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation.

    PubMed

    Guo, Jintao; Wang, Changwei; Han, Yi; Liu, Zhiyong; Wu, Tian; Liu, Yan; Liu, Yang; Tan, Yaoju; Cai, Xinshan; Cao, Yuanyuan; Wang, Bangxing; Zhang, Buchang; Liu, Chunping; Tan, Shouyong; Zhang, Tianyu

    2016-08-01

    Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies. PMID:27323652

  16. Aurora B is regulated by acetylation/deacetylation during mitosis in prostate cancer cells

    PubMed Central

    Fadri-Moskwik, Maria; Weiderhold, Kimberly N.; Deeraksa, Arpaporn; Chuang, Carol; Pan, Jing; Lin, Sue-Hwa; Yu-Lee, Li-Yuan

    2012-01-01

    Protein acetylation has been implicated in playing an important role during mitotic progression. Aurora B kinase is known to play a critical role in mitosis. However, whether Aurora B is regulated by acetylation is not known. Using IP with an anti-acetyl lysine antibody, we identified Aurora B as an acetylated protein in PC3 prostate cancer cells. Knockdown of HDAC3 or inhibiting HDAC3 deacetylase activity led to a significant increase (P<0.01 and P<0.05, respectively) in Aurora B acetylation as compared to siLuc or vehicle-treated controls. Increased Aurora B acetylation is correlated with a 30% reduction in Aurora B kinase activity in vitro and resulted in significant defects in Aurora B-dependent mitotic processes, including kinetochore-microtubule attachment and chromosome congression. Furthermore, Aurora B transiently interacts with HDAC3 at the kinetochore-microtubule interface of congressing chromosomes during prometaphase. This window of interaction corresponded with a transient but significant reduction (P=0.02) in Aurora B acetylation during early mitosis. Together, these results indicate that Aurora B is more active in its deacetylated state and further suggest a new mechanism by which dynamic acetylation/deacetylation acts as a rheostat to fine-tune Aurora B activity during mitotic progression.—Fadri-Moskwik, M., Weiderhold, K. N., Deeraksa, A., Chuang, C., Pan, J., Lin, S.-H., Yu-Lee, L.-Y. Aurora B is regulated by acetylation/deacetylation during mitosis in prostate cancer cells. PMID:22751009

  17. High polar organic-inorganic hybrid coating stir bar sorptive extraction combined with high performance liquid chromatography-inductively coupled plasma mass spectrometry for the speciation of seleno-amino acids and seleno-oligopeptides in biological samples.

    PubMed

    Mao, Xiangju; Hu, Bin; He, Man; Chen, Beibei

    2012-09-21

    In this work, partially sulfonated polystyrene-titania (PSP-TiO(2)) organic-inorganic hybrid stir bar coating was prepared by sol-gel and blending methods, and a new method of PSP-TiO(2) coating stir bar sorptive extraction (SBSE)-high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) was established for the analysis of seleno-amino acids (selenocystine (SeCys(2)), methylseleno-cysteine (MeSeCys), selenomethionine (SeMet) and selenoethionine (SeEt)) and seleno-oligopeptides (γ-glutamyl-Se-methyl-selenocysteine (γ-GluMeSeCys) and selenodiglutathione (GS-Se-SG)) in biological samples. The prepared high polar PSP-TiO(2) hybrid coating avoided the swelling of PSP and cracking of TiO(2) coating by combining the good film-forming property of PSP with the high mechanical strength of TiO(2). The scanning electron microscope (SEM) showed that no obvious swelling and damage occurred for the PSP-TiO(2) hybrid stir bar coating after 30 extraction/desorption cycles. The preparation reproducibility of PSP-TiO(2) coated stir bar, evaluated with the relative standard deviations (RSDs), was in the range of 6.7-12.6% (n=5) in one batch, and 9.9-17.6% (n=7) among different batches. The limits of detection (LODs) of the developed method for six target selenium species were in the range of 50.2-185.5 ngL(-1) (as (77)Se) and 45.9-158.8 ngL(-1) (as (82)Se) with the RSDs within 4.9-11.7%. The dynamic linear range was found to cover three orders of magnitude with correlation coefficient of 0.9995-0.9999. The developed method was applied for the analysis of Certified Reference Material SELM-1 selenium enriched yeast and the determined values were in good agreement with the certified values. The method has also been applied for the analysis of seleno-amino acids and seleno-oligopeptides in human urine and garlic samples. Different from the conventional organic polymer SBSE coatings (such as polydimethylsiloxane, PDMS), the extraction mechanism

  18. Microbial transformation of acetyl-11-keto-boswellic acid by Cunninghamella elegans.

    PubMed

    Xin, Xiu-Lan; Huo, Hua; Chen, Liang; Li, Jian; Sun, Jiang-Hao; Zheng, Peng-Wu; Sun, Yue; Wu, Zhi-Ming; Xiong, Ying-Hua

    2013-11-01

    Microbial biotransformation of acetyl-11-keto-boswellic acid by Cunninghamella elegans AS 3.1207 was carried out, and totally four transformed products were isolated. On the basis of the extensive spectral data, their structures were characterized as 7β-hydroxy-11-keto-boswellic acid (1), 7β,30-dihydroxy-11-keto-boswellic acid (2), 7β,16α-dihydroxy-3-acetyl-11-keto-boswellic acid (3), and 7β,15α,21β-trihydroxy-3-acetyl-11-keto-boswellic acid (4), respectively. Among them, products 1 and 2 are the new compounds.

  19. Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: overview and application.

    PubMed

    Yoshii, Yukie; Furukawa, Takako; Saga, Tsuneo; Fujibayashi, Yasuhisa

    2015-01-28

    Understanding cancer-specific metabolism is important for identifying novel targets for cancer diagnosis and therapy. Induced acetate/acetyl CoA metabolism is a notable feature that is related to fatty acid synthesis supporting tumor growth. In this review, we focused on the recent findings related to cancer acetate/acetyl CoA metabolism. We also introduce [1-¹¹C]acetate positron emission tomography (PET), which is a useful tool to visualize up-regulation of acetate/acetyl CoA metabolism in cancer, and discuss the utility of [1-¹¹C]acetate PET in cancer diagnosis and its application to personalized medicine.

  20. Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

    PubMed

    Young, Isaac A; Mittal, Chitvan; Shogren-Knaak, Michael A

    2016-02-01

    Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

  1. Sensitive gas chromatographic--mass spectrometric screening of acetylated benzodiazepines.

    PubMed

    Borrey, D; Meyer, E; Lambert, W; Van Calenbergh, S; Van Peteghem, C; De Leenheer, A P

    2001-02-23

    GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

  2. Oligopeptide cyclophilin inhibitors: a reassessment.

    PubMed

    Schumann, Michael; Jahreis, Günther; Kahlert, Viktoria; Lücke, Christian; Fischer, Gunter

    2011-11-01

    Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory interaction of linear proline-containing peptides with the active site of CypA can be increased through a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA, a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the context of the CsA framework a net charge of -7 clustered at the amino acid side chain of position 1 resulted in slightly improved CypA inhibition.

  3. Modifications of cell signalling and redox balance by targeting protein acetylation using natural and engineered molecules: implications in cancer therapy.

    PubMed

    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

    2014-01-01

    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-κB. 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

  4. Inter-laboratory study of an LC-MS/MS method for simultaneous determination of deoxynivalenol and its acetylated derivatives, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in wheat.

    PubMed

    Yoshinari, Tomoya; Tanaka, Toshitsugu; Ishikuro, Eiichi; Horie, Masakazu; Nagayama, Toshihiro; Nakajima, Masahiro; Naito, Shigehiro; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko

    2013-01-01

    To validate an LC-MS/MS method for simultaneous determination of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON), in wheat using a multifunctional column, an inter-laboratory study was performed in 9 laboratories using one blank wheat sample, three spiked wheat samples (10, 50, 150 µg/kg) and one naturally contaminated wheat sample. The recoveries ranged from 98.8 to 102.6% for DON, 89.3 to 98.7% for 3ADON, and from 84.9 to 90.0% for 15ADON. The relative standard deviations for repeatability (RSDR) and reproducibility (RSDR) of DON were in the ranges of 7.2-11.3% and 9.5-22.6%, respectively. For 3ADON, the RSDR ranged from 5.3 to 9.5% and the RSDR ranged from 16.1 to 18.0%, while for 15ADON, the RSDR ranged from 6.2 to 11.2% and the RSDR ranged from 17.0 to 27.2%. The HorRat values for the three analytes ranged from 0.4 to 1.2. These results validate this method for the simultaneous determination of DON and its acetylated derivatives, 3ADON and 15ADON.

  5. In vivo treatment by diallyl disulfide increases histone acetylation in rat colonocytes.

    PubMed

    Druesne-Pecollo, Nathalie; Chaumontet, Catherine; Pagniez, Anthony; Vaugelade, Pierre; Bruneau, Aurélia; Thomas, Muriel; Cherbuy, Claire; Duée, Pierre-Henri; Martel, Paule

    2007-03-01

    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.

  6. Histone Acetylation Modifiers in the Pathogenesis of Alzheimer’s Disease

    PubMed Central

    Lu, Xi; Wang, Li; Yu, Caijia; Yu, Daohai; Yu, Gang

    2015-01-01

    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

  7. In vivo treatment by diallyl disulfide increases histone acetylation in rat colonocytes

    SciTech Connect

    Druesne-Pecollo, Nathalie . E-mail: Nathalie.Pecollo@jouy.inra.fr; Chaumontet, Catherine; Pagniez, Anthony; Vaugelade, Pierre; Bruneau, Aurelia; Thomas, Muriel; Cherbuy, Claire; Duee, Pierre-Henri; Martel, Paule

    2007-03-02

    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.

  8. Effect of pulsed electric fields assisted acetylation on morphological, structural and functional characteristics of potato starch.

    PubMed

    Hong, Jing; Chen, Rujiao; Zeng, Xin-An; Han, Zhong

    2016-02-01

    Pulsed electric fields (PEF)-assisted acetylation of potato starch with different degree of substitution (DS) was prepared and effects of PEF strength, reaction time, starch concentration on DS were studied by response surface methodology. Results showed DS was increased from 0.054 (reaction time of 15 min) to 0.130 (reaction time of 60 min) as PEF strength increased from 3 to 5 kV/cm. External morphology revealed that acetylated starch with higher DS was aggravated more bulges and asperities. Fourier-transformed infrared spectroscopy confirmed the introduction of acetyl group through a band at 1730 cm(-1). The optimum sample (DS =0 .13) had lower retrogradation (39.1%), breakdown (155 BU) and setback value (149BU), while pasting temperature (62.2 °C) was slightly higher than non-PEF-assisted samples. These results demonstrated PEF treatment can be a potential and beneficial method for acetylation and achieve higher DS with shorter reaction time.

  9. Acetylation Enhances the Promoting Role of AIB1 in Breast Cancer Cell Proliferation

    PubMed Central

    You, Dingyun; Zhao, Hongbo; Wang, Yan; Jiao, Yang; Lu, Minnan; Yan, Shan

    2016-01-01

    The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator, which is overexpressed in various types of human cancers, including breast cancer. However, the molecular mechanisms regulating AIB1 function remain largely unknown. In this study, we present evidence demonstrating that AIB1 is acetylated by MOF in human breast cancer cells. Moreover, we also found that the acetylation of AIB1 enhances its function in promoting breast cancer cell proliferation. We further showed that the acetylation of AIB1 is required for its recruitment to E2F1 target genes by E2F1. More importantly, we found that the acetylation levels of AIB1 are greatly elevated in human breast cancer cells compared with that in non-cancerous cells. Collectively, our results shed light on the molecular mechanisms that regulate AIB1 function in breast cancer. PMID:27665502

  10. N-acetyl-L-phenylalanyl-L-phenylalaninol a metabolite of Emericellopsis salmosynnemata.

    PubMed

    Argoudelis, A D; Mizsak, S A; Baczynskyj, L

    1975-10-01

    A new metabolite N-acetyl-L-phenylalanyl-L-phenylalaninol was isolated from culture filtrates of Emericellopsis salmosynnemata which produces zervamicins I and II. The structure was assigned from spectral properties and degradative studies. PMID:1184465

  11. An autopsy case of acetyl fentanyl intoxication caused by insufflation of 'designer drugs'.

    PubMed

    Takase, Izumi; Koizumi, Takako; Fujimoto, Ihoko; Yanai, Aya; Fujimiya, Tatsuya

    2016-07-01

    We present a fatal case of intoxication due to insufflation of acetyl fentanyl. His blood concentration of acetyl fentanyl was 270ng/mL, and the manner of death was classified as an accident. This is the first report of an autopsy case of acetyl fentanyl delivered by insufflation, rather than intravenous administration. He had been snoring loudly for at least 12h prior to death, and transport to a hospital during this time and treatment with naloxone may have saved his life. In this sense, it can be said that his death was preventable. This case reemphasizes the risk of death associated with drug overdose and the narrow range of acetyl fentanyl between the effective dose (ED50) and lethal dose (LD50). The case should also raise awareness among medical professionals of the effectiveness of naloxone and the need to establish a comprehensive system for toxicological analysis while keeping the possibility of use of 'designer drugs' in mind. PMID:27497332

  12. Data for global lysine-acetylation analysis in rice (Oryza sativa).

    PubMed

    Xiong, Yehui; Zhang, Kai; Cheng, Zhongyi; Wang, Guo-Liang; Liu, Wende

    2016-06-01

    Rice is one of the most important crops for human consumption and is a staple food for over half of the world׳s population (Yu et al., 2002) [1]. A systematic identification of the lysine acetylome was performed by our research (Xiong et al., 2016) [2]. Rice plant samples were collected from 5 weeks old seedlings (Oryza sativa, Nipponbare). After the trypsin digestion and immunoaffinity precipitation, LC-MS/MS approach was used to identify acetylated peptides. After the collected MS/MS data procession and GO annotation, the InterProScan was used to annotate protein domain. Subcellular localization of the identified acetylated proteins was predicted by WoLF PSORT. The KEGG pathway database was used to annotate identified acetylated protein interactions, reactions, and relations. The data, supplied in this article, are related to "A comprehensive catalog of the lysine-acetylation targets in rice (O. sativa) based on proteomic analyses" by Xiong et al. (2016) [2]. PMID:26977447

  13. Manipulation of the host protein acetylation network by human immunodeficiency virus type 1

    PubMed Central

    Jeng, Mark Y.; Ali, Ibraheem; Ott, Melanie

    2016-01-01

    Over the last 15 years, protein acetylation has emerged as a globally important post-translational modification that fine-tunes major cellular processes in many life forms. This dynamic regulatory system is critical both for complex eukaryotic cells and for the viruses that infect them. HIV-1 accesses the host acetylation network by interacting with several key enzymes, thereby promoting infection at multiple steps during the viral life cycle. Inhibitors of host histone deacetylases and bromodomain-containing proteins are now being pursued as therapeutic strategies to enhance current antiretroviral treatment. As more acetylation-targeting compounds are reaching clinical trials, it is timely to review the role of reversible protein acetylation in HIV-infected CD4+ T cells. PMID:26329395

  14. Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

    1974-01-01

    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.

  15. Processing of Numerical and Proportional Quantifiers.

    PubMed

    Shikhare, Sailee; Heim, Stefan; Klein, Elise; Huber, Stefan; Willmes, Klaus

    2015-09-01

    Quantifier expressions like "many" and "at least" are part of a rich repository of words in language representing magnitude information. The role of numerical processing in comprehending quantifiers was studied in a semantic truth value judgment task, asking adults to quickly verify sentences about visual displays using numerical (at least seven, at least thirteen, at most seven, at most thirteen) or proportional (many, few) quantifiers. The visual displays were composed of systematically varied proportions of yellow and blue circles. The results demonstrated that numerical estimation and numerical reference information are fundamental in encoding the meaning of quantifiers in terms of response times and acceptability judgments. However, a difference emerges in the comparison strategies when a fixed external reference numerosity (seven or thirteen) is used for numerical quantifiers, whereas an internal numerical criterion is invoked for proportional quantifiers. Moreover, for both quantifier types, quantifier semantics and its polarity (positive vs. negative) biased the response direction (accept/reject). Overall, our results indicate that quantifier comprehension involves core numerical and lexical semantic properties, demonstrating integrated processing of language and numbers. PMID:25631283

  16. Induction of histone acetylation on the sucrase-isomaltase gene in the postnatal rat jejunum.

    PubMed

    Yorita, Satoko; Mochizuki, Kazuki; Goda, Toshinao

    2009-04-23

    The rapid induction of the sucrase-isomaltase (SI) gene in rat jejunum from the onset to final period of weaning was associated with increases of the acetylation of histones H3 and H4 on the promoter/transcriptional region of the gene, suggesting that an abrupt jejunal induction of histone acetylation changes on the SI gene during this period may be concerned with the expression of the gene.

  17. Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.

    PubMed

    Kuo, Yin-Ming; Andrews, Andrew J

    2013-01-01

    Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5. PMID:23437046

  18. Acetylation in vitro of constituent polypeptides by smooth endoplasmic reticulum (SER) and Golgi membrane fractions

    SciTech Connect

    Sambasivam, H.; Murray, R.K.

    1986-05-01

    Many polypeptides of the membranes of the ER are phosphorylated. To determine if any such polypeptides are acetylated, microsomal and other classical subcellular fractions were incubated with (/sup 3/H) acetyl-CoA; the specific activity of the microsomal fraction (MF) was the greatest. SDS-PAGE revealed that some 20 polypeptides of the MF were acetylated; 2-D electrophoretograms extended this number to approximately 60. Separation of the MF into smooth (S) and rough (R) fractions showed that the great majority of the labelled polypeptides belonged to the former. Isolation of a Golgi fraction revealed that its acetylation activity was approximately 3-fold greater than the SER fraction. Extensive proteolytic digestion of the MF followed by radiochromatography disclosed some 9 components whose precise nature (acetylated amino acids and/or sialic acids, etc.) is under study. Assuming that the majority of the radioactivity is in the former components and that a similar process occurs in vivo, the authors suggest that the Golgi apparatus may be a major site of acetylation of membrane and possibly other proteins.

  19. Modulation of histone deacetylase 6 (HDAC6) nuclear import and tubulin deacetylase activity through acetylation.

    PubMed

    Liu, Yuanjing; Peng, Lirong; Seto, Edward; Huang, Suming; Qiu, Yi

    2012-08-17

    The reversible acetylation of histones and non-histone proteins by histone acetyltransferases and deacetylases (HDACs) plays a critical role in many cellular processes in eukaryotic cells. HDAC6 is a unique histone deacetylase with two deacetylase domains and a C-terminal zinc finger domain. HDAC6 resides mainly in the cytoplasm and regulates many important biological processes, including cell migration and degradation of misfold proteins. HDAC6 has also been shown to localize in the nucleus to regulate transcription. However, how HDAC6 shuttles between the nucleus and cytoplasm is largely unknown. In addition, it is not clear how HDAC6 enzymatic activity is modulated. Here, we show that HDAC6 can be acetylated by p300 on five clusters of lysine residues. One cluster (site B) of acetylated lysine is in the N-terminal nuclear localization signal region. These lysine residues in site B were converted to glutamine to mimic acetylated lysines. The mutations significantly reduced HDAC6 tubulin deacetylase activity and further impaired cell motility, but had no effect on histone deacetylase activity. More interestingly, these mutations retained HDAC6 in the cytoplasm by blocking the interaction with the nuclear import protein importin-α. The retention of HDAC6 in the cytoplasm by acetylation eventually affects histone deacetylation. Thus, we conclude that acetylation is an important post-translational modification that regulates HDAC6 tubulin deacetylase activity and nuclear import.

  20. The energetics of the acetylation switch in p53-mediated transcriptional activation.

    PubMed

    Eichenbaum, Kenneth D; Rodríguez, Yoel; Mezei, Mihaly; Osman, Roman

    2010-02-01

    Targeted therapeutic intervention in receptor-ligand interactions of p53-mediated tumor suppression can impact progression of disease, aging, and variation in genetic expression. Here, we conducted a number of molecular simulations, based on structures of p53 in complex with its transcriptional coactivating CBP bromodomain, determined by NMR spectroscopy, to investigate the energetics of the binding complex. Building on the observation that acetylation of K382 in p53 serves as the essential triggering switch for a specific interaction with CBP, we assessed the differential effect of acetylation on binding from simulations of an octapeptide derived from p53 with acetylated and nonacetylated K382 (residues 379-386). Cluster analysis of the simulations shows that acetylation of the free peptide does not significantly change the population of the preferred conformation of the peptide in solution for binding to CBP. Conversion of the acetylated K382 to nonacetylated form with free energy perturbation (FEP) simulations of the p53 CBP complex and the free peptide showed that the relative contribution of the acetyl group to binding is 4.8 kcal/mol. An analysis of residue contributions to the binding energy using an MM-GBSA approach agrees with the FEP results and sheds additional light on the origin of selectivity in p53 binding to the CBP bromodomain.

  1. PPARα Activation Induces Nε-Lys-Acetylation of Rat Liver Peroxisomal Multifunctional Enzyme Type 1

    PubMed Central

    Contreras, Miguel A.; Alzate, Oscar; Singh, Avtar K.

    2013-01-01

    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 PPARα-agonist treated rat liver screened with an anti-Nε-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 (K155, K173, K190, and K583). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPARα-dependent proliferation of peroxisomes in rat liver. PMID:24092543

  2. Acetyl-coenzyme A: a metabolic master regulator of autophagy and longevity.

    PubMed

    Schroeder, Sabrina; Pendl, Tobias; Zimmermann, Andreas; Eisenberg, Tobias; Carmona-Gutierrez, Didac; Ruckenstuhl, Christoph; Mariño, Guillermo; Pietrocola, Federico; Harger, Alexandra; Magnes, Christoph; Sinner, Frank; Pieber, Thomas R; Dengjel, Jörn; Sigrist, Stephan J; Kroemer, Guido; Madeo, Frank

    2014-07-01

    As the major lysosomal degradation pathway, autophagy represents the guardian of cellular homeostasis, removing damaged and potentially harmful material and replenishing energy reserves in conditions of starvation. Given its vast physiological importance, autophagy is crucially involved in the process of aging and associated pathologies. Although the regulation of autophagy strongly depends on nutrient availability, specific metabolites that modulate autophagic responses are poorly described. Recently, we revealed nucleo-cytosolic acetyl-coenzyme A (AcCoA) as a phylogenetically conserved inhibitor of starvation-induced and age-associated autophagy. AcCoA is the sole acetyl-group donor for protein acetylation, explaining why pharmacological or genetic manipulations that modify the concentrations of nucleo-cytosolic AcCoA directly affect the levels of protein acetylation. The acetylation of histones and cytosolic proteins inversely correlates with the rate of autophagy in yeast and mammalian cells, respectively, despite the fact that the routes of de novo AcCoA synthesis differ across phyla. Thus, we propose nucleo-cytosolic AcCoA to act as a conserved metabolic rheostat, linking the cellular metabolic state to the regulation of autophagy via effects on protein acetylation.

  3. Histone H3 globular domain acetylation identifies a new class of enhancers

    PubMed Central

    Pradeepa, Madapura M; Grimes, Graeme R; Kumar, Yatendra; Olley, Gabrielle; Taylor, Gillian C A; Schneider, Robert; Bickmore, Wendy A

    2016-01-01

    Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes1. This includes acetylation of H3 on lysine 27 (H3K27ac), which blocks the deposition of polycomb mediated H3K27me32. H3K27ac is also widely used to identify active enhancers3,4, and the assumption has been that profiling of H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of H3 (H3K64ac and H3K122ac) marks active gene promoters and also a subset of active enhancers. Moreover, we find a novel class of active functional enhancers that are marked by H3K122ac but lack H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than was previously considered. PMID:27089178

  4. Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity

    PubMed Central

    Park, Joo-Man; Kim, Tae-Hyun; Jo, Seong-Ho; Kim, Mi-Young; Ahn, Yong-Ho

    2015-01-01

    Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD+-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies. PMID:26620281

  5. Argyrophilic grain disease differs from other tauopathies by lacking tau acetylation

    PubMed Central

    Grinberg, Lea Tenenholz; Wang, Xuehua; Wang, Chao; Sohn, Peter Dongmin; Theofilas, Panos; Sidhu, Manu; Arevalo, John Benjamin; Heinsen, Helmut; Huang, Eric J.; Rosen, Howard; Miller, Bruce L.; Gan, Li; Seeley, William W.

    2013-01-01

    Post-translational modifications play a key role in tau protein aggregation and related neurodegeneration. Because hyperphosphorylation alone does not necessarily cause tau aggregation, other post-translational modifications have been recently explored. Tau acetylation promotes aggregation and inhibits tau’s ability to stabilize microtubules. Recent studies have shown co-localization of acetylated and phosphorylated tau in AD and some 4R tauopathies. We developed a novel monoclonal antibody against acetylated tau at lysine residue 274, which recognizes both 3R and 4R tau, and used immunohistochemistry and immunofluorescence to probe 22 cases, including AD and another eight familial or sporadic tauopathies. Acetylated tau was identified in all tauopathies except argyrophilic grain disease (AGD). AGD is an age-associated, common but atypical 4R tauopathy, not always associated with clinical progression. Pathologically, AGD is characterized by neuropil grains, pre-neurofibrillary tangles, and oligodendroglial coiled bodies, all recognized by phospho-tau antibodies. The lack of acetylated tau in these inclusions suggests that AGD represents a distinctive tauopathy. Our data converge with previous findings to raise the hypothesis that AGD could play a protective role against the spread of AD-related tau pathology. Tau acetylation as a key modification for the propagation tau toxicity deserves further investigation. PMID:23371364

  6. Inhibition of acetyl-coenzyme A carboxylase by two classes of grass-selective herbicides

    SciTech Connect

    Rendina, A.R.; Craig-Kennard, A.C.; Beaudoin, J.D.; Breen, M.K. )

    1990-05-01

    The selective grass herbicides diclofop, haloxyfop, and trifop (((aryloxy)phenoxy)propionic acids) and alloxydim, sethoxydim, and clethodim (cyclohexanediones) are potent, reversible inhibitors of acetyl-coenzyme A carboxylase (ACC) partially purified from barley, corn, and wheat. Although inhibition of the wheat enzyme by clethodim and diclofop is noncompetitive versus each of the substrates adenosine triphosphate (ATP), HCO{sub 3}{sup {minus}}, and acetyl-coenzyme A (acetyl-CoA), diclofop and clethodim are nearly competitive versus acetyl-CoA since the level of inhibition is most sensitive to the concentration of acetyl-CoA (K{sub is} < K{sub ii}). To conclusively show whether the herbicides interact at the biotin carboxylation site or the carboxyl transfer site, the inhibition of isotope exchange and partial reactions catalyzed at each site was studied with the wheat enzyme. Only the ({sup 14}C)acetyl-CoA-malonyl-CoA exchange and decarboxylation of ({sup 14}C)malonyl-CoA reactions are strongly inhibited by clethodim and diclofop, suggesting that the herbicides interfere with the carboxyl transfer site rather than the biotin carboxylation site of the enzyme. Double-inhibition studies with diclofop and clethodim suggest that the ((aryloxy)phenoxy)propionic acid and cyclohexanedione herbicides may bind to the same region of the enzyme.

  7. Histone H3 globular domain acetylation identifies a new class of enhancers.

    PubMed

    Pradeepa, Madapura M; Grimes, Graeme R; Kumar, Yatendra; Olley, Gabrielle; Taylor, Gillian C A; Schneider, Robert; Bickmore, Wendy A

    2016-06-01

    Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.

  8. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    PubMed Central

    Pathak, Ravi; Philizaire, Marc; Mujtaba, Shiraz

    2015-01-01

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets. PMID:26295410

  9. Urinary mutagenicity and N-acetylation phenotype in textile industry workers exposed to arylamines

    SciTech Connect

    Sinues, B.; Perez, J.; Bernal, M.L.; Saenz, M.A.; Lanuza, J.; Bartolome, M. )

    1992-09-15

    Primary aromatic amines have been identified epidemiologically as human carcinogens. It has been suggested that the target organ affected by aromatic amines is dependent on the rate of metabolic activation. Epidemiological studies have shown an association between low acetyl transferase activity and bladder cancer risk. On this basis, our working hypothesis was that the slow acetylators could follow in a higher extent the metabolic pathway independent of N-acetylation, leading to the excretion of conjugates of electrophyles with glucuronic acid. The instability of these glucuronides could be responsible for the association between arylamine-induced bladder cancer and slow acetylator phenotype. A total of 153 individuals were included in this study: 70 exposed to arylamines (working in textile industry) and 83 nonexposed. The following parameters were determined in urine: mutagenic index in the absence of metabolic activation, S9; mutagenic index in the presence of S9; and the mutagenic index after incubation of the urine with beta-glucuronidase. All individuals were phenotyped according to their capacity of N-acetylation by using isoniazid as drug test. The results show that the mutagenic index after incubation of the urine with beta-glucuronidase is statistically higher in exposed subjects when compared with nonexposed individuals (P less than 0.001), this parameter being statistically higher among exposed subjects who were slow acetylators than among rapid metabolizers, independent of the fact that they were smokers or nonsmokers. There were no significant differences between groups for the mutagenicity in urine not incubated with beta-glucuronidase.

  10. The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

    PubMed Central

    Wurm, Torsten; Wright, Diana G.; Polakowski, Nicholas; Mesnard, Jean-Michel; Lemasson, Isabelle

    2012-01-01

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection. PMID:22434882

  11. Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

    PubMed Central

    Yildirim, Ferah; Ji, Shengbo; Kronenberg, Golo; Barco, Angel; Olivares, Roman; Benito, Eva; Dirnagl, Ulrich; Gertz, Karen; Endres, Matthias

    2014-01-01

    Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)–binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons. PMID:24748101

  12. Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding

    PubMed Central

    Holmes, William M.; Mannakee, Brian K.; Gutenkunst, Ryan N.; Serio, Tricia R.

    2014-01-01

    N-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. While loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI+], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910

  13. Novel mechanism of surface catalysis of protein adduct formation. NMR studies of the acetylation of ubiquitin.

    PubMed

    Macdonald, J M; Haas, A L; London, R E

    2000-10-13

    Reactivity of surface lysyl residues of proteins with a broad range of chemical agents has been proposed to be dependent on the catalytic microenvironment of the residue. We have investigated the acetylation of wild type ubiquitin and of the UbH68N mutant to evaluate the potential contribution of His-68 to the reactivity of Lys-6, which is about 4 A distant. These studies were performed using [1-(13)C]acetyl salicylate or [1,1'-(13)C(2)]acetic anhydride, and the acetylated products were detected by two-dimensional heteronuclear multiple quantum coherence spectroscopy. The results demonstrate that His-68 makes a positive contribution to the rate of acetylation of Lys-6 by labeled aspirin. Additionally, a pair of transient resonances is observed after treatment of wild type ubiquitin with the labeled acetic anhydride but not upon treatment of the H68N mutant. These resonances are assigned to the acetylated His-68 residue. The loss of intensity of the acetylhistidine resonances is accompanied by an increase in intensity of the acetyl-Lys-6 peak, supporting the existence of a transacetylation process between the acetylhistidine 68 and lysine 6 residues located on the protein surface. Hence, this may be the first direct demonstration of a catalytic intermediate forming on the protein surface. PMID:10906321

  14. Autoregulation of the Rsc4 Tandem Bromodomain by Gcn5 Acetylation

    SciTech Connect

    VanDemark,A.; Kasten, M.; Ferris, E.; Heroux, A.; Hill, C.; Cairns, B.

    2007-01-01

    An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.

  15. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat

    PubMed Central

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

  16. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation

    PubMed Central

    Cohen, Todd J.; Constance, Brian H.; Hwang, Andrew W.; James, Michael; Yuan, Chao-Xing

    2016-01-01

    Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies. PMID:27383765

  17. The oncoprotein HBXIP promotes migration of breast cancer cells via GCN5-mediated microtubule acetylation.

    PubMed

    Li, Leilei; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2015-03-13

    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 α-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.

  18. Nuclear factor κB-dependent histone acetylation is specifically involved in persistent forms of memory.

    PubMed

    Federman, Noel; de la Fuente, Verónica; Zalcman, Gisela; Corbi, Nicoletta; Onori, Annalisa; Passananti, Claudio; Romano, Arturo

    2013-04-24

    Memory consolidation requires gene expression regulation by transcription factors, which eventually may induce chromatin modifications as histone acetylation. This mechanism is regulated by histone acetylases and deacetylases. It is not yet clear whether memory consolidation always recruits histone acetylation or it is only engaged in more persistent memories. To address this question, we used different strength of training for novel object recognition task in mice. Only strong training induced a long-lasting memory and an increase in hippocampal histone H3 acetylation. Histone acetylase inhibition in the hippocampus during consolidation impaired memory persistence, whereas histone deacetylase inhibition caused weak memory to persist. Nuclear factor κB (NF-κB) transcription factor inhibition impaired memory persistence and, concomitantly, reduced the general level of H3 acetylation. Accordingly, we found an important increase in H3 acetylation at a specific NF-κB-regulated promoter region of the Camk2d gene, which was reversed by NF-kB inhibition. These results show for the first time that histone acetylation is a specific molecular signature of enduring memories.

  19. Histone acetylation is recruited in consolidation as a molecular feature of stronger memories.

    PubMed

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

    2009-10-01

    Gene expression is a key process for memory consolidation. Recently, the participation of epigenetic mechanisms like histone acetylation was evidenced in long-term memories. However, until now the training strength required and the persistence of the chromatin acetylation recruited are not well characterized. Here we studied whether histone acetylation is involved in consolidation in invertebrates, whether it depends on the training strength, and whether it is a permanent or transient mechanism. We used a well-characterized memory model in invertebrates, the context-signal memory in crabs. Our results show no changes in histone 3 (H3) acetylation during consolidation of a standard training protocol. However, strong training induced a significant increase in H3 acetylation 1-h post-training, returning to basal levels afterward. Accordingly, the administration of histone deacetylase inhibitors sodium butyrate (NaB) and trichostatin A allowed a weak training to induce long-term memory. NaB enhanced memory in two phases during consolidation. These findings support that H3 acetylation (1) is involved in consolidation, (2) occurs only after strong training, (3) is a transient process, and (4) memory is enhanced in two phases. The coincidence of these phases with other mechanisms of gene expression is discussed.

  20. Processing of Numerical and Proportional Quantifiers

    ERIC Educational Resources Information Center

    Shikhare, Sailee; Heim, Stefan; Klein, Elise; Huber, Stefan; Willmes, Klaus

    2015-01-01

    Quantifier expressions like "many" and "at least" are part of a rich repository of words in language representing magnitude information. The role of numerical processing in comprehending quantifiers was studied in a semantic truth value judgment task, asking adults to quickly verify sentences about visual displays using…

  1. Ambident Reactivity of Acetyl- and Formyl-Stabilized Phosphonium Ylides.

    PubMed

    Byrne, Peter A; Karaghiosoff, Konstantin; Mayr, Herbert

    2016-09-01

    The kinetics and mechanism of the reactions of formyl-stabilized ylide Ph3P═CHCHO (1) and acetyl-stabilized ylide Ph3P═CHCOMe (2) with benzhydrylium ions (Ar2CH(+), 3) were investigated by UV-vis and NMR spectroscopy. As ambident nucleophiles, ylides 1 and 2 can react at oxygen as well as at the α-carbon. For some reactions, it was possible to determine the second-order rate constant for O-attack as well as for C-attack and to derive the nucleophile-specific parameters N and sN according to the correlation lg k (20 °C) = sN(E + N) for both nucleophilic sites. Generally, O-attack of benzhydrylium ions is faster than C-attack. However, the initially formed benzhydryloxyvinylphosphonium ions can only be observed by NMR spectroscopy when benzhydryl cations with high Lewis acidity are employed. In other cases, rearrangement to the thermodynamically more stable products arising from C-attack occurs. The results derived from our investigations are employed to rationalize the behavior of ambident nucleophiles 1 and 2 in reactions with carbon-centered electrophiles in general. It is shown that the principle of hard and soft acids and bases (HSAB) and the related Klopman-Salem concept of charge and orbital control lead to incorrect predictions of regioselectivity. We also show that the rate of the Wittig reaction of ylide 2 with aldehyde 14 is significantly faster than the rate of either C- or O-attack calculated using lg k (20 °C) = sN(E + N), thus indicating that the oxaphosphetane is formed by a concerted [2 + 2] cycloaddition. PMID:27499510

  2. Acetyl salicylic acid attenuates cardiac hypertrophy through Wnt signaling.

    PubMed

    Gitau, Samuel Chege; Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Liang, Haihai; Qian, Ming; Lv, Lifang; Li, Tianshi; Xu, Bozhi; Wang, Zhiguo; Zhang, Yong; Xu, Chaoqian; Lu, Yanjie; Du, Zhiming; Shan, Hongli; Yang, Baofeng

    2015-12-01

    Ventricular hypertrophy is a powerful and independent predictor of cardiovascular morbid events. The vascular properties of low-dose acetyl salicylic acid (aspirin) provide cardiovascular benefits through the irreversible inhibition of platelet cyclooxygenase 1; however, the possible anti-hypertrophic properties and potential mechanism of aspirin have not been investigated in detail. In this study, healthy wild-type male mice were randomly divided into three groups and subjected to transverse aortic constriction (TAC) or sham operation. The TAC-operated mice were treated with the human equivalent of low-dose aspirin (10 mg·kg(-1)·d(-1)); the remaining mice received an equal amount of phosphate buffered saline with 0.65% ethanol, which was used as a vehicle. A cardiomyocyte hypertrophy model induced by angiotensin II (10 nmol·L(-1)) was treated with the human equivalent of low (10 or 100 μmol·L(-1)) and high (1000 μmol·L(-1)) aspirin concentrations in plasma. Changes in the cardiac structure and function were assessed through echocardiography and transmission electron microscopy. Gene expression was determined through RT-PCR and western blot analysis. Results indicated that aspirin treatment abrogated the increased thickness of the left ventricular anterior and posterior walls, the swelling of mitochondria, and the increased surface area in in vivo and in vitro hypertrophy models. Aspirin also normalized the upregulated hypertrophic biomarkers, β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and b-type natriuretic peptide (BNP). Aspirin efficiently reversed the upregulation of β-catenin and P-Akt expression and the TAC- or ANG II-induced downregulation of GSK-3β. Therefore, low-dose aspirin possesses significant anti-hypertrophic properties at clinically relevant concentrations for anti-thrombotic therapy. The downregulation of β-catenin and Akt may be the underlying signaling mechanism of the effects of aspirin. PMID:26626190

  3. Acetyl salicylic acid attenuates cardiac hypertrophy through Wnt signaling.

    PubMed

    Gitau, Samuel Chege; Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Liang, Haihai; Qian, Ming; Lv, Lifang; Li, Tianshi; Xu, Bozhi; Wang, Zhiguo; Zhang, Yong; Xu, Chaoqian; Lu, Yanjie; Du, Zhiming; Shan, Hongli; Yang, Baofeng

    2015-12-01

    Ventricular hypertrophy is a powerful and independent predictor of cardiovascular morbid events. The vascular properties of low-dose acetyl salicylic acid (aspirin) provide cardiovascular benefits through the irreversible inhibition of platelet cyclooxygenase 1; however, the possible anti-hypertrophic properties and potential mechanism of aspirin have not been investigated in detail. In this study, healthy wild-type male mice were randomly divided into three groups and subjected to transverse aortic constriction (TAC) or sham operation. The TAC-operated mice were treated with the human equivalent of low-dose aspirin (10 mg·kg(-1)·d(-1)); the remaining mice received an equal amount of phosphate buffered saline with 0.65% ethanol, which was used as a vehicle. A cardiomyocyte hypertrophy model induced by angiotensin II (10 nmol·L(-1)) was treated with the human equivalent of low (10 or 100 μmol·L(-1)) and high (1000 μmol·L(-1)) aspirin concentrations in plasma. Changes in the cardiac structure and function were assessed through echocardiography and transmission electron microscopy. Gene expression was determined through RT-PCR and western blot analysis. Results indicated that aspirin treatment abrogated the increased thickness of the left ventricular anterior and posterior walls, the swelling of mitochondria, and the increased surface area in in vivo and in vitro hypertrophy models. Aspirin also normalized the upregulated hypertrophic biomarkers, β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and b-type natriuretic peptide (BNP). Aspirin efficiently reversed the upregulation of β-catenin and P-Akt expression and the TAC- or ANG II-induced downregulation of GSK-3β. Therefore, low-dose aspirin possesses significant anti-hypertrophic properties at clinically relevant concentrations for anti-thrombotic therapy. The downregulation of β-catenin and Akt may be the underlying signaling mechanism of the effects of aspirin.

  4. Resistance to acetyl-CoA carboxylase-inhibiting herbicides.

    PubMed

    Kaundun, Shiv S

    2014-09-01

    Resistance to acetyl-CoA carboxylase herbicides is documented in at least 43 grass weeds and is particularly problematic in Lolium, Alopecurus and Avena species. Genetic studies have shown that resistance generally evolves independently and can be conferred by target-site mutations at ACCase codon positions 1781, 1999, 2027, 2041, 2078, 2088 and 2096. The level of resistance depends on the herbicides, recommended field rates, weed species, plant growth stages, specific amino acid changes and the number of gene copies and mutant ACCase alleles. Non-target-site resistance, or in essence metabolic resistance, is prevalent, multigenic and favoured under low-dose selection. Metabolic resistance can be specific but also broad, affecting other modes of action. Some target-site and metabolic-resistant biotypes are characterised by a fitness penalty. However, the significance for resistance regression in the absence of ACCase herbicides is yet to be determined over a practical timeframe. More recently, a fitness benefit has been reported in some populations containing the I1781L mutation in terms of vegetative and reproductive outputs and delayed germination. Several DNA-based methods have been developed to detect known ACCase resistance mutations, unlike metabolic resistance, as the genes remain elusive to date. Therefore, confirmation of resistance is still carried out via whole-plant herbicide bioassays. A growing number of monocotyledonous crops have been engineered to resist ACCase herbicides, thus increasing the options for grass weed control. While the science of ACCase herbicide resistance has progressed significantly over the past 10 years, several avenues provided in the present review remain to be explored for a better understanding of resistance to this important mode of action.

  5. Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

    PubMed Central

    Dresel, H A; Friedrich, E; Via, D P; Schettler, G; Sinn, H

    1985-01-01

    Acetylated low density lipoprotein (acetyl-LDL) binding to hepatic membrane proteins of rats was analysed in vitro by ligand blotting. Specific binding could be demonstrated to two hepatic proteins with an apparent mol. wt. of 250 kd and 220 kd. Polyanionic competitors and maleylated bovine serum albumin inhibited the binding of acetyl-LDL effectively. To determine the sites of the catabolism of acetyl-LDL, [131I]-acetyl-LDL was injected intravenously into control rats and rats pre-treated with the known competitors of the acetyl-LDL binding. Distribution of the radiolabelled acetyl-LDL was followed by a scintillation camera. Six minutes after injection, the radioactivity was concentrated in the liver. The competitors and unlabelled acetyl-LDL but not native LDL reduced the hepatic uptake of [131I]acetyl-LDL dramatically. Thus, the sensitivity of the 220- and 250-kd membrane binding sites to the competitors for the acetyl-LDL binding resembled that of the hepatic compartment in vivo. Finally, an application of scintigraphy with radiolabelled low density lipoproteins for diagnostic evaluation of tumor compartments is presented. Images Fig. 2. Fig. 4. Fig. 5. PMID:4006910

  6. Acetylated α-Tubulin Regulated by N-Acetyl-Seryl-Aspartyl-Lysyl-Proline(Ac-SDKP) Exerts the Anti-fibrotic Effect in Rat Lung Fibrosis Induced by Silica.

    PubMed

    Xiaojun, Wang; Yan, Liu; Hong, Xu; Xianghong, Zhang; Shifeng, Li; Dingjie, Xu; Xuemin, Gao; Lijuan, Zhang; Bonan, Zhang; Zhongqiu, Wei; Ruimin, Wang; Brann, Darrell; Fang, Yang

    2016-01-01

    Silicosis is the most serious occupational disease in China. The objective of this study was to screen various proteins related to mechanisms of the pathogenesis of silicosis underlying the anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) using proteomic profile analysis. We also aimed to explore a potential mechanism of acetylated α-tubulin (α-Ac-Tub) regulation by Ac-SDKP. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to assess the different protein expression profiles between control and silicosis rats treated with or without Ac-SDKP. Twenty-nine proteins were identified to be potentially involved in the progression of silicosis and the anti-fibrotic effect of Ac-SDKP. Our current study finds that 1) the lost expression of Ac-Tub-α may be a new mechanism in rat silicosis; 2) treatment of silicotic rats with N-acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is related with deacetylase family member 6 (HDAC6) and α-tubulin acetyl transferase (α-TAT1). Our data suggest that α-Ac-Tub regulation by Ac-SDKP may potentially be a new anti-fibrosis mechanism. PMID:27577858

  7. Acetylated α-Tubulin Regulated by N-Acetyl-Seryl-Aspartyl-Lysyl-Proline(Ac-SDKP) Exerts the Anti-fibrotic Effect in Rat Lung Fibrosis Induced by Silica.

    PubMed

    Xiaojun, Wang; Yan, Liu; Hong, Xu; Xianghong, Zhang; Shifeng, Li; Dingjie, Xu; Xuemin, Gao; Lijuan, Zhang; Bonan, Zhang; Zhongqiu, Wei; Ruimin, Wang; Brann, Darrell; Fang, Yang

    2016-08-31

    Silicosis is the most serious occupational disease in China. The objective of this study was to screen various proteins related to mechanisms of the pathogenesis of silicosis underlying the anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) using proteomic profile analysis. We also aimed to explore a potential mechanism of acetylated α-tubulin (α-Ac-Tub) regulation by Ac-SDKP. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to assess the different protein expression profiles between control and silicosis rats treated with or without Ac-SDKP. Twenty-nine proteins were identified to be potentially involved in the progression of silicosis and the anti-fibrotic effect of Ac-SDKP. Our current study finds that 1) the lost expression of Ac-Tub-α may be a new mechanism in rat silicosis; 2) treatment of silicotic rats with N-acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is related with deacetylase family member 6 (HDAC6) and α-tubulin acetyl transferase (α-TAT1). Our data suggest that α-Ac-Tub regulation by Ac-SDKP may potentially be a new anti-fibrosis mechanism.

  8. Acetylated α-Tubulin Regulated by N-Acetyl-Seryl-Aspartyl-Lysyl-Proline(Ac-SDKP) Exerts the Anti-fibrotic Effect in Rat Lung Fibrosis Induced by Silica

    PubMed Central

    Xiaojun, Wang; Yan, Liu; Hong, Xu; Xianghong, Zhang; Shifeng, Li; Dingjie, Xu; Xuemin, Gao; Lijuan, Zhang; Bonan, Zhang; Zhongqiu, Wei; Ruimin, Wang; Brann, Darrell; Fang, Yang

    2016-01-01

    Silicosis is the most serious occupational disease in China. The objective of this study was to screen various proteins related to mechanisms of the pathogenesis of silicosis underlying the anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) using proteomic profile analysis. We also aimed to explore a potential mechanism of acetylated α-tubulin (α-Ac-Tub) regulation by Ac-SDKP. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to assess the different protein expression profiles between control and silicosis rats treated with or without Ac-SDKP. Twenty-nine proteins were identified to be potentially involved in the progression of silicosis and the anti-fibrotic effect of Ac-SDKP. Our current study finds that 1) the lost expression of Ac-Tub-α may be a new mechanism in rat silicosis; 2) treatment of silicotic rats with N-acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is related with deacetylase family member 6 (HDAC6) and α-tubulin acetyl transferase (α-TAT1). Our data suggest that α-Ac-Tub regulation by Ac-SDKP may potentially be a new anti-fibrosis mechanism. PMID:27577858

  9. Acetylation and succinylation contribute to maturational alterations in energy metabolism in the newborn heart.

    PubMed

    Fukushima, Arata; Alrob, Osama Abo; Zhang, Liyan; Wagg, Cory S; Altamimi, Tariq; Rawat, Sonia; Rebeyka, Ivan M; Kantor, Paul F; Lopaschuk, Gary D

    2016-08-01

    Dramatic maturational changes in cardiac energy metabolism occur in the newborn period, with a shift from glycolysis to fatty acid oxidation. Acetylation and succinylation of lysyl residues are novel posttranslational modifications involved in the control of cardiac energy metabolism. We investigated the impact of changes in protein acetylation/succinylation on the maturational changes in energy metabolism of 1-, 7-, and 21-day-old rabbit hearts. Cardiac fatty acid β-oxidation rates increased in 21-day vs. 1- and 7-day-old hearts, whereas glycolysis and glucose oxidation rates decreased in 21-day-old hearts. The fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD) and β-hydroxyacyl-CoA dehydrogenase (β-HAD), were hyperacetylated with maturation, positively correlated with their activities and fatty acid β-oxidation rates. This alteration was associated with increased expression of the mitochondrial acetyltransferase, general control of amino acid synthesis 5 like 1 (GCN5L1), since silencing GCN5L1 mRNA in H9c2 cells significantly reduced acetylation and activity of LCAD and β-HAD. An increase in mitochondrial ATP production rates with maturation was associated with the decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator-1α, a transcriptional regulator for mitochondrial biogenesis. In addition, hypoxia-inducible factor-1α, hexokinase, and phosphoglycerate mutase expression declined postbirth, whereas acetylation of these glycolytic enzymes increased. Phosphorylation rather than acetylation of pyruvate dehydrogenase (PDH) increased in 21-day-old hearts, accounting for the low glucose oxidation postbirth. A maturational increase was also observed in succinylation of PDH and LCAD. Collectively, our data are the first suggesting that acetylation and succinylation of the key metabolic enzymes in newborn hearts play a crucial role in cardiac energy metabolism with maturation. PMID:27261364

  10. Cohesin’s ATPase Activity Couples Cohesin Loading onto DNA with Smc3 Acetylation

    PubMed Central

    Ladurner, Rene; Bhaskara, Venugopal; Huis in ’t Veld, Pim J.; Davidson, Iain F.; Kreidl, Emanuel; Petzold, Georg; Peters, Jan-Michael

    2014-01-01

    Summary Background Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin’s Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which “locks” cohesin on DNA by inactivating the cohesin release factor Wapl. Results Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis. Conclusions Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin’s ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA. PMID:25220052

  11. IDENTIFICATION OF HISTONE H3 LYSINE 36 ACETYLATION AS A HIGHLY CONSERVED HISTONE MODIFICATION*

    PubMed Central

    Morris, Stephanie A.; Rao, Bhargavi; Garcia, Benjamin A.; Hake, Sandra B.; Diaz, Robert L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Allis, C. David; Lieb, Jason D.; Strahl, Brian D.

    2010-01-01

    Histone lysine (K) acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an “acetyl/methyl switch” governing chromatin function along transcription units. PMID:17189264

  12. Reconsolidation involves histone acetylation depending on the strength of the memory.

    PubMed

    Federman, N; Fustiñana, M S; Romano, A

    2012-09-01

    Gene expression is a necessary step for memory re-stabilization after retrieval, a process known as reconsolidation. Histone acetylation is a fundamental mechanism involved in epigenetic regulation of gene expression and has been implicated in memory consolidation. However, few studies are available in reconsolidation, all of them in vertebrate models. Additionally, the recruitment of histone acetylation as a function of different memory strengths has not been systematically analyzed before. Here we studied the role of histone acetylation in reconsolidation using a well-characterized memory model in invertebrate, the context-signal memory in the crab Chasmagnathus. Firstly, we found an increase in histone H3 acetylation 1h after memory reactivation returning to basal levels at 3 h. Strikingly, this increment was only detected during reconsolidation of a long-term memory induced by a strong training of 30 trials, but not for a short-term memory formed by a weak training of five trials or for a long-term memory induced by a standard training of 15 trials. Furthermore, we showed that a weak memory which was enhanced during consolidation by histone deacetylases inhibition, also recruited histone H3 acetylation in reconsolidation as the strong training does. Accordingly, we found the first evidence that the administration of a histone acetyl transferase inhibitor during memory reconsolidation impairs long-term memory re-stabilization. Finally, we found that strong training memory, at variance with the standard training memory, was resistant to extinction, indicating that such strong training induced in fact a stronger memory. In conclusion, the results presented here support that the participation of histone acetylation during reconsolidation is an evolutionary conserved feature and constitutes a specific molecular characteristic of strong memories.

  13. Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva).

    PubMed

    Song, Yi; Yang, Yang; Zhang, Yuyu; Duan, Liusheng; Zhou, Chunli; Ni, Yuanying; Liao, Xiaojun; Li, Quanhong; Hu, Xiaosong

    2013-10-15

    Acetylation of pumpkin (Cucurbita pepo, lady godiva variety) polysaccharide using acetic anhydride with pyridines as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale. Furthermore, antioxidant activities and cytoprotective effects of pumpkin polysaccharide and its acetylated derivatives were investigated employing various established in vitro systems. Results showed that addition of pyridine as catalyst could increase the degree of substitution, whereas volume of acetic anhydride had little effect. The acetylated polysaccharides in DPPH scavenging radical activity assay, superoxide anion radical activity assay and reducing power assay exhibited higher antioxidant activity than that of unmodified polysaccharide. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by pumpkin polysaccharide and its acetylated derivatives and the derivatives presented higher protective effects. On the whole, acetylated polysaccharide showed relevant antioxidant activity both in vitro and in a cell system.

  14. Behavioral Neuroadaptation to Alcohol: From Glucocorticoids to Histone Acetylation

    PubMed Central

    Mons, Nicole; Beracochea, Daniel

    2016-01-01

    neuroadaptive changes during withdrawal from chronic alcohol intake. It then highlights the role of cAMP–PKA–CREB signaling cascade and histone acetylation within the PFC and limbic structures in alcohol-induced anxiety and behavioral impairments, and how an understanding of functional alterations of these pathways might lead to better treatments for neuropsychiatric disorders. PMID:27766083

  15. Evaluation of the developmental toxicity of acetyl cedrene.

    PubMed

    Lapczynski, Aurelia; Isola, Daniel A; Christian, Mildred S; Diener, Robert M; Api, Anne Marie

    2006-01-01

    The developmental toxicity of acetyl cedrene (AC), a widely used fragrance ingredient, was evaluated in pregnant Sprague-Dawley rats (25/group). Gavaged dosages of 0 (corn oil), 25, 50, or 100 mg/kg/day were administered on days 7 through 17 of gestation (GDs 7 to 17). First and last day dosing suspensions were analyzed for AC content. All rats were observed daily for viability, clinical signs, abortions, and premature deliveries. Body weights were recorded at frequent intervals. Cesarean-sectioning and necropsy examinations were performed on GD 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. The number of corpora lutea in each ovary was also recorded. Fetuses were weighed and examined for gender and gross external changes and soft tissue or skeletal alterations. Totals of 25, 23, 21, and 24 rats became pregnant in the 0 (control), 25, 50 and 100 mg/kg/day groups, respectively, and analysis of dosage preparations verified that administered dosages reflected calculated dosages +/-10%. No deaths or premature deliveries occurred in the study. Clinical signs included excessive salivation, which was attributed to the administration of AC. When compared to controls, significant reductions in feed consumption and body weight gains occurred only at 100 mg/kg/day. Both absolute (g/day) and relative (g/kg/day) feed consumption values were significantly decreased on GDs 7 to 12. Relative values were decreased significantly on GDs 15 to 18. Body weight gains were significantly reduced on GDs 7 to 10. Mean maternal body weights remained significantly lower than controls on GDs 9 to 14, but a marked compensatory increase in feed consumption on GDs 15 to 18 prevented further deterioration in body weight gains. No cesarean-sectioning or litter parameters were affected by dosages of AC and necropsy of the dams after cesarean section did not reveal any gross changes attributable to AC. No gross external

  16. Quantifying drug-protein binding in vivo.

    SciTech Connect

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-02-17

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS.

  17. DNA-damage-responsive acetylation of pRb regulates binding to E2F-1

    PubMed Central

    Markham, Douglas; Munro, Shonagh; Soloway, Judith; O'Connor, Darran P; La Thangue, Nicholas B

    2006-01-01

    The pRb (retinoblastoma protein) tumour suppressor protein has a crucial role in regulating the G1- to S-phase transition, and its phosphorylation by cyclin-dependent kinases is an established and important mechanism in controlling pRb activity. In addition, the targeted acetylation of lysine (K) residues 873/874 in the carboxy-terminal region of pRb located within a cyclin-dependent kinase-docking site hinders pRb phosphorylation and thereby retains pRb in an active state of growth suppression. Here, we report that the acetylation of pRb K873/874 occurs in response to DNA damage and that acetylation regulates the interaction between the C-terminal E2F-1-specific domain of pRb and E2F-1. These results define a new role for pRb acetylation in the DNA damage signalling pathway, and suggest that the interaction between pRb and E2F-1 is controlled by DNA-damage-dependent acetylation of pRb. PMID:16374512

  18. Histone H4 lysine 20 acetylation is associated with gene repression in human cells

    PubMed Central

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  19. Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

    PubMed

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  20. Boric acid-dependent decrease in regulatory histone H3 acetylation is not mutagenic in yeast.

    PubMed

    Pointer, Benjamin R; Schmidt, Martin

    2016-07-01

    Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations. The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast. PMID:27190149

  1. Studies of conformation and interaction of the cyclohexenone and acetyl group of progesterone with liposomes.

    PubMed

    Sanchez-Bueno, A; Watanabe, S; Sancho, M J; Saito, T

    1991-02-01

    The conformations of the A-ring and the 17-acetyl groups of progesterone were examined within liposomes, which were prepared from L-alpha-phosphatidylcholine in the presence or absence of cholesterol in the buffer, using qualitative nuclear magnetic resonance and circular dichroism of the progesterone spectra in the wavelength regions of 260-360 nm. The preferred conformational assignments, in the rotational conformations of the 17-acetyl group and invertible conformations of the cyclohexenone of progesterone were discussed on the basis of the elliptical strength of the Cotton effect and an energy estimation of the preferred conformers. Energetically unstable conformers of the acetyl group and alpha,beta-unsaturated cyclohexenone of progesterone remarkably increased with an increase in the concentration of the liposomes. The liposomes containing 10% cholesterol were similar to the effect of the liposomes lacking cholesterol on the 17-acetyl group and the alpha,beta-unsaturated cyclohexenone but those containing 50% cholesterol showed an increase in the number of energetically stable conformers of the alpha,beta-unsaturated cyclohexenone. The nuclear magnetic resonance signal from liposomes together with the progesterone indicated the existence of the progesterone adjacent to a double bond or ester moiety in the lipid molecule. Therefore, it was apparent that the liposomes and the cholesterol within the liposomes regulated the conformational populations of both the cyclohexone and acetyl groups of the progesterone molecule. PMID:2004040

  2. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects

    PubMed Central

    Myklebust, Line M.; Van Damme, Petra; Støve, Svein I.; Dörfel, Max J.; Abboud, Angèle; 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

    2015-01-01

    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

  3. Boric acid-dependent decrease in regulatory histone H3 acetylation is not mutagenic in yeast.

    PubMed

    Pointer, Benjamin R; Schmidt, Martin

    2016-07-01

    Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations. The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast.

  4. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development.

    PubMed

    Sachs, L M; Shi, Y B

    2000-11-21

    Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

  5. A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3.

    PubMed

    Remoroza, C; Wagenknecht, M; Gu, F; Buchholt, H C; Moerschbacher, B M; Schols, H A; Gruppen, H

    2014-07-17

    A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0-7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, (1)H NMR of polymer and LC-HILIC-MS(n) after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

  6. 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)

    1988-01-01

    Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [3H]-5-indole-3-acetyl-myo-inositol and [U-14C]-D-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [3H]indole-3-acetic acid, derived from the applied [3H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total 3H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol.

  7. Changed histone acetylation patterns in normal appearing white matter and early MS lesions

    PubMed Central

    Pedre, X; Mastronardi, F.; Bruck, W.; López-Rodas, G; Kuhlmann, T; Casaccia, P

    2011-01-01

    The epigenetic identity of oligodendrocytes is modulated by post-translational modifications of histones. Acetylation of histone H3 results from the balance between the activity of histone-acetyltransferases (HATs) and histone deacetylases (HDACs) and modulates transcriptional activation. We have previously shown that in rodents histone deacetylation favors oligodendrocyte differentiation, while acetylation is associated with increased levels of transcriptional inhibitors of oligodendrocyte differentiation. Here we report in humans brains, a shift towards histone acetylation in the white matter of the frontal lobes of aged subjects and in patients with chronic multiple sclerosis (MS). Increased immunoreactivity for acetylated histone H3 was observed in the nuclei of NogoA+ oligodendrocytes in a subset of MS samples. These changes were associated with high levels of transcriptional inhibitors of oligodendrocyte differentiation (i.e. TCF7L2, ID2 and SOX2) and higher HAT transcript levels (i.e. CBP, P300) in female MS patients compared to non-neurological controls and correlated with disease duration. Chromatin immunoprecipitation from samples of MS patients revealed enrichment of acetyl-histone H3 at the promoter of the increased target genes (i.e. TCF7L2). The data in chronic lesions contrasted with findings in early MS lesions, where a marked oligodendroglial histone deacetylation was observed. Together these data suggest that histone deacetylation is a process that occurs at the early stages of the disease and whose efficiency decreases with disease duration. PMID:21368055

  8. Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

    PubMed

    Knudsen, C T; Immerdal, L; Grunnet, N; Quistorff, B

    1992-02-15

    Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.

  9. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

    PubMed

    Myklebust, Line M; Van Damme, Petra; Støve, Svein I; Dörfel, Max J; Abboud, Angèle; 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

    2015-04-01

    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.

  10. Effect of (L-Carnitine) on acetyl-L-carnitine production by heart mitochondria

    SciTech Connect

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

    1986-05-01

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

  11. Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA

    PubMed Central

    Taschner, Michael; Vetter, Melanie; Lorentzen, Esben

    2012-01-01

    Acetylation of lysine residues is an important posttranslational modification found in all domains of life. α-tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on α-tubulin acetyltransferases. Here, we present the structure of the human α-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, α-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative α-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of α-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date. PMID:23071318

  12. Effects of histone acetylation on superoxide dismutase 1 gene expression in the pathogenesis of senile cataract

    PubMed Central

    Rong, Xianfang; Qiu, Xiaodi; Jiang, Yongxiang; Li, Dan; Xu, Jie; Zhang, Yinglei; Lu, Yi

    2016-01-01

    Histone acetylation plays key roles in gene expression, but its effects on superoxide dismutase 1 (SOD1) expression in senile cataract remains unknown. To address this problem, the study was to investigate the influence of histone acetylation on SOD1 expression and its effects in the pathogenesis of senile cataract. Senile cataract was classified into three types—nuclear cataract (NC), cortical cataract (CC), and posterior subcapsular cataract (SC)—using the Lens Opacities Classification System III. In senile cataracts, SOD1 expression decreased significantly. Both H3 and H4 were deacetylated at −600 bp of the SOD1 promoter of cataract lenses, and hypoacetylated at −1500, −1200, and −900 bp. In hypoacetylated histones, the hypoacetylation pattern differed among the cataracts. In vitro, anacardic acid (AA) significantly reduced H3 and H4 acetylation at the SOD1 promoter, decreased protein expression, and induced cataract formation in rabbits. AA also inhibited HLEC viability and increased cell apoptosis. In contrast, trichostatin A (TSA) was able to efficaciously stop AA’s effects on both rabbit lenses and HLECs. Decreased histone acetylation at the SOD1 promoter is associated with declined SOD1 expression in senile cataracts. Histone acetylation plays an essential role in the regulation of SOD1 expression and in the pathogenesis of senile cataracts. PMID:27703255

  13. Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus

    PubMed Central

    Chuang, Peter Y.; Dai, Yan; Liu, Ruijie; He, Helen; Kretzler, Matthias; Jim, Belinda; Cohen, Clemens D.; He, John C.

    2011-01-01

    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

  14. Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment.

    PubMed

    Duan, Ruifeng; Liu, Xiaohua; Wang, Tianhui; Wu, Lei; Gao, Xiujie; Zhang, Zhiqing

    2016-09-01

    Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions. PMID:27161370

  15. Global proteomic analysis of protein acetylation affecting metabolic regulation in Daphnia pulex.

    PubMed

    Kwon, Oh Kwang; Sim, Juhee; Kim, Sun Ju; Oh, Hye Ryeung; Nam, Doo Hyun; Lee, Sangkyu

    2016-02-01

    Daphnia (Daphnia pulex) is a small planktonic crustacean and a key constituent of aquatic ecosystems. It is generally used as a model organism to study environmental toxic problems. In the past decade, genomic and proteomic datasets of Daphnia have been developed. The proteomic dataset allows for the investigation of toxicological effects in the context of "Daphnia proteomics," resulting in greater insights for toxicological research. To exploit Daphnia for ecotoxicological research, information on the post-translational modification (PTM) of proteins is necessary, as this is a critical regulator of biological processes. Acetylation of lysine (Kac) is a reversible and highly regulated PTM that is associated with diverse biological functions. However, a comprehensive description of Kac in Daphnia is not yet available. To understand the cellular distribution of lysine acetylation in Daphnia, we identified 98 acetylation sites in 65 proteins by immunoprecipitation using an anti-acetyllysine antibody and a liquid chromatography system supported by mass spectroscopy. We identified 28 acetylated sites related to metabolic proteins and six acetylated enzymes associated with the TCA cycle in Daphnia. From GO and KEGG enrichment analyses, we showed that Kac in D. pulex is highly enriched in proteins associated with metabolic processes. Our data provide the first global analysis of Kac in D. pulex and is an important resource for the functional analysis of Kac in this organism. PMID:26700148

  16. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    SciTech Connect

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo; Neto Paiva, Claudia; Torres Bozza, Marcelo; Rosado Fantappie, Marcelo

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  17. Solvent-free acetylation of cellulose nanofibers for improving compatibility and dispersion.

    PubMed

    Ashori, Alireza; Babaee, Mehran; Jonoobi, Mehdi; Hamzeh, Yahya

    2014-02-15

    Cellulose nanofibers (CNFs), as bio-materials derived from wood or non-wood plants, have the advantages of being biodegradable, renewable, low cost, and having good mechanical properties compared to synthetic nanofibers. CNFs have been used as reinforcement in polymeric matrices, however, due to their polar surface, their dispersibility in non-polar solvents and compatibility with hydrophobic matrices are poor. In this work, the chemical modification of CNFs, using acetic anhydride in the presence of pyridine as a catalyst, was studied with the aim of changing the surface properties. Native and chemically modified CNFs were characterized in terms of dynamic absorption, thermal stability, surface chemistry, morphology, and crystal structure. The reaction of acetylation between the acetyl groups and the hydroxyl groups of the CNFs was examined using Fourier transform infrared (FT-IR) analysis, while its extent was assessed by titration. The ester content of CNFs was higher for the acetylated samples compared to the control samples. It was also shown that the crystallinity decreased moderately as a result of esterification. Thermal stability of the modified nanofibers was slightly increased. Unlike native CNFs, a stable aqueous suspension was obtained with the modified nanofibers in both ethanol and acetone. The contact angle measurements confirmed that the surface characteristics of acetylated CNFs were changed from hydrophilic to more hydrophobic. In addition, the obtained acetylated CNFs showed more hydrophobic surface, which is in favor of enhancing the hydrophobic non-polar mediums. PMID:24507293

  18. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle

    SciTech Connect

    Cederblad, G.; Carlin, J.I.; Constantin-Teodosiu, D.; Harper, P.; Hultman, E. )

    1990-03-01

    Radioisotopic assays for the determination of acetyl-CoA, CoASH, and acetylcarnitine have been modified for application to the amount of human muscle tissue that can be obtained by needle biopsy. In the last step common to all three methods, acetyl-CoA is condensed with (14C)oxaloacetate by citrate synthase to give (14C)-citrate. For determination of CoASH, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. In the assay for acetylcarnitine, acetylcarnitine is reacted with CoASH in a reaction catalyzed by carnitine acetyltransferase to form acetyl-CoA. Inclusion of new simple steps in the acetylcarnitine assay and conditions affecting the reliability of all three methods are also described. Acetylcarnitine and free carnitine levels in human rectus abdominis muscle were 3.0 +/- 1.5 (SD) and 13.5 +/- 4.0 mumol/g dry wt, respectively. Values for acetyl-CoA and CoASH were about 500-fold lower, 6.7 +/- 1.8 and 21 +/- 8.9 nmol/g dry wt, respectively. A strong correlation between acetylcarnitine (y) and short-chain acylcarnitine (x), determined as the difference between total and free carnitine, was found in biopsies from the vastus lateralis muscle obtained during intense muscular effort, y = 1.0x + 0.5; r = 0.976.

  19. MEC-17 deficiency leads to reduced α-tubulin acetylation and impaired migration of cortical neurons.

    PubMed

    Li, Lei; Wei, Dan; Wang, Qiong; Pan, Jing; Liu, Rong; Zhang, Xu; Bao, Lan

    2012-09-12

    Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of α-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has been found to play a major role in the acetylation of α-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of cortical neurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to mimic acetylated α-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of α-tubulin, appears to control the migration and morphological transition of cortical neurons. This finding reveals the importance of MEC-17 and α-tubulin acetylation in cortical development.

  20. The Acetylation Landscape of the H4 Histone Tail: Disentangling the Interplay between the Specific and Cumulative Effects.

    PubMed

    Winogradoff, David; Echeverria, Ignacia; Potoyan, Davit A; Papoian, Garegin A

    2015-05-20

    Histone tails, the intrinsically disordered terminal regions of histone proteins, are key modulators of the structure and dynamics of chromatin and, consequently, are central to many DNA template-directed processes including replication, repair, and transcription. Acetylation of histone tails is a major post-translational modification (PTM) involved in regulating chromatin, yet it remains unclear how acetylation modifies the disordered state of histone tails and affects their function. We investigated the consequences of increasing acetylation on the isolated H4 histone tail by characterizing the conformational ensembles of unacetylated, mono-, di-, tri-, and tetra-acetylated H4 histone tails using Replica Exchange Molecular Dynamics (REMD) simulations. We found that progressive acetylation has a cumulative effect on the H4 tail, decreasing conformational heterogeneity, increasing helical propensity, and increasing hydrogen bond occupancies. The monoacetylation of lysine 16, however, has unique and specific effects: drastically decreasing the conformational heterogeneity of the H4 tail and leading to highly localized helical secondary structure and elongated conformations. We describe how the cumulative effects of acetylation arise from the charge reduction and increased hydrophobicity associated with adding acetyl groups, while the specific effects are a consequence of steric interactions that are sequence specific. Additionally, we found that increasing the level of acetylation results in the formation of spatially clustered lysines that could serve as recognition patches for binding of chromatin regulating proteins. Hence, we explore the mechanisms by which different acetylation patterns may result in specific recognition of the H4 histone tails by protein or DNA binding partners.

  1. Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples.

    PubMed

    Fukushima, Romualdo S; Hatfield, Ronald D

    2004-06-16

    Present analytical methods to quantify lignin in herbaceous plants are not totally satisfactory. A spectrophotometric method, acetyl bromide soluble lignin (ABSL), has been employed to determine lignin concentration in a range of plant materials. In this work, lignin extracted with acidic dioxane was used to develop standard curves and to calculate the derived linear regression equation (slope equals absorptivity value or extinction coefficient) for determining the lignin concentration of respective cell wall samples. This procedure yielded lignin values that were different from those obtained with Klason lignin, acid detergent acid insoluble lignin, or permanganate lignin procedures. Correlations with in vitro dry matter or cell wall digestibility of samples were highest with data from the spectrophotometric technique. The ABSL method employing as standard lignin extracted with acidic dioxane has the potential to be employed as an analytical method to determine lignin concentration in a range of forage materials. It may be useful in developing a quick and easy method to predict in vitro digestibility on the basis of the total lignin content of a sample.

  2. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation. PMID:26990986

  3. Inhibition of acetyl phosphate-dependent transcription by an acetylatable lysine on RNA polymerase.

    PubMed

    Lima, Bruno P; Thanh Huyen, Tran Thi; Bäsell, Katrin; Becher, Dörte; Antelmann, Haike; Wolfe, Alan J

    2012-09-14

    The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment. PMID:22829598

  4. Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase*

    PubMed Central

    Lima, Bruno P.; Thanh Huyen, Tran Thi; Bäsell, Katrin; Becher, Dörte; Antelmann, Haike; Wolfe, Alan J.

    2012-01-01

    The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment. PMID:22829598

  5. Cellulose acetate from oil palm empty fruit bunch via a one step heterogeneous acetylation.

    PubMed

    Wan Daud, Wan Rosli; Djuned, Fauzi Muhammad

    2015-11-01

    Acetone soluble oil palm empty fruit bunch cellulose acetate (OPEFB-CA) of DS 2.52 has been successfully synthesized in a one-step heterogeneous acetylation of OPEFB cellulose without necessitating the hydrolysis stage. This has only been made possible by the mathematical modeling of the acetylation process by manipulating the variables of reaction time and acetic anhydride/cellulose ratio (RR). The obtained model was verified by experimental data with an error of less than 2.5%. NMR analysis showed that the distribution of the acetyl moiety among the three OH groups of cellulose indicates a preference at the C6 position, followed by C3 and C2. XRD revealed that OPEFB-CA is highly amorphous with a degree of crystallinity estimated to be ca. 6.41% as determined from DSC. The OPEFB-CA films exhibited good mechanical properties being their tensile strength and Young's modulus higher than those of the commercial CA. PMID:26256348

  6. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  7. Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group.

    PubMed

    Yabushita, Mizuho; Kobayashi, Hirokazu; Kuroki, Kyoichi; Ito, Shogo; Fukuoka, Atsushi

    2015-11-01

    Chitin, a polymer of N-acetylglucosamine units with β-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53% yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70%. Our process can greatly reduce the use of acid compared to the conventional process. PMID:26538108

  8. Beta-endorphin and alpha-n-acetyl beta-endorphin; synthesis, conformation and binding parameter

    SciTech Connect

    Lovegren, E.S.

    1986-01-01

    Beta-endorphin (EP) is a 31-residue opioid peptide found in many tissues, including the pituitary, brain and reproductive tract. Alpha-amino-acetyl beta-endorphin (AcEP) was characterized spectroscopically by proton nuclear magnetic resonance (NMR) and circular dichroism in deuterated water and trifluoroethanol (TFE). Both EP and AcEP bind to neuroblastoma N2a cells. This binding was not mediated through opiate receptors, and both peptides seemed to bind at common sites. Ovarian immunoreactive-EP levels were determined for immature and mature rates. These levels were found to be responsive to exogenous gonadotropin treatment in immature animals. A large percentage of the immunoreactive-EP is present in follicular fluid, and most of the endorphin-like peptides were acetylated, as measured by radioimmunoassay. Chromatogaphic analysis suggested at least three EP-like species: EP, a carboxy-terminally cleaved and an amino-terminally acetylated EP.

  9. Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards.

    PubMed

    Liu, Dasheng; Wei, Yuanan; Yao, Pingjia; Jiang, Linbin

    2006-05-01

    Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards is investigated. The UV absorbance of a pure chitosan solution is contributed additively by the N-acetylglucosamine and glucosamine residues; the absorbance divided by the total molar concentration of the residues (A/c(t)) is linearly related to the degree of acetylation (DA). Using acetyl glucosamine and glucosamine hydrochloride as standards in 0.1M hydrochloric acid solution, the equation obtained by linear regression is A/c(t)=3.3615 DA+0.0218, R(2)=0.9958. The DA of the analytical sample (m milligram of chitosan in V liters solution) can be calculated by.

  10. Cellulose acetate from oil palm empty fruit bunch via a one step heterogeneous acetylation.

    PubMed

    Wan Daud, Wan Rosli; Djuned, Fauzi Muhammad

    2015-11-01

    Acetone soluble oil palm empty fruit bunch cellulose acetate (OPEFB-CA) of DS 2.52 has been successfully synthesized in a one-step heterogeneous acetylation of OPEFB cellulose without necessitating the hydrolysis stage. This has only been made possible by the mathematical modeling of the acetylation process by manipulating the variables of reaction time and acetic anhydride/cellulose ratio (RR). The obtained model was verified by experimental data with an error of less than 2.5%. NMR analysis showed that the distribution of the acetyl moiety among the three OH groups of cellulose indicates a preference at the C6 position, followed by C3 and C2. XRD revealed that OPEFB-CA is highly amorphous with a degree of crystallinity estimated to be ca. 6.41% as determined from DSC. The OPEFB-CA films exhibited good mechanical properties being their tensile strength and Young's modulus higher than those of the commercial CA.

  11. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

    NASA Astrophysics Data System (ADS)

    Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

    2015-05-01

    DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

  12. Peptidoglycan of Rhodopseudomonas viridis: partial lack of N-acetyl substitution of glucosamine.

    PubMed Central

    Schmelzer, E; Weckesser, J; Warth, R; Mayer, H

    1982-01-01

    A lack of at least 70% of N-acetyl substitution of glucosamine in the glycan strands of the peptidoglycan from the gram-negative bacterium Rhodopseudomonas viridis is reported. A disaccharide, very likely GlcN beta(1 leads to 4) Mur, was observed in hydrolysates of the isolated peptidoglycan. The disaccharide was not observed when peptidoglycan was N-acetylated before hydrolysis. The peptidoglycan of R. viridis was resistant to lysozyme but became sensitive after N-acetylation with acetic anhydride. The disaccharide was found with peptidoglycan from all R. viridis strains investigated, as well as with R. sulfoviridis P1 and R. palustris strains, but not with peptidoglycan from R. gelatinosa, Rhodospirillum tenue, and Pseudomonas diminuta NCTC 8545. PMID:7054141

  13. In vivo measurement of the acetylation state of sirtuin substrates as a proxy for sirtuin activity.

    PubMed

    Dominy, John; Puigserver, Pere; Cantó, Carles

    2013-01-01

    Evaluating the precise catalytic activity of sirtuin proteins in vivo is a challenging endeavor. Enzymological methods, including those employed in commercially available kits, require the isolation of immunopurified protein from cells or tissues, which can perturb regulatory protein-protein interactions as well as remove the enzyme from the reaction-altering effects of intracellular NAD(+), nicotinamide, and O-acetyl-ADP ribose concentrations. As such, the measurement of the steady state acetylation status of select sirtuin substrates in vivo remains an important tool for evaluating changes in sirtuin activity. Here, we describe how to perform the analysis of the acetylation status of key SIRT1 and SIRT3 targets in rodent tissues and cultured cells.

  14. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  15. Regulation of acetylation restores proteolytic function of diseased myocardium in mouse and human.

    PubMed

    Wang, Ding; Fang, Caiyun; Zong, Nobel C; Liem, David A; Cadeiras, Martin; Scruggs, Sarah B; Yu, Hongxiu; Kim, Allen K; Yang, Pengyuan; Deng, Mario; Lu, Haojie; Ping, Peipei

    2013-12-01

    Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

  16. Genome-wide analysis of H4K5 acetylation associated with fear memory in mice

    PubMed Central

    2013-01-01

    Background Histone acetylation has been implicated in learning and memory in the brain, however, its function at the level of the genome and at individual genetic loci remains poorly investigated. This study examines a key acetylation mark, histone H4 lysine 5 acetylation (H4K5ac), genome-wide and its role in activity-dependent gene transcription in the adult mouse hippocampus following contextual fear conditioning. Results Using ChIP-Seq, we identified 23,235 genes in which H4K5ac correlates with absolute gene expression in the hippocampus. However, in the absence of transcription factor binding sites 150 bp upstream of the transcription start site, genes were associated with higher H4K5ac and expression levels. We further establish H4K5ac as a ubiquitous modification across the genome. Approximately one-third of all genes have above average H4K5ac, of which ~15% are specific to memory formation and ~65% are co-acetylated for H4K12. Although H4K5ac is prevalent across the genome, enrichment of H4K5ac at specific regions in the promoter and coding region are associated with different levels of gene expression. Additionally, unbiased peak calling for genes differentially acetylated for H4K5ac identified 114 unique genes specific to fear memory, over half of which have not previously been associated with memory processes. Conclusions Our data provide novel insights into potential mechanisms of gene priming and bookmarking by histone acetylation following hippocampal memory activation. Specifically, we propose that hyperacetylation of H4K5 may prime genes for rapid expression following activity. More broadly, this study strengthens the importance of histone posttranslational modifications for the differential regulation of transcriptional programs in cognitive processes. PMID:23927422

  17. Endo-N-acetyl-beta-D-glucosaminidase and peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activities during germination of Raphanus sativus.

    PubMed

    Berger, S; Menudier, A; Julien, R; Karamanos, Y

    1995-06-01

    Endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) activities were monitored during germination and postgerminative development in Raphanus sativus. The PNGase activity was found in dry seeds and its level was constant during germination and postgermination. The ENGase activity was first detected about 18 hr after the start of imbibition (HAI) and displayed a maximum level at 36 HAI. After 36 HAI the production of both enzymes was constant until days 4-5. Both enzymes displayed substrate specificities corresponding to the potential glycoprotein substrates found in plants. They are in agreement (i) with the hypothesis that ENGase and PNGase are at the origin of the production of 'unconjugated N-glycans' and (ii) with the possibility that protein activity could be regulated by the removal of N-glycans.

  18. Conformational studies of bacterial peptidoglycan: structure and stereochemistry of N-acetyl-β- D-glucosamine and N-acetyl-β- D-muramic acid

    NASA Astrophysics Data System (ADS)

    Yadav, P. N. S.; Rai, D. K.; Yadav, J. S.

    1989-03-01

    The energies of various conformations of N-acetyl-β- D-glucosamine (NAG) and its 3-O- D-lactic acid derivative N-acetyl-β- 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.

  19. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

    DOE PAGES

    James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; Mouttaki, Housna; Sieber, Jessica R.; Sheik, Cody S.; Nguyen, Hong H.; Yang, Yanan; Xie, Yongming; Erde, Jonathan; et al

    2016-08-16

    Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

  20. Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

    PubMed Central

    Fauvet, Bruno; Fares, Mohamed-Bilal; Samuel, Filsy; Dikiy, Igor; Tandon, Anurag; Eliezer, David; Lashuel, Hilal A.

    2012-01-01

    N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively Nα-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of Nα-acetylation on the biophysical and biological properties of α-syn, we produced Nα-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909–3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and Nα-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing Nα-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both Nα-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating Nα-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn Nα-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells. PMID:22718772

  1. Quantifying renewable groundwater stress with GRACE

    PubMed Central

    Richey, Alexandra S.; Thomas, Brian F.; Lo, Min‐Hui; Reager, John T.; Voss, Katalyn; Swenson, Sean; Rodell, Matthew

    2015-01-01

    Abstract Groundwater is an increasingly important water supply source globally. Understanding the amount of groundwater used versus the volume available is crucial to evaluate future water availability. We present a groundwater stress assessment to quantify the relationship between groundwater use and availability in the world's 37 largest aquifer systems. We quantify stress according to a ratio of groundwater use to availability, which we call the Renewable Groundwater Stress ratio. The impact of quantifying groundwater use based on nationally reported groundwater withdrawal statistics is compared to a novel approach to quantify use based on remote sensing observations from the Gravity Recovery and Climate Experiment (GRACE) satellite mission. Four characteristic stress regimes are defined: Overstressed, Variable Stress, Human‐dominated Stress, and Unstressed. The regimes are a function of the sign of use (positive or negative) and the sign of groundwater availability, defined as mean annual recharge. The ability to mitigate and adapt to stressed conditions, where use exceeds sustainable water availability, is a function of economic capacity and land use patterns. Therefore, we qualitatively explore the relationship between stress and anthropogenic biomes. We find that estimates of groundwater stress based on withdrawal statistics are unable to capture the range of characteristic stress regimes, especially in regions dominated by sparsely populated biome types with limited cropland. GRACE‐based estimates of use and stress can holistically quantify the impact of groundwater use on stress, resulting in both greater magnitudes of stress and more variability of stress between regions. PMID:26900185

  2. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    PubMed Central

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  3. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism.

    PubMed

    Chen, Yun; Daviet, Laurent; Schalk, Michel; Siewers, Verena; Nielsen, Jens

    2013-01-01

    Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of α-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.

  4. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  5. Design of interior-functionalized fully acetylated dendrimers for anticancer drug delivery.

    PubMed

    Hu, Jingjing; Su, Yunzhang; Zhang, Hongfeng; Xu, Tongwen; Cheng, Yiyun

    2011-12-01

    In this study, dendrimers was synthesized by introducing functional groups into the interior pockets of fully acetylated dendrimers. NMR techniques including COSY and 2D-NOESY revealed the molecular structures of the synthesized dendrimers and the encapsulation of guest molecule such as methotrexate within their interior pockets. The synthesized polymeric nanocarriers showed much lower cytotoxicity on two cell lines than cationic dendrimers, and exhibited better performance than fully acetylated dendrimers in the sustained release of methotrexate. The results provided a new strategy in the design of non-toxic dendrimers with high performance in the delivery of anti-cancer drugs for clinical applications.

  6. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  7. Two-year serial whole-brain N-acetyl-l-aspartate in patients with relapsing-remitting multiple sclerosis

    PubMed Central

    Rigotti, D.J.; Inglese, M.; Kirov, I.I.; Gorynski, E.; Perry, N.N.; Babb, J.S.; Herbert, J.; Grossman, R.I.

    2012-01-01

    Objectives: To test the hypotheses that 1) patients with relapsing-remitting multiple sclerosis (RR-MS) exhibit a quantifiable decline in their whole-brain concentration of the neural marker N-acetyl-l-aspartate (WBNAA), that is 2) more sensitive than clinical changes and 3) may provide a practical outcome measure for proof-of-concept and larger phase III clinical trials. Methods: Nineteen patients (5 men and 14 women) with clinically definite RR-MS, who were 33 ± 5 years old (mean ± SD), had a disease duration of 47 ± 28 months, and had a median Expanded Disability Status Scale (EDSS) score of 1.0 (range 0–5.5), underwent MRI and proton magnetic resonance spectroscopy (1H-MRS) semiannually for 2 years (5 time points). Eight matched control subjects underwent the protocol annually (3 time points). Their global N-acetyl-l-aspartate 1H-MRS signal was converted into absolute amounts by phantom replacement and into WBNAA by dividing with the brain parenchymal volume, VB, from MRI segmentation. Results: The baseline WBNAA of the patients (10.5 ± 1.7 mM) was significantly lower than that of the controls (12.3 ± 1.3 mM; p < 0.002) and declined significantly (5%/year, p < 0.002) vs that for the controls who did not show a decline (0.4%/year, p > 0.7). Likewise, VB values of the patients also declined significantly (0.5%/year, p < 0.0001), whereas those of the controls did not (0.2%/year, p = 0.08). The mean EDSS score of the patients increased insignificantly from 1.0 to 1.5 (range 0–6.0) and did not correlate with VB or WBNAA. Conclusions: WBNAA of patients with RR-MS declined significantly at both the group and individual levels over a 2-year time period common in clinical trials. Because of the small sample sizes required to establish power, WBNAA can be incorporated into future studies. PMID:22517095

  8. Interpolating Quantifier-Free Presburger Arithmetic

    NASA Astrophysics Data System (ADS)

    Kroening, Daniel; Leroux, Jérôme; Rümmer, Philipp

    Craig interpolation has become a key ingredient in many symbolic model checkers, serving as an approximative replacement for expensive quantifier elimination. In this paper, we focus on an interpolating decision procedure for the full quantifier-free fragment of Presburger Arithmetic, i.e., linear arithmetic over the integers, a theory which is a good fit for the analysis of software systems. In contrast to earlier procedures based on quantifier elimination and the Omega test, our approach uses integer linear programming techniques: relaxation of interpolation problems to the rationals, and a complete branch-and-bound rule tailored to efficient interpolation. Equations are handled via a dedicated polynomial-time sub-procedure. We have fully implemented our procedure on top of the SMT-solver OpenSMT and present an extensive experimental evaluation.

  9. Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth

    PubMed Central

    Carabetta, Valerie J.; Greco, Todd M.; Tanner, Andrew W.; Cristea, Ileana M.; Dubnau, David

    2016-01-01

    Nε-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. IMPORTANCE The past decade highlighted Nε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the

  10. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    ERIC Educational Resources Information Center

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  11. Surface modification of bacterial cellulose nanofibers for property enhancement of optically transparent composites: dependence on acetyl-group DS.

    PubMed

    Ifuku, Shinsuke; Nogi, Masaya; Abe, Kentaro; Handa, Keishin; Nakatsubo, Fumiaki; Yano, Hiroyuki

    2007-06-01

    Bacterial cellulose (BC) nanofibers were acetylated to enhance the properties of optically transparent composites of acrylic resin reinforced with the nanofibers. A series of BC nanofibers acetylated from degree-of-substitution (DS) 0 to 1.76 were obtained. X-ray diffraction profiles indicated that acetylation proceeded from the surface to the core of BC nanofibers, and scanning electron microscopy images showed that the volume of nanofibers increases by the bulky acetyl group. Since acetylation decreased the refractive index of cellulose, regular transmittance of composites comprised of 63% BC nanofiber was improved, and deterioration at 580 nm because of fiber reinforcement was suppressed to only 3.4%. Acetylation of nanofibers changed their surface properties and reduced the moisture content of the composite to about one-third that of untreated composite, although excessive acetylation increased hygroscopicity. Furthermore, acetylation was found to reduce the coefficient of thermal expansion of a BC sheet from 3 x 10(-6) to below 1 x 10(-6) 1/K.

  12. Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

    PubMed

    Henry, Ryan A; Singh, Tanu; Kuo, Yin-Ming; Biester, Alison; O'Keefe, Abigail; Lee, Sandy; Andrews, Andrew J; O'Reilly, Alana M

    2016-03-22

    Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.

  13. Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

    PubMed

    Henry, Ryan A; Singh, Tanu; Kuo, Yin-Ming; Biester, Alison; O'Keefe, Abigail; Lee, Sandy; Andrews, Andrew J; O'Reilly, Alana M

    2016-03-22

    Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation. PMID:26836402

  14. The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall polysaccharide O-acetylation.

    PubMed

    Schultink, Alex; Naylor, Dan; Dama, Murali; Pauly, Markus

    2015-04-01

    A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.

  15. Roles of Klf5 Acetylation in the Self-Renewal and the Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Chen, Lingyi

    2015-01-01

    Transcription factor Krüppel-like factor 5 (Klf5) plays important roles in the formation of the inner cell mass (ICM) and the trophectoderm during embryogenesis, as well as the self-renewal and the differentiation of mouse embryonic stem cells (ESCs). Acetylation of KLF5 has been shown to reverse the transcriptional activity of KLF5 in human epidermal cells and prostate cancer cells. Whether Klf5 acetylation contributes to the lineage specification in the blastocyst and pluripotency maintenance in ESCs remains unexplored. Here, we showed the ubiquitous expression of acetylated Klf5 in the ICM and the trophectoderm, ruling out the possibility that differential acetylation status of Klf5 leads to the lineage specification in the blastocyst. We found that K358Q mutation, mimicking acetylation, enhances the transcriptional activity of Klf5 for pluripotency genes in ESCs, and that K358Q Klf5 is more potent in pluripotency maintenance and in somatic cell reprogramming, compared to K358R Klf5. In ESCs, Klf5 acetylation, stimulated by TGF-β signaling, is involved in enhancing Sox2 expression. Moreover, upon ESC differentiation, acetylation of Klf5 facilitates the suppression of many differentiation genes, except for that K358Q Klf5 activates Cdx2, promoting trophectodermal differentiation. In summary, our results revealed the regulatory functions of Klf5 acetylation in ESC self-renewal and differentiation. PMID:26372456

  16. The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure*

    PubMed Central

    Iyer, Aditya; Roeters, Steven J.; Schilderink, Nathalie; Hommersom, Bob; Heeren, Ron M. A.; Woutersen, Sander; Claessens, Mireille M. A. E.

    2016-01-01

    Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on αS aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of αS in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-αS in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated αS. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in αS, thus predisposing αS toward specific interactions with other binding partners or alternatively decrease nonspecific interactions. PMID:27531743

  17. Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence.

    PubMed

    Ren, Jie; Sang, Yu; Tan, Yongcong; Tao, Jing; Ni, Jinjing; Liu, Shuting; Fan, Xia; Zhao, Wei; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2016-03-01

    The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix-turn-helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in