S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

40 CFR 721.10174 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. 721.10174 Section 721.10174 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. (a) Chemical substance...-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts (PMN P-04-139...

Progress toward Understanding Protein S-acylation: Prospective in Plants

PubMed Central

Li, Yaxiao; Qi, Baoxiu

2017-01-01

S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs). Since the discovery of the first PAT in yeast in 2002 research in S-acylation has accelerated in the mammalian system and followed by in plant. However, it is still a difficult field to study due to the large number of PATs and even larger number of putative S-acylated substrate proteins they modify in each genome. This is coupled with drawbacks in the techniques used to study S-acylation, leading to the slower progress in this field compared to protein phosphorylation, for example. In this review we will summarize the discoveries made so far based on knowledge learnt from the characterization of protein S-acyltransferases and the S-acylated proteins, the interaction mechanisms between PAT and its specific substrate protein(s) in yeast and mammals. Research in protein S-acylation and PATs in plants will also be covered although this area is currently less well studied in yeast and mammalian systems. PMID:28392791

40 CFR 721.10193 - 1-Butanaminium, N-(3-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. 721.10193 Section 721.10193 Protection of...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. (a) Chemical substance and...-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts (PMN P-06-263, Chemical B; CAS No...

Ground-State Distortion in N-Acyl-tert-butyl-carbamates (Boc) and N-Acyl-tosylamides (Ts): Twisted Amides of Relevance to Amide N-C Cross-Coupling.

PubMed

Szostak, Roman; Shi, Shicheng; Meng, Guangrong; Lalancette, Roger; Szostak, Michal

2016-09-02

Amide N-C(O) bonds are generally unreactive in cross-coupling reactions employing low-valent transition metals due to nN → π*C═O resonance. Herein we demonstrate that N-acyl-tert-butyl-carbamates (Boc) and N-acyl-tosylamides (Ts), two classes of acyclic amides that have recently enabled the development of elusive amide bond N-C cross-coupling reactions with organometallic reagents, are intrinsically twisted around the N-C(O) axis. The data have important implications for the design of new amide cross-coupling reactions with the N-C(O) amide bond cleavage as a key step.

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...

40 CFR 721.10055 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. 721.10055 Section 721.10055 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts. (a) Chemical substance and...

40 CFR 721.10174 - 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. 721.10174 Section 721.10174 Protection of...-amino-N-(carboxymethyl)-N,N-dimethyl-, N-peanut-oil acyl derivs., inner salts. (a) Chemical substance...

N-Acyl derivatives of Asn, new bacterial N-acyl D-amino acids with surfactant activity.

PubMed

Peypoux, F; Laprévote, O; Pagadoy, M; Wallach, J

2004-03-01

New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.

Identical acyl transfer reactions between pyridine N-oxides and their N-acylonium salts

NASA Astrophysics Data System (ADS)

Rybachenko, V. I.; Shroeder, G.; Chotii, K. Yu.; Kovalenko, V. V.; Red'Ko, A. N.; Gierzyk, B.

2007-10-01

28 identical acyl exchange reactions R-CO-Nu+, X- + Nu between pyridine N-oxides in acetonitrile were studied. Here, X- = BPh{4/-} and R = methyl, N,N-dimethylamino, N,N-diethylamino, 4-morpholino, 1-piperidino, N-methyl, N-phenylamino, or N,N-diphenylamino group. The IR and NMR spectroscopic characteristics of acyloxypyridinium salts were determined, and the quantum-chemical parameters of all reagents calculated. The results were subjected to correlation analysis. It was found that the rate of identical acyl transfer reactions was controlled by the interaction of frontier orbitals in the transition state.

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

PubMed Central

Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

2013-01-01

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

An efficient system for the asymmetric acylation of (R,S)-3-n-butylphthalide catalyzed by novozyme 435.

PubMed

Li, Cuiqin; He, Laping; Qiu, Baoquan; Gao, Bing

2010-01-01

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran-hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee(S)), enantiomeric excess of product (ee(P)), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30 °C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee(S) and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10056 Benzenemethanaminium, N-(3...

40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10056 Benzenemethanaminium, N-(3...

Friedel-Crafts Acylation with Amides

PubMed Central

Raja, Erum K.; DeSchepper, Daniel J.; Nilsson Lill, Sten O.; Klumpp, Douglas A.

2012-01-01

Friedel-Crafts acylation has been known since the 1870s and it is an important organic synthetic reaction leading to aromatic ketone products. Friedel-Crafts acylation is usually done with carboxylic acid chlorides or anhydrides while amides are generally not useful substrates in these reactions. Despite being the least reactive carboxylic acid derivative, we have found a series of amides capable of providing aromatic ketones in good yields (55–96%, 17 examples). We propose a mechanism involving diminished C-N resonance through superelectrophilic activation and subsequent cleavage to acyl cations. PMID:22690740

α-Amidoalkylating agents from N-acyl-α-amino acids: 1-(N-acylamino)alkyltriphenylphosphonium salts.

PubMed

Mazurkiewicz, Roman; Adamek, Jakub; Październiok-Holewa, Agnieszka; Zielińska, Katarzyna; Simka, Wojciech; Gajos, Anna; Szymura, Karol

2012-02-17

N-Acyl-α-amino acids were efficiently transformed in a two-step procedure into 1-N-(acylamino)alkyltriphenylphosphonium salts, new powerful α-amidoalkylating agents. The effect of the α-amino acid structure, the base used [MeONa or a silica gel-supported piperidine (SiO(2)-Pip)], and the main electrolysis parameters (current density, charge consumption) on the yield and selectivity of the electrochemical decarboxylative α-methoxylation of N-acyl-α-amino acids (Hofer-Moest reaction) was investigated. For most proteinogenic and all studied unproteinogenic α-amino acids, very good results were obtained using a substoichiometric amount of SiO(2)-Pip as the base. Only in the cases of N-acylated cysteine, methionine, and tryptophan, attempts to carry out the Hofer-Moest reaction in the applied conditions failed, probably because of the susceptibility of these α-amino acids to an electrochemical oxidation on the side chain. The methoxy group of N-(1-methoxyalkyl)amides was effectively displaced with the triphenylphosphonium group by dissolving an equimolar amount of N-(1-methoxyalkyl)amide and triphenylphosphonium tetrafluoroborate in CH(2)Cl(2) at room temperature for 30 min, followed by the precipitation of 1-N-(acylamino)alkyltriphenylphosphonium salt with Et(2)O.

40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.7270 1-propanaminium, 3-amino-, N,N,N-trimethyl-N...

40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.7270 1-propanaminium, 3-amino-, N,N,N-trimethyl-N...

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Enantioselective N-Heterocyclic Carbene Catalysis via the Dienyl Acyl Azolium.

PubMed

Gillard, Rachel M; Fernando, Jared E M; Lupton, David W

2018-04-16

Herein we report the enantioselective N-heterocyclic carbene catalyzed (4+2) annulation of the dienyl acyl azolium with enolates. The reaction exploits readily accessible acyl fluorides and TMS enol ethers to give a range of highly enantio- and diastereo-enriched cyclohexenes (most >97:3 er and >20:1 dr). The reaction was found to require high nucleophilicity NHC catalysts with mechanistic studies supporting a stepwise 1,6-addition/β-lactonization. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novozyme 435-catalyzed efficient acylation of 3-n-butylphthalide in organic medium.

PubMed

He, Laping; Sun, Jiong; Xu, Yan; Sun, Zhihao; Zheng, Changge

2008-01-01

Novozyme 435 could catalyze efficient acylation of 3-n-butylphthalide in organic medium. The conversion of 3-n-butylphthalide increased with the increase of hydrophobicity of solvent below that of hexane. The more available solvent was hexane. Salt hydride could control fixed water activity. The optimum water activity was 0.62. And the optimum of reaction time, velocity of agitation, dosage of Novozyme 435 and acetic anhydride to 3-n-butylphtrhalide molar ratio were 48 hours, 150 rpm, 8 mg/mL and 8:1, respectively. The conversion of 48.9% could be obtained at a water activity of 0.62 in hexane. Furthermore, Novozyme 435 had an enantioselective acylation of racemic 3-n-butylphthalide by original analysis.

Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

PubMed Central

Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

2012-01-01

We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

PubMed

Kite, Geoffrey C; Larsson, Sonny; Veitch, Nigel C; Porter, Elaine A; Ding, Ning; Simmonds, Monique S J

2013-04-10

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

40 CFR 721.10193 - 1-Butanaminium, N-(3-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Butanaminium, N-(3-aminopropyl)-N-butyl-N-(2-carboxyethyl)-, N-coco acyl derivs., inner salts. 721.10193 Section 721.10193 Protection of... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10193 1-Butanaminium, N-(3...

pHP-Tethered N-Acyl Carbamate: A Photocage for Nicotinamide.

PubMed

Salahi, Farbod; Purohit, Vatsal; Ferraudi, Guillermo; Stauffacher, Cynthia; Wiest, Olaf; Helquist, Paul

2018-05-04

The synthesis of a new photocaged nicotinamide having an N-acyl carbamate linker and a p-hydroxyphenacyl (pHP) chromophore is described. The photophysical and photochemical studies showed an absorption maximum at λ = 330 nm and a quantum yield for release of 11% that are dependent upon both pH and solvent. While the acyl carbamate releases nicotinamide efficiently, a simpler amide linker was inert to photocleavage. This photocaged nicotinamide has significant advantages with respect to quantum yield, absorbance wavelength, rate of release, and solubility that make it the first practical example of a photocaged amide.

N-Cinnamoylation of Antimalarial Classics: Effects of Using Acyl Groups Other than Cinnamoyl toward Dual-Stage Antimalarials.

PubMed

Gomes, Ana; Machado, Marta; Lobo, Lis; Nogueira, Fátima; Prudêncio, Miguel; Teixeira, Cátia; Gomes, Paula

2015-08-01

In a follow-up study to our reports of N-cinnamoylated chloroquine and quinacrine analogues as promising dual-stage antimalarial leads with high in vitro potency against both blood-stage Plasmodium falciparum and liver-stage Plasmodium berghei, we decided to investigate the effect of replacing the cinnamoyl moiety with other acyl groups. Thus, a series of N-acylated analogues were synthesized, and their activities against blood- and liver-stage Plasmodium spp. were assessed along with their in vitro cytotoxicities. Although the new N-acylated analogues were found to be somewhat less active and more cytotoxic than their N-cinnamoylated counterparts, they equally displayed nanomolar activities in vitro against blood-stage drug-sensitive and drug-resistant P. falciparum, and significant in vitro liver-stage activity against P. berghei. Therefore, it is demonstrated that simple N-acylated surrogates of classical antimalarial drugs are promising dual-stage antimalarial leads. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

PubMed Central

Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

2014-01-01

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

PubMed Central

Bakken, A M; Farstad, M

1992-01-01

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium. PMID:1471991

Atypical cleavage of protonated N-fatty acyl amino acids derived from aspartic acid evidenced by sequential MS3 experiments.

PubMed

Boukerche, Toufik Taalibi; Alves, Sandra; Le Faouder, Pauline; Warnet, Anna; Bertrand-Michel, Justine; Bouchekara, Mohamed; Belbachir, Mohammed; Tabet, Jean-Claude

2016-12-01

Lipidomics calls for information on detected lipids and conjugates whose structural elucidation by mass spectrometry requires to rationalization of their gas phase dissociations toward collision-induced dissociation (CID) processes. This study focused on activated dissociations of two lipoamino acid (LAA) systems composed of N-palmitoyl acyl coupled with aspartic and glutamic acid mono ethyl esters (as LAA (*D) and LAA (*E) ). Although in MS/MS, their CID spectra show similar trends, e.g., release of water and ethanol, the [(LAA (*D/*E) +H)-C 2 H 5 OH] + product ions dissociate via distinct pathways in sequential MS 3 experiments. The formation of all the product ions is rationalized by charge-promoted cleavages often involving stepwise processes with ion isomerization into ion-dipole prior to dissociation. The latter explains the maleic anhydride or ketene neutral losses from N-palmitoyl acyl aspartate and glutamate anhydride fragment ions, respectively. Consequently, protonated palmitoyl acid amide is generated from LAA (*D), whereas LAA (*E) leads to the [*E+H-H 2 O] + anhydride. The former releases ammonia to provide acylium, which gives the C n H (2n-1) and C n H (2n-3) carbenium series. This should offer structural information, e.g., to locate either unsaturation(s) or alkyl group branching present on the various fatty acyl moieties of lipo-aspartic acid in further studies based on MS n experiments.

A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material

PubMed Central

Wu, Min-Hui; Wan, Liang-Ze; Zhang, Yu-Qing

2014-01-01

A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry. PMID:24651079

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

PubMed

Kumar, Manoj; Wightman, Raymond; Atanassov, Ivan; Gupta, Anjali; Hurst, Charlotte H; Hemsley, Piers A; Turner, Simon

2016-07-08

Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. Copyright © 2016, American Association for the Advancement of Science.

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

PubMed

Moriya, Koko; Kimoto, Mayumi; Matsuzaki, Kanako; Kiwado, Aya; Takamitsu, Emi; Utsumi, Toshihiko

2016-10-15

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus. Copyright © 2016 Elsevier Inc. All rights reserved.

S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

PubMed

Antinone, Sarah E; Ghadge, Ghanashyam D; Ostrow, Lyle W; Roos, Raymond P; Green, William N

2017-01-25

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects

PubMed Central

Antinone, Sarah E.; Ghadge, Ghanashyam D.; Ostrow, Lyle W.; Roos, Raymond P.; Green, William N.

2017-01-01

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord. PMID:28120938

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by Apolipoprotein N-Acyltransferase BCG_2070c

PubMed Central

2013-01-01

Background Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. Results We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Conclusions Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria. PMID:24093492

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

PubMed

Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

2013-10-05

Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

Pantoea sp. Isolated from Tropical Fresh Water Exhibiting N-Acyl Homoserine Lactone Production

PubMed Central

Tan, Wen-Si; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

2014-01-01

N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry. PMID:25197715

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

PubMed

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

PubMed Central

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

PubMed

Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

2018-02-28

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography

PubMed Central

Shaw, Paul D.; Ping, Gao; Daly, Sean L.; Cha, Chung; Cronan, John E.; Rinehart, Kenneth L.; Farrand, Stephen K.

1997-01-01

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-l-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-l-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-l-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules. PMID:9177164

Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation.

PubMed

Sakamoto, Ken; Sato, Kohei; Shigenaga, Akira; Tsuji, Kohei; Tsuda, Shugo; Hibino, Hajime; Nishiuchi, Yuji; Otaka, Akira

2012-08-17

N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

Chemically Modified N-Acylated Hyaluronan Fragments Modulate Proinflammatory Cytokine Production by Stimulated Human Macrophages*

PubMed Central

Babasola, Oladunni; Rees-Milton, Karen J.; Bebe, Siziwe; Wang, Jiaxi; Anastassiades, Tassos P.

2014-01-01

Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30–214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on 1H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule. PMID:25053413

N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

PubMed

Wakabayashi, H; Matsumoto, H; Hashimoto, K; Teraguchi, S; Takase, M; Hayasawa, H

1999-05-01

N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

PubMed

Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

2009-11-01

Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

PubMed Central

Wakabayashi, Hiroyuki; Matsumoto, Hiroshi; Hashimoto, Koichi; Teraguchi, Susumu; Takase, Mitsunori; Hayasawa, Hirotoshi

1999-01-01

N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B. PMID:10223949

Flow chemistry and polymer-supported pseudoenantiomeric acylating agents enable parallel kinetic resolution of chiral saturated N-heterocycles

NASA Astrophysics Data System (ADS)

Kreituss, Imants; Bode, Jeffrey W.

2017-05-01

Kinetic resolution is a common method to obtain enantioenriched material from a racemic mixture. This process will deliver enantiopure unreacted material when the selectivity factor of the process, s, is greater than 1; however, the scalemic reaction product is often discarded. Parallel kinetic resolution, on the other hand, provides access to two enantioenriched products from a single racemic starting material, but suffers from a variety of practical challenges regarding experimental design that limit its applications. Here, we describe the development of a flow-based system that enables practical parallel kinetic resolution of saturated N-heterocycles. This process provides access to both enantiomers of the starting material in good yield and high enantiopurity; similar results with classical kinetic resolution would require selectivity factors in the range of s = 100. To achieve this, two immobilized quasienantiomeric acylating agents were designed for the asymmetric acylation of racemic saturated N-heterocycles. Using the flow-based system we could efficiently separate, recover and reuse the polymer-supported reagents. The amide products could be readily separated and hydrolysed to the corresponding amines without detectable epimerization.

Gas Phase Dissociation Behavior of Acyl-Arginine Peptides.

PubMed

McGee, William M; McLuckey, Scott A

2013-11-15

The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N -hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed "ornithine effect". Path 2 involves the coordinated losses of H 2 O and NH 3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

Design, synthesis, characterisation, conformation and biological investigation of N-acyl r-2,c-6-bis (4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones

NASA Astrophysics Data System (ADS)

Mohanraj, V.; Ponnuswamy, S.

2017-09-01

In a wide research programme towards the study of piperidin-4-ones with efficient pharmacological effect, a new series of N-acyl r-2,c-6-bis(4-methoxyphenyl)-c-3,t-3-dimethylpiperidin-4-ones 2-5 are synthesized and characterized by IR spectra, 1H, 13C, DEPT - 135 and 2D (COSY and HSQC) NMR and mass spectra. The parent compound 1 prefers to exist in a chair conformation whereas the extracted coupling constant, chemical shifts and estimated dihedral angles show that the N-acyl piperdine-4-ones 2-5 prefer to exist in a distorted boat conformation B1 (with C2 and C5 in prow and stern positions) with coplanar orientation of Nsbnd Cdbnd O moiety. The existence of a fast Nsbnd CO rotational equilibrium between the boat conformations B (I) and B (II) has also been observed. Anti bacterial activity of the above test compounds 1-5 is determined against pseudomonas sp. and salmonella sp. The antioxidant activities are determined by the ABTS, DPPH and superoxide assays. Furthermore, molecular docking studies have been carried out for the compounds 1-5 with target protein CHK1.

Versatility of acyl-acyl carrier protein synthetases

DOE PAGES

Beld, Joris; Finzel, Kara; Burkart, Michael D.

2014-10-09

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

PubMed Central

Saroj, Sunil D.; Holmer, Linda; Berengueras, Júlia M.; Jonsson, Ann-Beth

2017-01-01

Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence. PMID:28303956

Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165.

PubMed

Saroj, Sunil D; Holmer, Linda; Berengueras, Júlia M; Jonsson, Ann-Beth

2017-03-17

Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence.

N-Acyl amino acids and their impact on biological processes.

PubMed

Hanuš, Lumír; Shohami, Esther; Bab, Itai; Mechoulam, Raphael

2014-01-01

Over the last two decades a large number of N-long-chain acyl amino acids have been identified in the mammalian body. The pharmacological activities of only a few of them have been investigated and some have been found to be of considerable interest. Thus arachidonoyl serine is vasodilatory and neuroprotective, arachidonoyl glycine is antinociceptive, and oleoyl serine rescues bone loss. However, the pathophysiological/biochemical roles of these amides are mostly unknown. © 2014 International Union of Biochemistry and Molecular Biology.

Identification and characterization of a novel N-acyl-homoserine lactonase gene in Sphingomonas ursincola isolated from industrial cooling water systems.

PubMed

Morohoshi, Tomohiro; Sato, Niina; Iizumi, Taro; Tanaka, Airi; Ikeda, Tsukasa

2017-05-01

Biofilm formation by bacteria is one of the main causes of fouling in industrial cooling water systems. In many gram-negative bacteria, biofilm formation is regulated by N-acyl-homoserine lactone (AHL)-mediated quorum sensing. In this study, we isolated three AHL-degrading bacteria from cooling water systems and identified them as Sphingomonas ursincola. The draft genome sequence of S. ursincola A1 revealed the presence of an AHL-degrading gene homolog, designated qsdS. The qsdS region was also amplified by PCR from the genomes of the other two S. ursincola strains, SF1 and SF8. Escherichia coli DH5α harboring a QsdS-expressing plasmid showed high degradative activity against AHLs with short and 3-oxo-substituted acyl chains. High-performance liquid chromatography analysis revealed that QsdS is an AHL lactonase, an enzyme that catalyzes AHL ring opening. Furthermore, heterologous expression of QsdS in Pseudomonas aeruginosa PAO1 resulted in degradation of endogenous AHLs and interfered with the quorum-sensing-regulated phenotype. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

In vivo metabolism of fumonisin B1 to N-acylated ceramide-like compounds

USDA-ARS?s Scientific Manuscript database

Fumonisins are toxic and carcinogenic mycotoxins found in corn-based foods. Fumonisin B1 (FB1) metabolism to ceramide-like cytotoxic N-acylated FB1 (NAFB1) compounds has been shown in vitro, but in vivo metabolism has not been reported. Therefore, male Sprague-Dawley rats (2/group) were given 5 da...

Miscibility and Phase Behavior of N-Acylethanolamine/Diacylphosphatidylethanolamine Binary Mixtures of Matched Acyl Chainlengths (n = 14, 16)

PubMed Central

Kamlekar, Ravi Kanth; Satyanarayana, S.; Marsh, Derek; Swamy, Musti J.

2007-01-01

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress. PMID

Polymorphism in 'L' shaped lipids: structure of N-, O-diacylethanolamines with mixed acyl chains.

PubMed

Tarafdar, Pradip K; Swamy, Musti J

2009-11-01

Although solid state polymorphism in lipids has been established by spectroscopic and calorimetric studies long ago, only in a few cases crystal structures of different polymorphs of the same compound have been reported, possibly due to difficulties in obtaining high quality single crystals of individual polymorphs. Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of the stress-related lipids, the N-acylethanolamines under physiological conditions. In this study, two DAEs with mixed acyl chains, namely N-palmitoyl, O-octanoylethanolamine and N-palmitoyl, O-decanoylethanolamine have been synthesized and their three-dimensional structures were determined. Both the compounds were found to adopt 'L' shaped structures and exist in two polymorphic forms, alpha and beta. In the alpha form a mixed-type chain packing has been observed whereas in the beta form the chain packing is symmetric. Similar polymorphic forms are likely to exist in other 'L' shaped lipids such as 1,3-diacylglycerols and ceramides, where polymorphism has been detected earlier, but three-dimensional structures - which can give precise information about the packing at atomic resolution - have not been reported.

Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

PubMed

Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-03-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

PubMed Central

See-Too, Wah Seng; Ee, Robson; Lim, Yan-Lue; Convey, Peter; Pearce, David A.; Yin, Wai-Fong; Chan, Kok-Gan

2017-01-01

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions. PMID:28225085

Inverse association of des-acyl ghrelin with worksite blood pressure in overweight/obese male workers.

PubMed

Narisada, Akihiko; Hasegawa, Tomomi; Nakahigashi, Maki; Hirobe, Takaaki; Ikemoto, Tatsunori; Ushida, Takahiro; Kobayashi, Fumio

2015-05-01

Job strain, defined as a combination of high job demands and low job control, has been reported to elevate blood pressure (BP) during work. Meanwhile, a recent experimental study showed that ghrelin blunted the BP response to such mental stress. In the present study, we examined the hypothesis that des-acyl ghrelin may have some beneficial effects on worksite BP through modulating the BP response to work-related mental stress, i.e., job strain. Subjects were 34 overweight/obese male day-shift workers (mean age 41.7 ± 6.7 years). No subjects had received any anti-hypertensive medication. A 24-h ambulatory BP monitoring was recorded every 30 min on a regular working day. The average BP was calculated for Work BP, Morning BP, and Home BP. Job strain was assessed using the short version of the Japanese Job Content Questionnaire. Des-acyl ghrelin showed significant inverse correlations with almost all BPs except Morning SBP, Morning DBP, and Home DBP. In multiple regression analysis, des-acyl ghrelin inversely correlated with Work SBP after adjusting for confounding factors. Des-acyl ghrelin was also negatively associated with BP changes from Sleep to Morning, Sleep to Work, and Sleep to Home. Des-acyl ghrelin was inversely associated with Worksite BP, suggesting a unique beneficial effect of des-acyl ghrelin on Worksite BP in overweight/obese male day-shift workers.

N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.

PubMed

Weiss, Stefan; Keller, Max; Bernhardt, Günther; Buschauer, Armin; König, Burkhard

2010-09-01

N(G)-Acylated argininamides, covering a broad range of lipophilicity (calculated logD values: -1.8-12.5), were synthesized and investigated for NPY Y(1) receptor (Y(1)R) antagonism, Y(1)R affinity and stability in buffer (N(G)-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K(i) (binding) and K(b) values (Y(1)R antagonism) varied from low nM to one-digit muM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism. Copyright 2010 Elsevier Ltd. All rights reserved.

In silico prediction of acyl glucuronide reactivity

NASA Astrophysics Data System (ADS)

Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

2011-11-01

Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

Oxonitriles: A Grignard Addition-Acylation Route to Enamides

PubMed Central

Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W.

2008-01-01

Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents, or a trialkyl zincate, effectively assembling highly substituted, cyclic enamides. PMID:17020332

Oxonitriles: a grignard addition-acylation route to enamides.

PubMed

Fleming, Fraser F; Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W

2006-10-12

[reaction: see text] Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by an in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents or a trialkylzincate, effectively assembling highly substituted, cyclic enamides.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs.

PubMed

Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M; Simons, Brigitte; Zhang, Guo-Fang

2014-03-01

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

PubMed Central

Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-01-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

Acylation-dependent protein export in Leishmania.

PubMed

Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

2000-04-14

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase.

PubMed

Sorek, Nadav; Poraty, Limor; Sternberg, Hasana; Bar, Enat; Lewinsohn, Efraim; Yalovsky, Shaul

2007-03-01

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His(6)-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His(6)-GFP-Atrop6(CA) mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPgammaS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His(6)-GFP-Atrop6(CA)mS(156) in which cysteine(156) was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.

N-Sulfanylethylaminooxybutyramide (SEAoxy): A Crypto-Thioester Compatible with Fmoc Solid-Phase Peptide Synthesis.

PubMed

Tsuda, Shugo; Mochizuki, Masayoshi; Sakamoto, Ken; Denda, Masaya; Nishio, Hideki; Otaka, Akira; Yoshiya, Taku

2016-11-18

An N-sulfanylethylaminooxybutyramide (SEAoxy) has been developed as a novel thioester equivalent for native chemical ligation. SEAoxy peptide was straightforwardly synthesized by conventional Fmoc solid-phase peptide synthesis without a problem. Moreover, SEAoxy peptide could be directly applied to native chemical ligation owing to the intramolecular N-to-S acyl shift that releases the peptide-thioester in situ. This methodology was successfully applied to the synthesis of two bioactive peptides.

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

PubMed

Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

1995-01-01

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

Nε-Fatty acylation of Rho GTPases by a MARTX toxin effector.

PubMed

Zhou, Yan; Huang, Chunfeng; Yin, Li; Wan, Muyang; Wang, Xiaofei; Li, Lin; Liu, Yanhua; Wang, Zhao; Fu, Panhan; Zhang, Ni; Chen, She; Liu, Xiaoyun; Shao, Feng; Zhu, Yongqun

2017-10-27

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are a family of large toxins that are extensively distributed in bacterial pathogens. MARTX toxins are autocatalytically cleaved to multiple effector domains, which are released into host cells to modulate the host signaling pathways. The Rho guanosine triphosphatase (GTPase) inactivation domain (RID), a conserved effector domain of MARTX toxins, is implicated in cell rounding by disrupting the host actin cytoskeleton. We found that the RID is an N ε -fatty acyltransferase that covalently modifies the lysine residues in the C-terminal polybasic region of Rho GTPases. The resulting fatty acylation inhibited Rho GTPases and disrupted Rho GTPase-mediated signaling in the host. Thus, RID can mediate the lysine N ε -fatty acylation of mammalian proteins and represents a family of toxins that harbor N-fatty acyltransferase activities in bacterial pathogens. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

PubMed

Smith, Dennis A; Hammond, Timothy; Baillie, Thomas A

2018-06-01

While simple O - (ether-linked) and N -glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

PubMed Central

Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

2016-01-01

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

PubMed Central

Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

1995-01-01

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

NASA Astrophysics Data System (ADS)

Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

2017-03-01

We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

A General and Selective Rhodium-Catalyzed Reduction of Amides, N-Acyl Amino Esters, and Dipeptides Using Phenylsilane.

PubMed

Das, Shoubhik; Li, Yuehui; Lu, Liang-Qiu; Junge, Kathrin; Beller, Matthias

2016-05-17

This article describes a selective reduction of functionalized amides, including N-acyl amino esters and dipeptides, to the corresponding amines using simple [Rh(acac)(cod)]. The catalyst shows excellent chemoselectivity in the presence of different sensitive functional moieties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress

PubMed Central

Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A.; Williams, Todd D.; Welti, Ruth

2014-01-01

Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

Oxidative acylation using thioacids

NASA Technical Reports Server (NTRS)

Liu, R.; Orgel, L. E.

1997-01-01

Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

Acylation of Ferrocene: A Greener Approach

ERIC Educational Resources Information Center

Birdwhistell, Kurt R.; Nguyen, Andy; Ramos, Eric J.; Kobelja, Robert

2008-01-01

The acylation of ferrocene is a common reaction used in organic laboratories to demonstrate Friedel-Crafts acylation and the purification of compounds using column chromatography. This article describes an acylation of ferrocene experiment that is more eco-friendly than the conventional acylation experiment. The traditional experiment was modified…

Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

PubMed Central

Faergeman, N J; Knudsen, J

1997-01-01

The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

ZnS-Based ZnSTe:N/n-ZnS Light-Emitting Diodes

NASA Astrophysics Data System (ADS)

Ichino, Kunio; Kojima, Takahiro; Obata, Shunsuke; Kuroyanagi, Takuma; Nakazawa, Shoichi; Kashiyama, Shota

2013-11-01

ZnS1-xTex:N/n-ZnS diodes have been fabricated in an attempt to convert ZnS into p-type by Te incorporation and the resulting upward shift of the valence band maximum. The diodes exhibit clear rectification in the current-voltage characteristic and a peak of the electron-beam-induced current at the ZnS1-xTex:N/n-ZnS interface. Furthermore, a ZnS0.85Te0.15:N/n-ZnS diode exhibits blue-green electroluminescence due to self-activated emission in n-ZnS at 290 K under a forward current. These results suggest p-type conduction in ZnS1-xTex:N, and thus the LED operation of a ZnS-based pn-junction.

Pharmacological Evaluation and Preparation of Nonsteroidal Anti-Inflammatory Drugs Containing an N-Acyl Hydrazone Subunit

PubMed Central

de Melo, Thais Regina Ferreira; Chelucci, Rafael Consolin; Pires, Maria Elisa Lopes; Dutra, Luiz Antonio; Barbieri, Karina Pereira; Bosquesi, Priscila Longhin; Trossini, Gustavo Henrique Goulart; Chung, Man Chin; dos Santos, Jean Leandro

2014-01-01

A series of anti-inflammatory derivatives containing an N-acyl hydrazone subunit (4a–e) were synthesized and characterized. Docking studies were performed that suggest that compounds 4a–e bind to cyclooxygenase (COX)-1 and COX-2 isoforms, but with higher affinity for COX-2. The compounds display similar anti-inflammatory activities in vivo, although compound 4c is the most effective compound for inhibiting rat paw edema, with a reduction in the extent of inflammation of 35.9% and 52.8% at 2 and 4 h, respectively. The anti-inflammatory activity of N-acyl hydrazone derivatives was inferior to their respective parent drugs, except for compound 4c after 5 h. Ulcerogenic studies revealed that compounds 4a–e are less gastrotoxic than the respective parent drug. Compounds 4b–e demonstrated mucosal damage comparable to celecoxib. The in vivo analgesic activities of the compounds are higher than the respective parent drug for compounds 4a–b and 4d–e. Compound 4a was more active than dipyrone in reducing acetic-acid-induced abdominal constrictions. Our results indicate that compounds 4a–e are anti-inflammatory and analgesic compounds with reduced gastrotoxicity compared to their respective parent non-steroidal anti-inflammatory drugs. PMID:24714090

Glycogen phosphorylase as a target for type 2 diabetes: synthetic, biochemical, structural and computational evaluation of novel N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors.

PubMed

Kantsadi, Anastassia L; Parmenopoulou, Vanessa; Bakalov, Dimitar N; Snelgrove, Laura; Stravodimos, George A; Chatzileontiadou, Demetra S M; Manta, Stella; Panagiotopoulou, Angeliki; Hayes, Joseph M; Komiotis, Dimitri; Leonidas, Demetres D

2015-01-01

Glycogen phosphorylase (GP), a validated target for the development of anti-hyperglycaemic agents, has been targeted for the design of novel glycopyranosylamine inhibitors. Exploiting the two most potent inhibitors from our previous study of N-acyl-β-D-glucopyranosylamines (Parmenopoulou et al., Bioorg. Med. Chem. 2014, 22, 4810), we have extended the linking group to -NHCONHCO- between the glucose moiety and the aliphatic/aromatic substituent in the GP catalytic site β-cavity. The N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors were synthesized and their efficiency assessed by biochemical methods, revealing inhibition constant values of 4.95 µM and 2.53 µM. Crystal structures of GP in complex with these inhibitors were determined and analyzed, providing data for further structure based design efforts. A novel Linear Response - Molecular Mechanics Coulomb Surface Area (LR-MM-CBSA) method has been developed which relates predicted and experimental binding free energies for a training set of N-acyl-N´-(β-D-glucopyranosyl) urea ligands with a correlation coefficient R(2) of 0.89 and leave-one-out cross-validation (LOO-cv) Q(2) statistic of 0.79. The method has significant applications to direct future lead optimization studies, where ligand entropy loss on binding is revealed as a key factor to be considered. ADMET property predictions revealed that apart from potential permeability issues, the synthesized N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors have drug-like potential without any toxicity warnings.

Structural Basis for Substrate Fatty Acyl Chain Specificity

PubMed Central

McAndrew, Ryan P.; Wang, Yudong; Mohsen, Al-Walid; He, Miao; Vockley, Jerry; Kim, Jung-Ja P.

2008-01-01

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ∼400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an α-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial β-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. PMID:18227065

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determiningmore » the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.« less

Synergistic N-Heterocyclic Carbene/Palladium-Catalyzed Reactions of Aldehyde Acyl Anions with either Diarylmethyl or Allylic Carbonates.

PubMed

Yasuda, Shigeo; Ishii, Takuya; Takemoto, Shunsuke; Haruki, Hiroki; Ohmiya, Hirohisa

2018-03-05

Benzylation and allylation of aldehyde acyl anions were enabled by the merger of a thiazolium N-heterocyclic carbene (NHC) catalyst and a palladium/bisphosphine catalyst in a synergistic manner. Owing to the mildness of the reaction conditions, various functional groups were tolerated in the substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein.

PubMed

Dörmann, P; Spener, F; Ohlrogge, J B

1993-03-01

Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

PubMed

Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-10-04

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

Association of acylated ghrelin profiles with chronic inflammatory markers in overweight and obese postmenopausal women: a MONET study.

PubMed

St-Pierre, David H; Bastard, Jean-Philippe; Coderre, Lise; Brochu, Martin; Karelis, Antony D; Lavoie, Marie-Eve; Malita, Florin; Fontaine, Jonathan; Mignault, Diane; Cianflone, Katherine; Imbeault, Pascal; Doucet, Eric; Rabasa-Lhoret, Rémi

2007-10-01

Recent reports have suggested that the existence of associations between hormonal dysregulation and chronic upregulation of inflammatory markers, which may cause obesity-related disturbances. Thus, we examined whether acylated ghrelin (AcylG) and total ghrelin (TotG) levels could be associated with the following inflammatory markers: C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), and soluble TNF receptor 1 (sTNF-R1). Cross-sectional study consisting of 50 overweight and obese postmenopausal women. AcylG and TotG levels were assessed at 0, 60, 160, 170, and 180 min of the euglycemic/hyperinsulinemic clamp (EHC). We evaluated insulin sensitivity, body composition, and blood lipid profiles as well as fasting concentrations of CRP, TNF-alpha, and sTNF-R1. In fasting conditions, sTNF-R1 was negatively correlated with AcylG (r = -0.48, P < 0.001) levels. In addition, AcylG/TotG was associated negatively with sTNF-R1 (r = -0.44, P = 0.002) and positively with TNF-alpha (r = 0.38, P = 0.009) values. During the EHC, TotG (at all time points) and AcylG (at 60 and 160 min) values were significantly decreased from fasting concentrations. AcylG maximal reduction and area under the curve (AUC) values were correlated to sTNF-R1 (r = -0.35, P = 0.02 and r = -0.34, P = 0.02, respectively). Meanwhile, the AcylG/TotG AUC ratio was associated negatively with sTNF-R1 (r = -0.29, P < 0.05) and positively with TNF-alpha (r = 0.36, P = 0.02). Following adjustments for total adiposity, sTNF-R1 remained correlated with fasting and maximal reduction AcylG values. Similarly, AcylG/TotG ratios remained significantly correlated with sTNF-R1 and TNF-alpha. Importantly, 23% of the variation in sTNF-R1 was independently predicted by fasting AcylG. These results are the first to suggest that both fasting and EHC-induced AcylG profiles are correlated with fasting values of sTNF-R1, a component of the TNF-alpha system. Thus, AcylG may act, at least in part, as one mediator of

N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens.

PubMed

Hernández-Reyes, Casandra; Schenk, Sebastian T; Neumann, Christina; Kogel, Karl-Heinz; Schikora, Adam

2014-11-01

The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Diversion of a thioglycoligase for the synthesis of 1-O-acyl arabinofuranoses.

PubMed

Pavic, Quentin; Tranchimand, Sylvain; Lemiègre, Loïc; Legentil, Laurent

2018-05-15

An arabinofuranosylhydrolase from the GH51 family was transformed into an acyl transferase by mutation of the catalytic acid/base amino acid. The resulting enzyme was able to transfer carboxylic acid onto the anomeric position of arabinose with complete chemo- and stereoselectivity. A wide range of acyl α-l-arabinofuranoses was obtained with yields ranging from 25 to 83%. Using this method, ibuprofen and N-Boc phenylalanine were successfully transformed into their corresponding acyl conjugates, expanding the scope of the reaction to drugs and amino acids.

Are the endocannabinoid-like compounds N-acyl aminoacids neuroprotective after traumatic brain injury?

PubMed

Mann, Aniv; Cohen-Yeshurun, Ayelet; Trembovler, Victoria; Mechoulam, Raphael; Shohami, Esther

2016-05-01

In recent years, a library of approx. 70 N-acyl aminoacids (NAAAs) was discovered in the rat brain. A particular member of this family of compounds is arachidonoyl serine (AraS), which has generated special interest as a potential therapy for traumatic brain injury (TBI). This is due to its structural similarity to the endocannabinoid (eCB) 2-arachidonoyl glycerol (2-AG), which was previously shown to be beneficial in the recovery in a closed head injury model of TBI. Indeed, AraS exerted eCB-mediated neuroprotection, which was evident in numerous aspects related to the secondary damage characterizing TBI. These findings promoted broadening of the research to additional compounds of the NAAA family that share a structural similarity to AraS, namely, palmitoyl serine (PalmS) and oleoyl serine. The latter did not exhibit any improvement in recovery, whereas the former displayed some neuroprotection, albeit inferior to 2-AG and AraS, via unknown mechanisms. Interestingly, when a combined treatment of 2-AG, AraS and PalmS was tested, the overall effect on the severity score was inferior to their individual effects, suggesting not only a lack of direct or indirect synergism, but also possibly some spatial hindrance. Taken together, the complexity of the damage caused by TBI and the many open questions concerning the role of the eCB system in health and disease, the findings so far may serve as a small trace to the understanding of the eCB system, as well as of the mechanisms underlying TBI.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

Muoniated acyl and thioacyl radicals

NASA Astrophysics Data System (ADS)

McKenzie, Iain; Brodovitch, Jean-Claude; Ghandi, Khashayar; Percival, Paul W.

2006-03-01

The product of the reaction of muonium with tert-butylisocyanate was previously assigned as the muoniated tert-butylaminyl radical (I. McKenzie, J.-C. Brodovitch, K. Ghandi, S. Kecman, P. W. Percival, Physica B 326 (2003) 76). This assignment is incorrect since the muon and 14N hyperfine-coupling constants (hfcc) of this radical would have the opposite sign, which is in conflict with the experimental results. The radical is now reassigned as the muoniated N-tert-butylcarbamoyl radical, based on the similarities between the experimental muon and 14N hfcc and hfcc calculated at the UB3LYP/6-311G(d,p)//UB3LYP/EPR-III level. The large zero-point energy in the N-Mu bond results in the dissociation barrier of the muoniated N-tert-butylcarbamoyl radical being above the combined energy of the reactants, in contrast to the N-tert-butylcarbamoyl radical where the dissociation barrier lies below the combined energy of the reactants. The reaction of muonium with tert-butylisothiocyanate produced both conformers of the muoniated N-tert-butylthiocarbamoyl radical and their assignment was based on the similarities between the experimental and calculated muon hfcc. These are the first acyl and thioacyl radicals to be directly detected by muon spin spectroscopy.

The level of circulating octanoate does not predict ghrelin O-acyl transferase (GOAT)-mediated acylation of ghrelin during fasting.

PubMed

Nass, Ralf; Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S; Farhy, Leon S; Patrie, James; Gaylinn, Bruce D; Heiman, Mark; Thorner, Michael O

2015-01-01

Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

PubMed Central

Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-01-01

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

Identification of the Binding Region of the [2Fe-2S] Ferredoxin in Stearoyl-Acyl Carrier Protein Desaturase

PubMed Central

Sobrado, Pablo; Lyle, Karen S.; Kaul, Steven P.; Turco, Michelle M.; Arabshahi, Ida; Marwah, Ashok; Fox, Brian G.

2008-01-01

Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O2 and 2e- dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Δ9D complex by use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches and molecular docking studies. Treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Δ9D contribute to the cross-linking. The single substitutions of K60A, K56A, and K230A on Δ9D decreased the kcat/KM for Fd by 4-, 22- and 2,400-fold, respectively, as compared to wt Δ9D and a K41A substitution. The double substitution K56A/K60A decreased the kcat/KM for Fd by 250-fold, while the triple mutation K56A/K60A/K230A decreased the kcat/KM for Fd by at least 700,000-fold. These results strongly implicate the triad of K56, K60 and K230 of Δ9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd near to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix-7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Δ9D and other protein-protein complexes. PMID:16605252

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

PubMed

Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

2017-11-24

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

PubMed Central

Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

2017-01-01

SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers1[OPEN

PubMed Central

Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K.; Dyer, John M.; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Jenks, Matthew A.

2017-01-01

We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4’s principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation’s effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. PMID:28069670

Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.

PubMed

Fezza, Filomena; Gasperi, Valeria; Mazzei, Cinzia; Maccarrone, Mauro

2005-04-01

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

NASA Technical Reports Server (NTRS)

White, D. H.; Kennedy, R. M.; Macklin, J.

1984-01-01

Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

PubMed Central

Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

2009-01-01

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE PAGES

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; ...

2017-06-14

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a

Acyl transfer from membrane lipids to peptides is a generic process.

PubMed

Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

2013-11-15

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis1

PubMed Central

Jasieniecka-Gazarkiewicz, Katarzyna; Lager, Ida; Carlsson, Anders S.; Gutbrod, Katharina; Peisker, Helga; Dörmann, Peter; Stymne, Sten; Banaś, Antoni

2017-01-01

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE. The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC. The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells. PMID:28408542

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

PubMed Central

Kawelke, Steffen; Feussner, Ivo

2015-01-01

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity. PMID:26714272

Serum Levels of Acyl-Carnitines along the Continuum from Normal to Alzheimer's Dementia

PubMed Central

Sapere, Nadia; La Marca, Giancarlo; Angiolillo, Antonella; Vitale, Michela; Corbi, Graziamaria; Scapagnini, Giovanni; Intrieri, Mariano; Russo, Claudio

2016-01-01

This study aimed to determine the serum levels of free L-carnitine, acetyl-L-carnitine and 34 acyl-L-carnitine in healthy subjects and in patients with or at risk of Alzheimer’s disease. Twenty-nine patients with probable Alzheimer’s disease, 18 with mild cognitive impairment of the amnestic type, 24 with subjective memory complaint and 46 healthy subjects were enrolled in the study, and the levels of carnitine and acyl-carnitines were measured by tandem mass spectrometry. The concentrations of acetyl-L-carnitine progressively decreased passing from healthy subjects group (mean±SD, 5.6±1.3 μmol/L) to subjective memory complaint (4.3±0.9 μmol/L), mild cognitive impairment (4.0±0.53 μmol/L), up to Alzheimer’s disease (3.5±0.6 μmol/L) group (p<0.001). The differences were significant for the comparisons: healthy subjects vs. subjective memory complaint, mild cognitive impairment or Alzheimer’s disease group; and subjective memory complaint vs. Alzheimer’s disease group. Other acyl-carnitines, such as malonyl-, 3-hydroxyisovaleryl-, hexenoyl-, decanoyl-, dodecanoyl-, dodecenoyl-, myristoyl-, tetradecenoyl-, hexadecenoyl-, stearoyl-, oleyl- and linoleyl-L-carnitine, showed a similar decreasing trend, passing from healthy subjects to patients at risk of or with Alzheimer’s disease. These results suggest that serum acetyl-L-carnitine and other acyl-L-carnitine levels decrease along the continuum from healthy subjects to subjective memory complaint and mild cognitive impairment subjects, up to patients with Alzheimer’s disease, and that the metabolism of some acyl-carnitines is finely connected among them. These findings also suggest that the serum levels of acetyl-L-carnitine and other acyl-L-carnitines could help to identify the patients before the phenotype conversion to Alzheimer’s disease and the patients who would benefit from the treatment with acetyl-L-carnitine. However, further validation on a larger number of samples in a longitudinal

Acyl coenzyme A thioesterase 7 regulates neuronal fatty acid metabolism to prevent neurotoxicity.

PubMed

Ellis, Jessica M; Wong, G William; Wolfgang, Michael J

2013-05-01

Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7(N-/-), revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7(N-/-) mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7(N-/-) mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity.

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2013-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2010-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

PubMed

Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K; Tomasi, Pernell; Dyer, John M; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Parsons, Eugene P; Jenks, Matthew A; Lü, Shiyou

2017-02-01

We report n-6 monounsaturated primary alcohols (C 26:1 , C 28:1 , and C 30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. © 2017 American Society of Plant Biologists. All Rights Reserved.

A simple synthesis of N-perfluoroacylated and N-acylated glycals of neuraminic acid with a cyclic aminic substituent at the 4α position as possible inhibitors of sialidases.

PubMed

Rota, Paola; Allevi, Pietro; Agnolin, Irene S; Mattina, Roberto; Papini, Nadia; Anastasia, Mario

2012-04-14

A simple protocol for the synthesis of N-perfluoroacylated and N-acylated glycals of neuraminic acid, with a secondary cyclic amine (morpholine or piperidine) at the 4α position, has been set-up, starting from peracetylated N-acetylneuraminic acid methyl ester that undergoes, sequentially to its direct N-transacylation followed by a C-4 amination, a β-elimination, and a selective hydrolysis of the ester functions, without affecting the sensitive perfluorinated amide. This journal is © The Royal Society of Chemistry 2012

Pseudomonas cremoricolorata Strain ND07 Produces N-acyl Homoserine Lactones as Quorum Sensing Molecules

PubMed Central

Yunos, Nina Yusrina Muhamad; Tan, Wen-Si; Koh, Chong-Lek; Sam, Choon-Kook; Mohamad, Nur Izzati; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

2014-01-01

Quorum sensing (QS) is a bacterial cell-to-cell communication system controlling QS-mediated genes which is synchronized with the population density. The regulation of specific gene activity is dependent on the signaling molecules produced, namely N-acyl homoserine lactones (AHLs). We report here the identification and characterization of AHLs produced by bacterial strain ND07 isolated from a Malaysian fresh water sample. Molecular identification showed that strain ND07 is clustered closely to Pseudomonas cremoricolorata. Spent culture supernatant extract of P. cremoricolorata strain ND07 activated the AHL biosensor Chromobacterium violaceum CV026. Using high resolution triple quadrupole liquid chromatography-mass spectrometry, it was confirmed that P. cremoricolorata strain ND07 produced N-octanoyl-l-homoserine lactone (C8-HSL) and N-decanoyl-l-homoserine lactone (C10-HSL). To the best of our knowledge, this is the first documentation on the production of C10-HSL in P. cremoricolorata strain ND07. PMID:24984061

Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

PubMed

Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

2016-04-19

Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

PubMed

Nieto Penalver, Carlos G; Morin, Danièle; Cantet, Franck; Saurel, Olivier; Milon, Alain; Vorholt, Julia A

2006-01-23

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

Molecular structure, supramolecular organization and thermotropic phase behavior of N-acylglycine alkyl esters with matched acyl and alkyl chains.

PubMed

Reddy, S Thirupathi; Swamy, Musti J

2017-11-01

N-Acylglycines (NAGs), the endogenous single-tailed lipids present in rat brain and other mammalian tissues, play significant roles in cell physiology and exhibit interesting pharmacological properties. In the present study, a homologous series of N-acylglycine alkyl esters (NAGEs) with matched chains were synthesized and characterized. Results of differential scanning calorimetric studies revealed that all NAGEs exhibit a single sharp phase transition and that the transition enthalpy and entropy show a linear dependence on the N-acyl and ester alkyl chain length. The structure of N-myristoylglycine myristyl ester (NMGME), solved by single-crystal X-ray diffraction, showed that the molecule adopts a linear geometry and revealed that the structure of N-myristoyl glycyl moiety in NMGME is identical to that in N-myristoylglycine. The molecules are packed in layers with the polar functional groups of the ester and amide functionalities located at the center of the layer. The crystal packing is stabilized by NH⋯O hydrogen bonds between the amide CO and NH groups of adjacent molecules as well as by CH⋯O hydrogen bonds between the amide carbonyl and the methylene CH adjacent to the ester carbonyl of neighboring molecules as well as between ester carbonyl and methylene group of the glycine moiety of adjacent molecules. Powder X-ray diffraction studies showed a linear dependence of the d-spacings on the acyl chain length, suggesting that all NAGEs adopt a structure similar to the packing exhibited in the crystal lattice of NMGME. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164.

PubMed

Bruns, Hilke; Herrmann, Jennifer; Müller, Rolf; Wang, Hui; Wagner Döbler, Irene; Schulz, Stefan

2018-01-26

The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.

Synthesis of Polyheterocyclic Pyrrolo[3,4-b]pyridin-5-ones via a One-Pot (Ugi-3CR/aza Diels-Alder/N-acylation/aromatization/SN2) Process. A Suitable Alternative towards Novel Aza-Analogues of Falipamil.

PubMed

Zamudio-Medina, Angel; García-González, Ailyn N; Herrera-Carrillo, Genesis K; Zárate-Zárate, Daniel; Benavides-Macías, Adriana; Tamariz, Joaquín; Ibarra, Ilich A; Islas-Jácome, Alejandro; González-Zamora, Eduardo

2018-03-27

We describe the one-pot synthesis of twenty polyheterocyclic pyrrolo[3,4- b ]pyridin-5-ones via a cascade process (Ugi-3CR/aza Diels-Alder/ N -acylation/aromatization) in 20 to 95% overall yields, as well as four pharmacologically promising analogues via an improved cascade process (Ugi-3CR/aza Diels-Alder/ N -acylation/aromatization/S N 2): two piperazine-linked pyrrolo[3,4- b ]pyridin-5-ones in 33 and 34%, and a couple of Falipamil aza-analogues in 30 and 35% overall yields. It is worth highlighting the good substrate scope found, because final products are furnished with alkyl, aryl, and heterocyclic substituents. The use of chain-ring tautomerizable isocyanides (as key reagents for the Ugi-type three component reaction) allowed for a rapid and efficient assembly of the polysubstituted oxindoles, which were used in situ toward the complex products, conferring features like robustness, sustainability, and the one-pot approach to this synthetic methodology.

Global impacts of the 1980s regime shift.

PubMed

Reid, Philip C; Hari, Renata E; Beaugrand, Grégory; Livingstone, David M; Marty, Christoph; Straile, Dietmar; Barichivich, Jonathan; Goberville, Eric; Adrian, Rita; Aono, Yasuyuki; Brown, Ross; Foster, James; Groisman, Pavel; Hélaouët, Pierre; Hsu, Huang-Hsiung; Kirby, Richard; Knight, Jeff; Kraberg, Alexandra; Li, Jianping; Lo, Tzu-Ting; Myneni, Ranga B; North, Ryan P; Pounds, J Alan; Sparks, Tim; Stübi, René; Tian, Yongjun; Wiltshire, Karen H; Xiao, Dong; Zhu, Zaichun

2016-02-01

Despite evidence from a number of Earth systems that abrupt temporal changes known as regime shifts are important, their nature, scale and mechanisms remain poorly documented and understood. Applying principal component analysis, change-point analysis and a sequential t-test analysis of regime shifts to 72 time series, we confirm that the 1980s regime shift represented a major change in the Earth's biophysical systems from the upper atmosphere to the depths of the ocean and from the Arctic to the Antarctic, and occurred at slightly different times around the world. Using historical climate model simulations from the Coupled Model Intercomparison Project Phase 5 (CMIP5) and statistical modelling of historical temperatures, we then demonstrate that this event was triggered by rapid global warming from anthropogenic plus natural forcing, the latter associated with the recovery from the El Chichón volcanic eruption. The shift in temperature that occurred at this time is hypothesized as the main forcing for a cascade of abrupt environmental changes. Within the context of the last century or more, the 1980s event was unique in terms of its global scope and scale; our observed consequences imply that if unavoidable natural events such as major volcanic eruptions interact with anthropogenic warming unforeseen multiplier effects may occur. © 2015 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids.

PubMed

Ardhaoui, M; Falcimaigne, A; Ognier, S; Engasser, J M; Moussou, P; Pauly, G; Ghoul, M

2004-06-10

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and omega-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on omega-substituted fatty acid chain were unfavourable to these reactions. The 1H NMR and 13C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4"'-OH of the rhamnose residue of rutin. Copyright 2004 Elsevier B.V.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

PubMed

Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

2010-11-30

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Domino Acylation/Diels-Alder Synthesis of N-Alkyl-octahydroisoquinolin-1-one-8-carboxylic Acids under Low-Solvent Conditions.

PubMed

Slauson, Stephen R; Pemberton, Ryan; Ghosh, Partha; Tantillo, Dean J; Aubé, Jeffrey

2015-05-15

The development of the domino reaction between an aminoethyl-substituted diene and maleic anhydride to afford an N-substituted octahydroisoquinolin-1-one is described. A typical procedure involves the treatment of a 1-aminoethyl-substituted butadiene with maleic anhydride at 0 °C to room temperature for 20 min under low-solvent conditions, which affords a series of isoquinolinone carboxylic acids in moderate to excellent yields. NMR monitoring suggested that the reaction proceeded via an initial acylation step followed by an intramolecular Diels-Alder reaction. For the latter step, a significant rate difference was observed depending on whether the amino group was substituted by a phenyl or an alkyl (usually benzyl) substituent, with the former noted by NMR to be substantially slower. The Diels-Alder step was studied by density functional theory (DFT) methods, leading to the conclusion that the degree of preorganization in the starting acylated intermediate had the largest effect on the reaction barriers. In addition, the effect of electronics on the aromatic ring in N-phenyl substrates was studied computationally and experimentally. Overall, this protocol proved considerably more amenable to scale up compared to earlier methods by eliminating the requirement of microwave batch chemistry for this reaction as well as significantly reducing the quantity of solvent.

Plasma concentrations of acyl-ghrelin are associated with average daily gain and feeding behavior in grow-finish pigs.

PubMed

Lents, C A; Brown-Brandl, T M; Rohrer, G A; Oliver, W T; Freking, B A

2016-04-01

The objectives of this study were to determine the effect of sex, sire line, and litter size on concentrations of acyl-ghrelin and total ghrelin in plasma of grow-finish pigs and to understand the relationship of plasma concentrations of ghrelin with feeding behavior, average daily gain (ADG), and back fat in grow-finish swine. Yorkshire-Landrace crossbred dams were inseminated with semen from Yorkshire, Landrace, or Duroc sires. Within 24 h of birth, pigs were cross-fostered into litter sizes of normal (N; >12 pigs/litter) or small (S; ≤ 9 pigs/litter). At 8 wk of age, pigs (n = 240) were blocked by sire breed, sex, and litter size and assigned to pens (n = 6) containing commercial feeders modified with a system to monitor feeding behavior. Total time eating, number of daily meals, and duration of meals were recorded for each individual pig. Body weight was recorded every 4 wk. Back fat and loin eye area were recorded at the conclusion of the 12-wk feeding study. A blood sample was collected at week 7 of the study to quantify concentrations of acyl- and total ghrelin in plasma. Pigs from small litters weighed more (P < 0.05) and tended (P = 0.07) to be fatter than pigs from normal litters. Postnatal litter size did not affect ADG, feeding behavior, or concentrations of ghrelin in plasma during the grow-finish phase. Barrows spent more time eating (P < 0.001) than gilts, but the number of meals and concentrations of ghrelin did not differ with sex of the pig. Pigs from Duroc and Yorkshire sires had lesser (P < 0.0001) concentrations of acyl-ghrelin than pigs from Landrace sires, but plasma concentrations of total ghrelin were not affected by sire breed. Concentrations of acyl-ghrelin were positively correlated with the number of meals and negatively correlated with meal length and ADG (P < 0.05). A larger number of short-duration meals may indicate that pigs with greater concentrations of acyl-ghrelin consumed less total feed, which likely explains why they were

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

PubMed Central

Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

2015-01-01

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Miho; Nakahara, Keiko; Goto, Shintaro

Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [{sup 125}I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmedmore » that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.« less

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils†

PubMed Central

Wang, Ya-Juan; Leadbetter, Jared Renton

2005-01-01

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol · h−1 · g of fresh weight soil−1. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 × 105 cells · g of turf soil−1 degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems. PMID:15746331

Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase.

PubMed

Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D; Bogarin, Roberto; Haim, Alon; Thorner, Michael O; Chanoine, Jean-Pierre

2009-05-01

Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean +/- SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 +/- 0.2 vs. 10.2 +/- 1.9 ng.ml(-1).90 min(-1), P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 +/- 1.9 vs. 8.6 +/- 1.2 ng.ml(-1).90 min(-1), P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 +/- 0.5 vs. 4.5 +/- 0.6 microg/ml, P = 0.029) and cholinesterase activity (705 +/- 33 vs. 1,013 +/- 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy

Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans

PubMed Central

Sidani, Reem M.; Garcia, Anna E.; Antoun, Joseph; Isbell, James M.; Abumrad, Naji N.

2016-01-01

Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (iv) infusions of glucose. These “isoglycemic clamps” enabled us to determine the contribution of the GI tract and postabsorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared with gastric glucose administration (P < 0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P ≤ 0.01). However, gastric and isoglycemic iv glucose infusions resulted in similar degrees of acyl ghrelin suppression (P > 0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion. PMID:27279247

Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives.

PubMed

Bi, Huiping; Bai, Yanfen; Cai, Tao; Zhuang, Yibin; Liang, Xiaomei; Zhang, Xueli; Liu, Tao; Ma, Yanhe

2013-12-01

Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96 ± 0.06 mg/L chloramphenicol-3-isobutyrate and 3.94 ± 0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77 ± 3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.

Diverse profiles of N-acyl-homoserine lactones in biofilm forming strains of Cronobacter sakazakii.

PubMed

Singh, Niharika; Patil, Amrita; Prabhune, Asmita A; Raghav, Mamta; Goel, Gunjan

2017-04-03

The present study investigates the role of quorum sensing (QS) molecules expressed by C. sakazakii in biofilm formation and extracellular polysaccharide expression. The QS signaling was detected using Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4(pZLR4) based bioassay. Long chain N-acyl-homoserine lactones (AHLs) with C6- C18 chain length were identified using High Performance Liquid Chromatography and Liquid Chromatography-High Resolution Mass Spectrometry. A higher Specific Biofilm Formation (SBF) index (p < 0.05) with the presence of genes associated with cellulose biosynthesis (bcsA, bcsC and bcsG) was observed in the strains. AHLs and their mechanisms can serve as novel targets for developing technologies to eradicate and prevent biofilm formation by C. sakazakii.

Fluorescent high-performance liquid chromatographic analysis of vigabatrin enantiomers after derivatizing with naproxen acyl chloride.

PubMed

Hsieh, Chun-Yu; Wang, Shing-Yaw; Kwan, Aij-Lie; Wu, Hsin-Lung

2008-01-18

Vigabatrin is widely used as an anticonvulsant in the treatment of seizures. Vigabatrin is usually supplied as racemate in formulation, but only the (S)-(+)-enantiomer of vigabatrin is pharmacologically active. A simple and sensitive liquid chromatographic method is described for the separation and quantification of vigabatrin enantiomers. The method is based on derivatizing racemic vigabatrin with a fluorescent chiral reagent (naproxen acyl chloride). The resulting diastereomeric derivatives are highly responsive to a fluorimetric detector (lambda(ex)=230 nm, lambda(em)=350 nm). The lower quantitation limit of the method is attainable at 25 nM for (S)-(+)-vigabatrin or (R)-(-)-vigabatrin with a detection limit of about 2.5 nM (S/N=3 with 10 microl injected). Application of the method to the analysis of vigabatrin in serum of dosed patients proved feasible.

Age-dependent decline in acyl-ghrelin concentrations and reduced association of acyl-ghrelin and growth hormone in healthy older adults.

PubMed

Nass, Ralf; Farhy, Leon S; Liu, Jianhua; Pezzoli, Suzan S; Johnson, Michael L; Gaylinn, Bruce D; Thorner, Michael O

2014-02-01

Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001). These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

PubMed Central

Jones, A; Davies, H M; Voelker, T A

1995-01-01

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

PubMed

Jones, A; Davies, H M; Voelker, T A

1995-03-01

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

USDA-ARS?s Scientific Manuscript database

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

Dopaminergic Neurons Respond to Iron-Induced Oxidative Stress by Modulating Lipid Acylation and Deacylation Cycles

PubMed Central

Sánchez Campos, Sofía; Rodríguez Diez, Guadalupe; Oresti, Gerardo Martín; Salvador, Gabriela Alejandra

2015-01-01

Metal-imbalance has been reported as a contributor factor for the degeneration of dopaminergic neurons in Parkinson Disease (PD). Specifically, iron (Fe)-overload and copper (Cu) mis-compartmentalization have been reported to be involved in the injury of dopaminergic neurons in this pathology. The aim of this work was to characterize the mechanisms of membrane repair by studying lipid acylation and deacylation reactions and their role in oxidative injury in N27 dopaminergic neurons exposed to Fe-overload and Cu-supplementation. N27 dopaminergic neurons incubated with Fe (1mM) for 24 hs displayed increased levels of reactive oxygen species (ROS), lipid peroxidation and elevated plasma membrane permeability. Cu-supplemented neurons (10, 50 μM) showed no evidence of oxidative stress markers. A different lipid acylation profile was observed in N27 neurons pre-labeled with [3H] arachidonic acid (AA) or [3H] oleic acid (OA). In Fe-exposed neurons, AA uptake was increased in triacylglycerols (TAG) whereas its incorporation into the phospholipid (PL) fraction was diminished. TAG content was 40% higher in Fe-exposed neurons than in controls. This increase was accompanied by the appearance of Nile red positive lipid bodies. Contrariwise, OA incorporation increased in the PL fractions and showed no changes in TAG. Lipid acylation profile in Cu-supplemented neurons showed AA accumulation into phosphatidylserine and no changes in TAG. The inhibition of deacylation/acylation reactions prompted an increase in oxidative stress markers and mitochondrial dysfunction in Fe-overloaded neurons. These findings provide evidence about the participation of lipid acylation mechanisms against Fe-induced oxidative injury and postulate that dopaminergic neurons cleverly preserve AA in TAG in response to oxidative stress. PMID:26076361

Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase

PubMed Central

Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D.; Bogarin, Roberto; Haim, Alon; Thorner, Michael O.; Chanoine, Jean-Pierre

2009-01-01

Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean ± SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 ± 0.2 vs. 10.2 ± 1.9 ng·ml−1·90 min−1, P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 ± 1.9 vs. 8.6 ± 1.2 ng·ml−1·90 min−1, P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 ± 0.5 vs. 4.5 ± 0.6 μg/ml, P = 0.029) and cholinesterase activity (705 ± 33 vs. 1,013 ± 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy, likely

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

PubMed

Yurchenko, Olga; Singer, Stacy D; Nykiforuk, Cory L; Gidda, Satinder; Mullen, Robert T; Moloney, Maurice M; Weselake, Randall J

2014-06-01

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1 cisΔ11 ) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2 cisΔ9,12 ; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1 cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3 cisΔ9,12,15 ) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

PubMed

Martinelle, M; Hult, K

1995-09-06

The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

Strategies for Correcting Very Long Chain Acyl-CoA Dehydrogenase Deficiency*

PubMed Central

Tenopoulou, Margarita; Chen, Jie; Bastin, Jean; Bennett, Michael J.; Ischiropoulos, Harry; Doulias, Paschalis-Thomas

2015-01-01

Very long acyl-CoA dehydrogenase (VLCAD) deficiency is a genetic pediatric disorder presenting with a spectrum of phenotypes that remains for the most part untreatable. Here, we present a novel strategy for the correction of VLCAD deficiency by increasing mutant VLCAD enzymatic activity. Treatment of VLCAD-deficient fibroblasts, which express distinct mutant VLCAD protein and exhibit deficient fatty acid β-oxidation, with S-nitroso-N-acetylcysteine induced site-specific S-nitrosylation of VLCAD mutants at cysteine residue 237. Cysteine 237 S-nitrosylation was associated with an 8–17-fold increase in VLCAD-specific activity and concomitant correction of acylcarnitine profile and β-oxidation capacity, two hallmarks of the disorder. Overall, this study provides biochemical evidence for a potential therapeutic modality to correct β-oxidation deficiencies. PMID:25737446

The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constantsmore » for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.« less

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

PubMed

Smoum, Reem; Bar, Arik; Tan, Bo; Milman, Garry; Attar-Namdar, Malka; Ofek, Orr; Stuart, Jordyn M; Bajayo, Alon; Tam, Joseph; Kram, Vardit; O'Dell, David; Walker, Michael J; Bradshaw, Heather B; Bab, Itai; Mechoulam, Raphael

2010-10-12

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

PubMed Central

Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

2015-01-01

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides*

PubMed Central

Nemoto, Takayuki K.; Ohara-Nemoto, Yuko; Bezerra, Gustavo Arruda; Shimoyama, Yu; Kimura, Shigenobu

2016-01-01

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser615 and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm−1 s−1, optimal pH was 7–8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met16–Glu101). Three-dimensional modeling revealed the three domain structures (residues Met16–Ala126, which has no similar homologue with known structure; residues Leu127–Met495 (β-propeller domain); and residues Ala496–Phe736 (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides. PMID:26733202

Regioselective self-acylating cyclodextrins in organic solvent

NASA Astrophysics Data System (ADS)

Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

2016-03-01

Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.

The structure of S . lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain

DOE PAGES

Mitchell, Carter A.; Tucker, Alex C.; Escalante-Semerena, Jorge C.; ...

2014-12-09

The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ~110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. In this paper, the structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to othermore » members of this family. Finally, whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain.« less

Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

PubMed

Garg, Hari G; Mrabat, Hicham; Yu, Lunyin; Hales, Charles A; Li, Boyangzi; Moore, Casey N; Zhang, Fuming; Linhardt, Robert J

2011-08-01

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

Influence of fluorocarbon and hydrocarbon acyl groups at the surface of bovine carbonic anhydrase II on the kinetics of denaturation by sodium dodecyl sulfate.

PubMed

Lee, Andrew; Mirica, Katherine A; Whitesides, George M

2011-02-10

This paper examines the influence of acylation of the Lys-ε-NH(3)(+) groups of bovine carbonic anhydrase (BCA, EC 4.2.1.1) to Lys-ε-NHCOR (R = -CH(3), -CH(2)CH(3), and -CH(CH(3))(2), -CF(3)) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (k(Ac,n)) of each series of acylated derivatives depended on the number of acylations (n). Plots of log k(Ac,n) vs n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ∼7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ∼7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14, however, rates of denaturation stayed approximately constant; analysis suggested that these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ∼3 for n ∼ 14) in the order CH(3)CONH- < CH(3)CH(2)CONH- < (CH(3))(2)CHCONH- < CF(3)CONH-. These results suggested that the hydrophobicity of CF(3)CONH- is slightly greater (by a factor of <2) than that of RHCONH- with similar surface area.

Acylation, Diastereoselective Alkylation, and Cleavage of an Oxazolidinone Chiral Auxiliary: A Multistep Asymmetric Synthesis Experiment for Advanced Undergraduates

ERIC Educational Resources Information Center

Smith, Thomas E.; Richardson, David P.; Truran, George A.; Belecki, Katherine; Onishi, Megumi

2008-01-01

An introduction to the concepts and experimental techniques of diastereoselective synthesis using a chiral auxiliary is described. The 4-benzyl-2-oxazolidinone chiral auxiliary developed by Evans is acylated with propionic anhydride under mild conditions using DMAP as an acyl transfer catalyst. Deprotonation with NaN(TMS)[subscript 2] at -78…

MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia.

PubMed

Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom; Zhang, Xiao-Hua

2015-01-01

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 10(5) s(-1) M(-1). Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the "HXHXDH" motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Plasma levels of acylated ghrelin in patients with functional dyspepsia

PubMed Central

Kim, Yeon Soo; Lee, Joon Seong; Lee, Tae Hee; Cho, Joo Young; Kim, Jin Oh; Kim, Wan Jung; Kim, Hyun Gun; Jeon, Seong Ran; Jeong, Hoe Su

2012-01-01

AIM: To investigate the relationship between plasma acylated ghrelin levels and the pathophysiology of functional dyspepsia. METHODS: Twenty-two female patients with functional dyspepsia and twelve healthy volunteers were recruited for the study. The functional dyspepsia patients were each diagnosed based on the Rome III criteria. Eligible patients completed a questionnaire concerning the severity of 10 symptoms. Plasma acylated ghrelin levels before and after a meal were determined in the study participants using a commercial human acylated enzyme immunoassay kit; electrogastrograms were performed for 50 min before and after a standardized 10-min meal containing 265 kcal. RESULTS: There were no significant differences in plasma acylated ghrelin levels between healthy volunteers and patients with functional dyspepsia. However, in patients with functional dyspepsia, there was a negative correlation between fasting plasma acylated ghrelin levels and the sum score of epigastric pain (r = -0.427, P = 0.047) and a positive correlation between the postprandial/fasting plasma acylated ghrelin ratio and the sum score of early satiety (r = 0.428, P =0.047). Additionally, there was a negative correlation between fasting acylated ghrelin plasma levels and fasting normogastria (%) (r = -0.522, P = 0.013). Interestingly, two functional dyspepsia patients showed paradoxically elevated plasma acylated ghrelin levels after the meal. CONCLUSION: Abnormal plasma acylated ghrelin levels before or after a meal may be related to several of the dyspeptic symptoms seen in patients with functional dyspepsia. PMID:22611317

Anorexia in hemodialysis patients: the possible role of des-acyl ghrelin.

PubMed

Muscaritoli, Maurizio; Molfino, Alessio; Chiappini, Maria Grazia; Laviano, Alessandro; Ammann, Thomas; Spinsanti, Paola; Melchiorri, Daniela; Inui, Akio; Alegiani, Filippo; Rossi Fanelli, Filippo

2007-01-01

Anorexia is frequently found in end-stage renal disease and is a reliable predictor of morbidity and mortality in hemodialysis (HD) patients. The pathogenesis of anorexia is complex and the appetite-modulating hormone ghrelin could be involved. Two forms of circulating ghrelin have been described: acylated ghrelin (<10% of circulating ghrelin) which promotes food intake, and des-acyl ghrelin which induces a negative energy balance. The aim of this cross-sectional study is to clarify whether anorexia and body weight change in HD patients relate to plasma des-acyl ghrelin levels. 34 HD patients and 15 healthy controls were studied. The presence of anorexia was assessed by a questionnaire. Serum des-acyl ghrelin was measured in HD patients and in 15 body mass index-, sex- and age-matched controls by ELISA. Energy intake was assessed by a 3-day dietary diary, and fat-free mass (FFM) was evaluated by body impedance analysis. Data have been statistically analyzed and are presented as mean +/- SD. 14 patients (41%) were found to be anorexic, and 20 patients (59%) non-anorexic. Energy intake (kcal/day) was significantly lower in anorexic than in non-anorexic patients (1,682 +/- 241 vs. 1,972.50 +/- 490; p < 0.05). FFM (%) was lower in anorexic than in non-anorexic patients (65.8 +/- 4.4 vs. 70.9 +/- 8.7; p = 0.05). Plasma des-acyl ghrelin levels (fmol/ml) were significantly higher in HD patients than in controls (214.88 +/- 154.24 vs. 128.93 +/- 51.07; p < 0.05), and in anorexic HD patients than in non-anorexic (301.7 +/- 162.4 vs. 159.1 +/- 115.5; p < 0.01). Anorexia is highly prevalent among HD patients and des-acyl ghrelin could be involved in its pathogenesis. Copyright 2007 S. Karger AG, Basel.

The Influence of Fluorocarbon and Hydrocarbon Acyl Groups at the Surface of Bovine Carbonic Anhydrase II on the Kinetics of Denaturation by Sodium Dodecyl Sulfate

PubMed Central

Lee, Andrew; Mirica, Katherine A.; Whitesides, George M.

2011-01-01

This paper examines the influence of acylation of the Lys-ε-NH3+ groups of bovine carbonic anhydrase (BCA, E.C. 4.2.1.1) to Lys-ε-NHCOR (R = -CH3, -CH2CH3, and -CH(CH3)2, -CF3) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (kAc,n) of each series of acylated derivatives depended on the number of acylations (n). Plots of log kAc,n vs. n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ~7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ~7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14 however, rates of denaturation stayed approximately constant; analysis suggested these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ~3 for n ~ 14) in the order CH3CONH- < CH3CH2CONH- < (CH3)2CHCONH- < CF3CONH-. These results suggested that the hydrophobicity of CF3CONH- is slightly greater (by a factor of < 2) than that of RHCONH- similar in surface area. PMID:21182314

Do shifts in ectomycorrhizal fungal communities change the sources of N and S for Bishop pine trees?

NASA Astrophysics Data System (ADS)

Willing, C.; Roddy, A. B.; Glassman, S. I.; Dawson, T. E.

2016-12-01

Estimates predict that more than 90% percent of land plants are hosts to mycorrhizal fungi. As these ubiquitous symbionts interact with their plant hosts along a spectrum from parasitism to mutualism, it is important to determine how different communities of these fungi might function along this continuum. In this study, we investigated the consequences of Pinus muricata association with ectomycorrhizal fungi (EcM) sampled from different environments throughout North America. Pines are rarely, if ever, found in nature without their EcM associates. As such, it is important to consider the potential consequences of shifts in EcM communities on the trees themselves and how this may also change across different geographical areas occupied by the trees. Our focus was on quantifying how mycorrhizal (M) and non-mycorhizal (NM) trees may differ in their acquisition of the nutrient resources, N and S. N is essential to life, yet it is typically the most limiting nutrient in temperate terrestrial ecosystems. Though C:N ratios did not vary between our treatments, the stable N isotope ratio between M and NM treatments did differ. This result suggests plants could be using different sources (organic versus inorganic) of N. In addition, the relative dependence on organic N pools also appears to vary with latitude of M origin. S is required for the production of many amino-acids, vitamins, chlorophyll, and important antioxidants such as glutathionine, which has been shown to mitigate reactive oxygen species (ROS). In spite of its potential nutritional and ecological importance, sulfur dynamics and the influence of EcM on sulfur uptake is poorly understood. We found that all M treatments demonstrated S isotope enrichment of approximately 6‰ compared to NM treatments. We hypothesize that this apparent enrichment is due to the EcM reduction of soil-borne sulfate to sulfite and the subsequent transport of sulfite from the fungal associate into the tree's roots.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass

PubMed Central

Smoum, Reem; Bar, Arik; Tan, Bo; Milman, Garry; Attar-Namdar, Malka; Ofek, Orr; Stuart, Jordyn M.; Bajayo, Alon; Tam, Joseph; Kram, Vardit; O'Dell, David; Walker, Michael J.; Bradshaw, Heather B.; Bab, Itai; Mechoulam, Raphael

2010-01-01

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive. PMID:20876113

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions.

PubMed

Beld, Joris; Blatti, Jillian L; Behnke, Craig; Mendez, Michael; Burkart, Michael D

2014-08-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

PubMed Central

Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

2014-01-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

PubMed

Nieto-Peñalver, Carlos G; Bertini, Elisa V; de Figueroa, Lucía I C

2012-07-01

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)-Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis.

PubMed

Hemmerling, Franziska; Lebe, Karen E; Wunderlich, Johannes; Hahn, Frank

2018-03-08

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and validation of a general derivatization HPLC method for the trace analysis of acyl chlorides in lipophilic drug substances.

PubMed

Zheng, Xiangyuan; Luo, Lan; Zhou, Jie; Ruan, Xiaoling; Liu, Wenyuan; Zheng, Feng

2017-06-05

Acyl chlorides are important acylating agents in the synthesis of active pharmaceutical ingredients. Determining the residual acyl chlorides in drug substances is a challenge due to their high reactivity and the matrix interferences from drug substances and their related impurities. This paper describes a general derivatization HPLC method for the determination of aromatic and aliphatic acyl chlorides in lipophilic drug substances. Since most drug substances have weak absorptions in the visible range (above 380nm), the nitro-substituted anilines and nitro-substituted phenylhydrazines were selected as the derivatization reagents due to their weak basicity and red-shift of UV absorption spectra. The maximum wavelength and absorption intensity of nitro-substituted anilines decreased after derivatization with acyl chlorides, whereas the derivatization products of nitro-substituted phenylhydrazines showed the slight increases of maximum wavelength and absorbance intensity. Hence, 2-nitrophenylhydrazine was selected as the suitable derivatization reagent because the derivatives have the maximum UV wavelength absorbance at 395nm, which could largely minimize the matrix interferences. The optimization of the concentration of 2-nitrophenylhydrazine is important for the sensitivity and stability of derivatives. Other reaction conditions including reaction temperature, time and the influence of three competitive solvents (water, methanol and ethanol) on the reaction efficiency were also studied. After derivatization with 100μgmL -1 2-nitrophenylhydrazine at room temperature for 30min, the method was validated for high specificity and sensitivity with the detection limits in the range of 0.01-0.03μgmL -1 . The proposed method was applied as a generic method to determine the residual acyl chlorides in lipophilic drug substances. Copyright © 2017 Elsevier B.V. All rights reserved.

[The Effect of Introduction of the Heterologous Gene Encoding the N-acyl-homoserine Lactonase (aiiA) on the Properties of Burkholderia cenocepacia 370].

PubMed

Plyuta, V A; Lipasova, V A; Koksharova, O A; Veselova, M A; Kuznetsov, A E; Khmel, I A

2015-08-01

To study the role of Quorum Sensing (QS) regulation in the control of the cellular processes of Burkholderia cenocepacia 370, plasmid pME6863 was transferred into its cells. The plasmid contains a heterologous gene encoding for AiiA N-acyl-homoserine lactonase, which degrades the signaling molecules of the QS system of N-acyl-homoserine lactones (AHL). An absence or reduction of AHL in the culture was revealed with the biosensors Chromobacterium violaceum CV026 and Agrobacterium tumifaciens NT1/pZLR4, respectively. The presence of the aiiA gene, which was cloned from Bacillus sp. A24 in the cells of B. cenocepacia 370, resulted in a lack of hemolytic activity, which reduced the extracellular proteolytic activity and decreased the cells' ability to migration in swarms on the surface of the agar medium. The introduction of the aiiA gene did not affect lipase activity, fatty acids synthesis, HCN synthesis, or biofilm formation. Hydrogen peroxide was shown to stimulate biofilm formation by B. cenocepacia 370 in concentrations that inhibited or weakly suppressed bacterial growth. The introduction of the aiiA gene into the cells did not eliminate this effect but it did reduce it.

Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase.

PubMed

Sorek, Nadav; Poraty, Limor; Sternberg, Hasana; Buriakovsky, Ella; Bar, Einat; Lewinsohn, Efraim; Yalovsky, Shaul

2017-12-01

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His 6 -green fluorescent protein (GFP)- Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His 6 -GFP-Atrop6 CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His 6 -GFP-Atrop6 CA mS 156 , in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes. Copyright © 2017 American Society for Microbiology.

Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase

PubMed Central

Sorek, Nadav; Poraty, Limor; Sternberg, Hasana; Buriakovsky, Ella; Bar, Einat; Lewinsohn, Efraim

2017-01-01

ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes. PMID:28894027

Assembly of N,N-disubstituted hydrazines and 1-aryl-1H-indazoles via copper-catalyzed coupling reactions.

PubMed

Xiong, Xiaodong; Jiang, Yongwen; Ma, Dawei

2012-05-18

CuI-catalyzed coupling of N-acyl-N'-substituted hydrazines with aryl iodides takes place at 60-90 °C to afford N-acyl-N',N'-disubstituted hydrazines regioselectively and thereby gives a facile method for assembling N,N-diaryl hydrazines. N-Acyl-N'-substituted hydrazines can also react with 2-bromoarylcarbonylic compounds at 60-125 °C under the catalysis of CuI/4-hydroxy-l-proline to provide 1-aryl-1H-indazoles.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed

Stahl, U.; Banas, A.; Stymne, S.

1995-03-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

Identification, synthesis and regulatory function of the N-acylated homoserine lactone signals produced by Pseudomonas chlororaphis HT66.

PubMed

Peng, Huasong; Ouyang, Yi; Bilal, Muhammad; Wang, Wei; Hu, Hongbo; Zhang, Xuehong

2018-01-22

Pseudomonas chlororaphis HT66 isolated from the rice rhizosphere is an important plant growth-promoting rhizobacteria that produce phenazine-1-carboxamide (PCN) in high yield. Phenazine production is regulated by a quorum sensing (QS) system that involves the N-acylated homoserine lactones (AHLs)-a prevalent type of QS molecule. Three QS signals were detected by thin layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS), which identified to be N-(3-hydroxy hexanoyl)-L-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy octanoyl)-L-homoserine lactone (3-OH-C8-HSL) and N-(3-hydroxy decanoyl)-L-homoserine lactone (3-OH-C10-HSL). The signal types and methods of synthesis were different from that in other phenazine-producing Pseudomonas strains. By non-scar deletion and heterologous expression techniques, the biosynthesis of the AHL-signals was confirmed to be only catalyzed by PhzI, while other AHLs synthases i.e., CsaI and HdtS were not involved in strain HT66. In comparison to wild-type HT66, PCN production was 2.3-folds improved by over-expression of phzI, however, phzI or phzR mutant did not produce PCN. The cell growth of HT66∆phzI mutant was significantly decreased, and the biofilm formation in phzI or phzR inactivated strains of HT66 decreased to various extents. In conclusion, the results demonstrate that PhzI-PhzR system plays a critical role in numerous biological processes including phenazine production.

Design and synthesis of 2-nitroimidazoles with variable alkylating and acylating functionality.

PubMed

Winters, Thomas; Sercel, Anthony; Suto, Carla; Elliott, William; Leopold, Wilbur; Leopold, Judith; Showalter, Hollis

2014-01-01

The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C(1.6) values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed <1% of the alkylating activity of RSU-1069. In vivo, one analog showed an enhancement ratio of 1.6 relative to vehicle control when tested in B6C3F1 mice with an implanted KHT sarcoma. The data reinforce prior findings that there is a correlation between alkylation potential and in vivo activity.

Dynamic cost shifting in hospitals: evidence from the 1980s and 1990s.

PubMed

Clement, J P

The purpose of this paper is to determine whether dynamic cost shifting occurred among acute care hospitals during the period from the early 1980s to the early 1990s and, if so, whether market factors affected the ability to shift costs. Evidence from this study of California acute care hospitals during three time intervals shows that the hospital did practice dynamic cost shifting, but that their ability to shift costs decreased over time. Surprisingly, hospital competition and HMO penetration did not influence cost shifting. However, increasing HMO penetration (measured as the HMO percentage of hospital discharges) did decrease both net prices and costs for the early part of the study, but later was associated with increases in both.

Blunted Suppression of Acyl-Ghrelin in Response to Fructose Ingestion in Obese Adolescents: the Role of Insulin Resistance

PubMed Central

Van Name, Michelle; Giannini, Cosimo; Santoro, Nicola; Jastreboff, Ania; Kubat, Jessica; Li, Fangyong; Kursawe, Romy; Savoye, Mary; Duran, Elvira; Dziura, James; Sinha, Rajita; Sherwin, Robert; Cline, Gary; Caprio, Sonia

2015-01-01

Objective Fructose consumption has risen alongside obesity and diabetes. Gut hormones involved in hunger and satiety (ghrelin and PYY) may respond differently to fructose compared to glucose ingestion. We evaluated the effects of glucose and fructose ingestion on ghrelin and PYY in lean and obese adolescents with differing insulin sensitivity. Methods Adolescents were divided into lean (n=14), obese insulin sensitive (n=12) (OIS), and obese insulin resistant (n=15) (OIR). In a double-blind, cross-over design, subjects drank 75g of glucose or fructose in random order, serum was obtained every 10 minutes for 60 minutes. Results Baseline acyl-ghrelin was highest in lean and lowest in OIR (p=0.02). After glucose ingestion acyl-ghrelin decreased similarly in lean and OIS, but appeared lower in OIR (vs lean p=0.03). Suppression differences were more pronounced after fructose (lean vs. OIS p=0.008, lean vs. OIR p<0.001). OIS became significantly hungrier after fructose (p=0.015). PYY was not significantly different at baseline, varied minimally after glucose, and rose after fructose. Conclusion Compared to lean, OIS adolescents have impaired acyl-ghrelin responses to fructose but not glucose, whereas OIR adolescents have blunted responses to both. Diminished suppression of acyl-ghrelin in childhood obesity, particularly if accompanied by insulin resistance, may promote hunger and overeating. PMID:25645909

Understanding Acyl Chain and Glycerolipid Metabolism in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohlrogge, John B.

2013-11-05

Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed Central

Stahl, U.; Banas, A.; Stymne, S.

1995-01-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

PubMed Central

Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

2015-01-01

Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

Role of AlGaN/GaN interface traps on negative threshold voltage shift in AlGaN/GaN HEMT

NASA Astrophysics Data System (ADS)

Malik, Amit; Sharma, Chandan; Laishram, Robert; Bag, Rajesh Kumar; Rawal, Dipendra Singh; Vinayak, Seema; Sharma, Rajesh Kumar

2018-04-01

This article reports negative shift in the threshold-voltage in AlGaN/GaN high electron mobility transistor (HEMT) with application of reverse gate bias stress. The device is biased in strong pinch-off and low drain to source voltage condition for a fixed time duration (reverse gate bias stress), followed by measurement of transfer characteristics. Negative threshold voltage shift after application of reverse gate bias stress indicates the presence of more carriers in channel as compared to the unstressed condition. We propose the presence of AlGaN/GaN interface states to be the reason of negative threshold voltage shift, and developed a process to electrically characterize AlGaN/GaN interface states. We verified the results with Technology Computer Aided Design (TCAD) ATLAS simulation and got a good match with experimental measurements.

Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

1998-12-01

Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated

Acyl donors for native chemical ligation.

PubMed

Yan, Bingjia; Shi, Weiwei; Ye, Linzhi; Liu, Lei

2018-04-11

Native chemical ligation (NCL) has become one of the most important methods in chemical syntheses of proteins. Recently, in order to expand its scope, considerable effort has been devoted to tuning the C-terminal acyl donor thioesters used in NCL. This article reviews the recent advances in the design of C-terminal acyl donors, their precursors and surrogates, and highlights some noteworthy progress that may lead the future direction of protein chemical synthesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

PubMed

Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

2013-04-02

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synthesis, characterization stereochemistry and anti-bacterial evaluation of certain N-acyl-c-3,t-3-dimethyl-r-2,c-6-diphenylpiperidin-4-ones

NASA Astrophysics Data System (ADS)

Ponnuswamy, S.; Kayalvizhi, R.; Jamesh, M.; Uma Maheswari, J.; Thenmozhi, M.; Ponnuswamy, M. N.

2016-09-01

A new series of N-acyl-c-3,t-3-dimethyl-r-2,c-6-diphenylpiperidin-4-ones 2-6 has been synthesized and characterized using IR, mass, 1H, 13C, DEPT and 2D (COSY and HSQC) NMR spectral techniques. The NMR spectral data indicate that the N-acylpiperidin-4-ones 2-6 prefer to exist in a distorted boat conformation B1 with coplanar orientation of N-C=O moiety. The stereodynamics of these systems have been studied by recording the dynamic 1H NMR spectra of compound 4, and the energy barrier for N-CO rotation is determined to be 52.75 kJ/mol. Furthermore the compounds 1-5 show significant antibacterial activity.

Finite Element Analysis and Vibration Control of Lorry’s Shift Mechanism

NASA Astrophysics Data System (ADS)

Qiangwei, Li

2017-11-01

The transmission is one of the important parts of the automobile’s transmission system, Shift mechanism’s main function of transmission is to adjust the position of the shift fork, toggle the synchronizer’s tooth ring, so that the gears are separated and combined to achieve the shift. Therefore, in order to ensure the reliability and stability of the shift process, the vibration characteristics of the shift mechanism cannot be ignored. The static analysis of the shift fork is carried out, and the stress distribution of the shift fork is obtained according to the operating characteristics of the shift mechanism of the lorry transmission in this paper. The modal analysis of the shift mechanism shows the low-order vibration frequencies and the corresponding modal vibration shapes, and the vibration control analysis is carried out according to the simulation results. The simulation results provide the theoretical basis for the reasonable optimization design of the shift mechanism of the lorry transmission.

Regioselective Acylation of Diols and Triols: The Cyanide Effect.

PubMed

Peng, Peng; Linseis, Michael; Winter, Rainer F; Schmidt, Richard R

2016-05-11

Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation.

Acyl chain length and charge effect on Tamoxifen-lipid model membrane interactions

NASA Astrophysics Data System (ADS)

Bilge, Duygu; Kazanci, Nadide; Severcan, Feride

2013-05-01

Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pancreas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of multilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalmitoyl phosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycerol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concentration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%). However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The interactions between TAM and head group of multilamellar liposomes was monitored by analyzing the Cdbnd O stretching and PO2- antisymmetric double bond stretching bands. Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics.

Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

PubMed Central

Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

2016-01-01

An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Swaminathan, S.; Zhou, R.

2011-02-18

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Z Zhang; R Zhou; J Sauder

2011-12-31

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

New acylated clionasterol glycosides from Valeriana officinalis.

PubMed

Pullela, Srinivas V; Choi, Young Whan; Khan, Shabana I; Khan, Ikhlas A

2005-10-01

The chloroform extract of Valeriana officinalis led to the isolation of clionasterol-3-O-beta-D-glucopyranoside and a mixture of 6'-O-acyl-beta-D-glucosyl-clionasterols. The acyl moieties were identified as hexadecanoyl, 8 E,11 E-octadecadienoyl and 14-methylpentadecanoyl by alkaline hydrolysis followed by GC-MS analysis. The isolated compounds did not exhibit any anti-inflammatory, anticancer or cytotoxic activity when tested in a variety of in vitro cell-based assays.

α-melanocyte stimulating hormone modulates the central acyl ghrelin-induced stimulation of feeding, gastrointestinal motility, and colonic secretion.

PubMed

Huang, Hsien-Hao; Chen, Liang-Yu; Doong, Ming-Luen; Chang, Shi-Chuan; Chen, Chih-Yen

2017-01-01

Acyl ghrelin-induced intake depends on hypothalamic neuropeptide Y and agouti-related protein (AgRP) neurotransmitters. Intracerebroventricular (ICV) injection of AgRP increases feeding through competitive antagonism at melanocortin receptors. ICV administration of α-melanocyte stimulating hormone (α-MSH), a natural antagonist of AgRP, may modulate the acyl ghrelin-induced orexigenic effect. This study aimed to investigate the modulating effect of α-MSH on the central acyl ghrelin-induced food intake, gastrointestinal motility, and colonic secretion in rats. We examined the effects of α-MSH and acyl ghrelin on food intake, gastric emptying, small intestinal transit, colonic motility, and secretion in conscious rats with a chronic implant of ICV catheters. ICV injection of O - n -octanoylated ghrelin (0.1 nmol/rat) significantly increased the cumulative food intake up to 8 h ( P <0.01), enhanced non-nutrient semi-liquid gastric emptying ( P <0.001), increased the geometric center and running percentage of small intestinal transit ( P <0.001), accelerated colonic transit time ( P <0.05), and increased fecal pellet output ( P <0.01) and total fecal weight ( P <0.01). Pretreatment with ICV injection of α-MSH (1.0 and 2.0 nmol/rat) attenuated the acyl ghrelin-induced hyperphagic effect, fecal pellet output, and total fecal weight, while higher dose of α-MSH (2.0 nmol/rat) attenuated the increase in the geometric center of small intestinal transit ( P <0.01). However, neither dose of α-MSH altered acyl ghrelin-stimulated gastroprokinetic effect, increase in the running percentage of small intestinal transit, nor accelerated colonic transit time. α-MSH is involved in central acyl ghrelin-elicited feeding, small intestinal transit, fecal pellet output, and fecal weight. α-MSH does not affect central acyl ghrelin-induced acceleration of gastric emptying and colonic transit time in rats.

The I=2 ππ S-wave Scattering Phase Shift from Lattice QCD

DOE PAGES

Beane, S. R.; Chang, E.; Detmold, W.; ...

2012-02-16

The π +π + s-wave scattering phase-shift is determined below the inelastic threshold using Lattice QCD. Calculations were performed at a pion mass of m π ≈ 390 MeV with an anisotropic n f = 2+1 clover fermion discretization in four lattice volumes, with spatial extent L ≈ 2.0, 2.5, 3.0 and 3.9 fm, and with a lattice spacing of b s ≈ 0.123 fm in the spatial direction and b t b s/3.5 in the time direction. The phase-shift is determined from the energy-eigenvalues of π +π + systems with both zero and non-zero total momentum in the latticemore » volume using Luscher's method. Our calculations are precise enough to allow for a determination of the threshold scattering parameters, the scattering length a, the effective range r, and the shape-parameter P, in this channel and to examine the prediction of two-flavor chiral perturbation theory: m π 2 a r = 3+O(m π 2/Λ χ 2). Chiral perturbation theory is used, with the Lattice QCD results as input, to predict the scattering phase-shift (and threshold parameters) at the physical pion mass. Our results are consistent with determinations from the Roy equations and with the existing experimental phase shift data.« less

Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

PubMed

Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

2013-01-17

Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis.

Two fatty acyl reductases involved in moth pheromone biosynthesis

PubMed Central

Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

2016-01-01

Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

A chiral Brønsted acid-catalyzed highly enantioselective Mannich-type reaction of α-diazo esters with in situ generated N-acyl ketimines.

PubMed

Unhale, Rajshekhar A; Sadhu, Milon M; Ray, Sumit K; Biswas, Rayhan G; Singh, Vinod K

2018-04-03

A chiral phosphoric acid-catalyzed asymmetric Mannich-type reaction of α-diazo esters with in situ generated N-acyl ketimines, derived from 3-hydroxyisoindolinones has been demonstrated in this communication. A variety of isoindolinone-based α-amino diazo esters bearing a quaternary stereogenic center were afforded in high yields (up to 99%) with excellent enantioselectivities (up to 99% ee). Furthermore, the synthetic utility of the products has been depicted by the hydrogenation of the diazo moiety of adducts.

Fatty acyl-CoA reductases of birds

PubMed Central

2011-01-01

Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

PubMed

Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

2010-11-15

Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication. Published by Elsevier Ltd.

Synthesis and emulsifying properties of carbohydrate fatty acid esters produced from Agave tequilana fructans by enzymatic acylation.

PubMed

Casas-Godoy, Leticia; Arrizon, Javier; Arrieta-Baez, Daniel; Plou, Francisco J; Sandoval, Georgina

2016-08-01

Carbohydrate fatty acid esters are non-ionic surfactants with a broad spectrum of applications. These molecules are generally synthesized using short carbohydrates or linear fructans; however in this research carbohydrate fatty acid esters were produced for the first time with branched fructans from Agave tequilana. Using immobilized lipases we successfully acylated A. tequilana fructans with vinyl laurate, obtaining products with different degrees of polymerization (DP). Lipozyme 435 was the most efficient lipase to catalyze the transesterification reaction. HPLC and ESI-MS analysis proved the presence of a mixture of acylated products as a result of the chemical complexity of fructans in the A. tequilana. The ESI-MS spectra showed a molecular mass shift between 183 and 366g/mol for fructooligosaccharides with a DP lower than 6, which indicated the presence of Agave fructans that had been mono- and diacylated with lauric acid. The carbohydrate fatty acid esters (CFAE) obtained showed good emulsifying properties in W/O emulsions. Copyright © 2016 Elsevier Ltd. All rights reserved.

N-acyl-homoserine lactone-mediated quorum-sensing in Azospirillum: an exception rather than a rule.

PubMed

Vial, Ludovic; Cuny, Caroline; Gluchoff-Fiasson, Katia; Comte, Gilles; Oger, Phil M; Faure, Denis; Dessaux, Yves; Bally, René; Wisniewski-Dyé, Florence

2006-11-01

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

PubMed

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2016-08-01

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Acyl Meldrum's acid derivatives: application in organic synthesis

NASA Astrophysics Data System (ADS)

Janikowska, K.; Rachoń, J.; Makowiec, S.

2014-07-01

This review is focused on an important class of Meldrum's acid derivatives commonly known as acyl Meldrum's acids. The preparation methods of these compounds are considered including the recently proposed and rather rarely used ones. The chemical properties of acyl Meldrum's acids are described in detail, including thermal stability and reactions with various nucleophiles. The possible mechanisms of these transformations are analyzed. The bibliography includes 134 references.

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

PubMed

Sugimoto, H; Yamashita, S

1999-05-18

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

PubMed Central

Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

2016-01-01

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

PubMed

Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

2016-02-22

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

Identifying and Investigating the Late-1960s Interhemispheric SST Shift

NASA Astrophysics Data System (ADS)

Friedman, A. R.; Lee, S. Y.; Liu, Y.; Chiang, J. C. H.

2014-12-01

The global north-south interhemispheric sea surface temperature (SST) difference experienced a pronounced and rapid decrease in the late 1960s, which has been linked to drying in the Sahel, South Asia, and East Asia. However, some basic questions about the interhemispheric SST shift remain unresolved, including its scale and whether the constituent changes in different basins were coordinated. In this study, we systematically investigate the spatial and temporal behavior of the late-1960s interhemispheric SST shift using ocean surface and subsurface observations. We also evaluate potential mechanisms using control and specific-forcing CMIP5 simulations. Using a regime shift detection technique, we identify the late-1960s shift as the most prominent in the historical observational SST record. We additionally examine the corresponding changes in upper-ocean heat content and salinity associated with the shift. We find that there were coordinated upper-ocean cooling and freshening in the subpolar North Atlantic, the region of the largest-magnitude SST decrease during the interhemispheric shift. These upper-ocean changes correspond to a weakened North Atlantic thermohaline circulation (THC). However, the THC decrease does not fully account for the rapid global interhemispheric SST shift, particularly the warming in the extratropical Southern Hemisphere.

Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

PubMed

Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

2016-03-01

To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis.

PubMed

Abbadi, A; Brummel, M; Spener, F

2000-10-01

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction.

PubMed

Mitamura, Kuniko; Hori, Naohiro; Mino, Shiori; Iida, Takashi; Hofmann, Alan F; Ikegawa, Shigeo

2012-04-01

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

An annotated database of Arabidopsis mutants of acyl lipid metabolism

DOE PAGES

McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

2014-12-10

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

ERIC Educational Resources Information Center

Thomas, Jacob

2009-01-01

Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder

PubMed Central

Modi, Hiren R.; Basselin, Mireille; Taha, Ameer Y.; Li, Lei O.; Coleman, Rosalind A.; Bialer, Meir; Rapoport, Stanley I.

2013-01-01

Background Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action. Methods Rat Acsl4-flag protein was expressed in E. coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics. Results Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4 mM compared to a published Ki of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect. Conclusions PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients. PMID:23354024

N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

PubMed

Welch, M; Todd, D E; Whitehead, N A; McGowan, S J; Bycroft, B W; Salmond, G P

2000-02-15

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

PubMed

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

USDA-ARS?s Scientific Manuscript database

Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

PubMed

Fujiwara, Yuko; Hama, Kotaro; Tsukahara, Makoto; Izumi-Tsuzuki, Ryosuke; Nagai, Toru; Ohe-Yamada, Mihoko; Inoue, Keizo; Yokoyama, Kazuaki

2018-01-01

Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [ 14 C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [ 14 C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [ 14 C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Synthesis of 2-Ethenylcyclopropyl Aryl Ketones via Intramolecular SN2-like Displacement of an Ester.

PubMed

Jung, Michael E; Sun, Daniel L; Dwight, Timothy A; Yu, Peiyuan; Li, Wei; Houk, K N

2016-10-07

The efficient synthesis of trans-2-ethenylcyclopropyl aryl ketones via an intramolecular S N 2-like displacement of an allylic ester is reported. A novel 1,5-acyl shift process is also observed that contributes to the product mixture. Theoretical calculations provide a rationale for the observed product ratio.

Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status.

PubMed

El-Shehaby, Amal M; Obaia, Eman M; Alwakil, Sahar S; Hiekal, Ahmed A

2010-07-01

The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. The aim of the present study was to compare acylated and total ghrelin concentration in patients with liver cirrhosis and to investigate the possible relationship between ghrelin and clinical and nutritional parameters. Sixty patients with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twenty healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls. Fasting levels of total, acylated ghrelin, leptin, TNF-alpha and insulin were measured in all subjects, in addition, clinical and nutrition parameters were assessed. In cirrhotic patients, plasma levels of both acylated and total ghrelin were significantly higher than those in the controls. The mean plasma acylated ghrelin levels were significantly higher in Child C cirrhosis compared to Child A and B. Ghrelin (total and acylated) were negatively correlated with leptin in cirrhotic patients confirming the fact that leptin acts as a physiological counterpart of ghrelin. Nutritional and metabolic abnormalities in cirrhotic patients may be dependent on the changes in the ghrelin/leptin systems, mainly the acylated form of ghrelin.

Chlorogenic acids and the acyl-quinic acids: discovery, biosynthesis, bioavailability and bioactivity.

PubMed

Clifford, Michael N; Jaganath, Indu B; Ludwig, Iziar A; Crozier, Alan

2017-12-13

Covering: 2000 up to late 2017This review is focussed upon the acyl-quinic acids, the most studied group within the ca. 400 chlorogenic acids so far reported. The acyl-quinic acids, the first of which was characterised in 1846, are a diverse group of plant-derived compounds produced principally through esterification of an hydroxycinnamic acid and 1l-(-)-quinic acid. Topics addressed in this review include the confusing nomenclature, quantification and characterisation by NMR and MS, biosynthesis and role in planta, and the occurrence of acyl-quinic acids in coffee, their transformation during roasting and delivery to the beverage. Coffee is the major human dietary source world-wide of acyl-quinic acids and consideration is given to their absorption and metabolism in the upper gastrointestinal tract, and the colon where the microbiota play a key role in the formation of catabolites. Evidence on the potential of the in vivo metabolites and catabolites of acyl-quinic acids to promote the consumer's health is evaluated.

A novel series of N-acyl substituted indole-linked benzimidazoles and naphthoimidazoles as potential anti inflammatory, anti biofilm and anti microbial agents.

PubMed

Abraham, Rajan; Prakash, Periakaruppan; Mahendran, Karthikeyan; Ramanathan, Murugappan

2018-01-01

A novel N-acyl substituted indole-linked benzimidazoles and naphthoimidazoles were synthesized. Their chemical structures were confirmed using spectroscopic tools including 1 H NMR, 13 C NMR and CHN-elemental analyses. Anti inflammatory activity for all target compounds was evaluated in-vitro. The synthesized compounds hinder the biofilm formation and control the growth of the pathogen, Staphylococcus epidermis. Anti microbial activity of the compounds was evaluated against both Gram negative and Gram positive bacteria such as Staphylococcus aureus (MTCC 2940), Pseudomonas aeruginosa (MTCC424), Escherchia coli (MTCC 443) and Enterococcus fecalis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides.

PubMed

Ziaullah; Rupasinghe, H P Vasantha

2016-04-01

This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-2'-glucoside, using Candida antarctica lipase B (Novozyme 435(®)), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4-5 folds. Copyright © 2016 Elsevier Inc. All rights reserved.

Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides

PubMed Central

Chen, Haotong; Olson, Andrew S.; Su, Wei; Dussault, Patrick H.; Du, Liangcheng

2015-01-01

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs. PMID:26726302

Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides.

PubMed

Chen, Haotong; Olson, Andrew S; Su, Wei; Dussault, Patrick H; Du, Liangcheng

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on ( R )-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several ( R )-3-hydroxy fatty acids and their acyl SNAC ( N -acetylcysteamine) thioesters as substrates for the ACLs. Feeding ( R )-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro , ACL6 showed the highest catalytic efficiency in converting ( R )-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.

Theoretical study of hydrogen bonding interaction in nitroxyl (HNO) dimer: interrelationship of the two N-H...O blue-shifting hydrogen bonds.

PubMed

Liu, Ying; Liu, Wenqing; Li, Haiyang; Liu, Jianguo; Yang, Yong

2006-10-19

The hydrogen bonding interactions of the HNO dimer have been investigated using ab initio molecular orbital and density functional theory (DFT) with the 6-311++G(2d,2p) basis set. The natural bond orbital (NBO) analysis and atom in molecules (AIM) theory were applied to understand the nature of the interactions. The interrelationship between one N-H...O hydrogen bond and the other N-H...O hydrogen bond has been established by performing partial optimizations. The dimer is stabilized by the N-H...O hydrogen bonding interactions, which lead to the contractions of N-H bonds as well as the characteristic blue-shifts of the stretching vibrational frequencies nu(N-H). The NBO analysis shows that both rehybridization and electron density redistribution contribute to the large blue-shifts of the N-H stretching frequencies. A quantitative correlations of the intermolecular distance H...O (r(H...O)) with the parameters: rho at bond critical points (BCPs), s-characters of N atoms in N-H bonds, electron densities in the sigma*(N-H), the blue-shift degrees of nu(N-H) are presented. The relationship between the difference of rho (|Deltarho|) for the one hydrogen bond compared with the other one and the difference of interaction energy (DeltaE) are also illustrated. It indicates that for r(H...O) ranging from 2.05 to 2.3528 A, with increasing r(H...O), there is the descending tendency for one rho(H...O) and the ascending tendency for the other rho(H...O). r(H...O) ranging from 2.3528 to 2.85 A, there are descending tendencies for the two rho(H...O) with increasing r(H...O). On the potential energy surface of the dimer, the smaller the difference between one rho(H...O) and the other rho(H...O) is, the more stable the structure is. As r(H...O) increases, the blue-shift degrees of nu(N-H) decrease. The cooperative descending tendencies in s-characters of two N atoms with increasing r(H...O) contribute to the decreases in blue-shift degrees of nu(N-H). Ranging from 2.05 to 2.55 A

Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

NASA Astrophysics Data System (ADS)

Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

2017-06-01

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

Acyl hydrazides as acyl donors for the synthesis of diaryl and aryl alkyl ketones.

PubMed

Akhbar, Ahmed R; Chudasama, Vijay; Fitzmaurice, Richard J; Powell, Lyn; Caddick, Stephen

2014-01-21

In this communication we describe a novel strategy for the formation of valuable diaryl and aryl alkyl ketones from acyl hydrazides. A wide variety of ketones are prepared and the mild reaction conditions allow for the use of a range of functionalities, especially in the synthesis of diaryl ketones.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

PubMed

Karlen, Steven D; Free, Heather C A; Padmakshan, Dharshana; Smith, Bronwen G; Ralph, John; Harris, Philip J

2018-06-01

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p -coumarate. The Poaceae, or grass family, is a member of this group, and most of the p -coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p -coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. © 2018 American Society of Plant Biologists. All rights reserved.

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

PubMed

Périgaud, C; Gosselin, G; Girardet, J L; Korba, B E; Imbach, J L

1999-01-01

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

PubMed Central

Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

2017-01-01

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

LOCATION OF ACYL GROUPS ON TWO PARTLY ACYLATED GLYCOLIPIDS FROM STRAINS OF USTILAGO (SMUT FUNGI),

DTIC Science & Technology

erythritol from Ustilago sp. (probably U. nuda (Jens.) Rostr. = U. tritici (Pers.) Rostr.) PRL-627 were acetalated with methyl vinyl ether, deacylated...Partly acylated ustilagic acids 8 (from Ustilago maydis (DC) Corda (= U. zeae Unger) PRL-119), consisting of partially esterified beta-cellobiosyl

MomL, a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

PubMed Central

Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom

2014-01-01

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105 s−1 M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. PMID:25398866

Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).

PubMed

Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C

2011-04-01

The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

Evolution of the acyl-CoA binding protein (ACBP)

PubMed Central

Burton, Mark; Rose, Timothy M.; Færgeman, Nils J.; Knudsen, Jens

2005-01-01

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein that binds C12–C22 acyl-CoA esters with high affinity. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified in all four eukaryotic kingdoms, Animalia, Plantae, Fungi and Protista, and eleven eubacterial species. ACBP homologues were not detected in any other known bacterial species, or in archaea. Nearly all of the ACBP-containing bacteria are pathogenic to plants or animals, suggesting that an ACBP gene could have been acquired from a eukaryotic host by horizontal gene transfer. Many bacterial, fungal and higher eukaryotic species only harbour a single ACBP homologue. However, a number of species, ranging from protozoa to vertebrates, have evolved two to six lineage-specific paralogues through gene duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage-specific paralogues have evolved altered functions. The appearance of ACBP very early on in evolution points towards a fundamental role of ACBP in acyl-CoA metabolism, including ceramide synthesis and in signalling. PMID:16018771

Acylated and unacylated ghrelin confer neuroprotection to mesencephalic neurons.

PubMed

Wagner, Johanna; Vulinović, Franca; Grünewald, Anne; Unger, Marcus M; Möller, Jens C; Klein, Christine; Michel, Patrick P; Ries, Vincent; Oertel, Wolfgang H; Alvarez-Fischer, Daniel

2017-12-04

The polypeptide ghrelin is an endogenous ligand at the growth hormone secretagogue receptor 1a. To ghrelin multiple functions have been ascribed including promotion of gastrointestinal motility. Postprandial ghrelin levels have been reported to be reduced in patients suffering from Parkinson disease (PD). Experimental studies revealed neuroprotective effects of ghrelin in different PD models. The purpose of the present study was (i) to further elucidate the mechanism underlying the neuroprotective action of ghrelin and (ii) to determine whether these effects occur with both the acylated and the unacylated form. The study was conducted in primary mesencephalic cultures treated with mitochondrial complex I and complex II inhibitors. We show that protective effects of ghrelin against complex I inhibition with MPP + were independent of the acylation status of ghrelin, although acylated ghrelin appeared to be more potent. Protection by both forms was also observed when neurons were exposed to the complex II inhibitor 3-NP. Both forms led to higher oxygen consumption rates upon electron transport chain uncoupling, indicating that the two peptides may exert uncoupling effects themselves. We demonstrate that the rescue provided by ghrelin required calcium influx through L-type voltage-gated calcium channels. Whereas the protective effects of acylated ghrelin required receptor binding, effects of the unacylated form remained unaffected by treatment with a ghrelin receptor antagonist. Importantly, inhibition of ghrelin O-acyltransferase failed to reduce the activity of unacylated ghrelin. Overall, our data suggest that both acylated and unacylated ghrelin afford protection to dopamine neurons but through mechanisms that only partially overlap. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Regioselective and stereospecific acylation across oxirane- and silyloxy systems as a novel strategy to the synthesis of enantiomerically pure mono-, di- and triglycerides.

PubMed

Stamatov, Stephan D; Stawinski, Jacek

2007-12-07

A trifluoroacetate-catalyzed opening of the oxirane ring of glycidyl derivatives bearing allylic acyl or alkyl functionalities with trifluoroacetic anhydride (TFAA), provides an efficient entry to configurationally homogeneous 1(3)-acyl- or 1(3)-O-alkyl-sn-glycerols. Selective introduction of tert-butyldimethylsilyl- (TBDMS), or triisopropylsilyl- (TIPS) transient protections at the terminal sites within these key intermediates secures 1(3)-acyl- or 1(3)-O-alkyl-3(1)-O-TBDMS (or TIPS)-sn-glycerols as general bifunctional precursors to 1,2(2,3)-diacyl-, 1(3)-O-alkyl-2-acyl- and 1,3-diacyl-sn-glycerols and hence triester isosters. Incorporation of a requisite acyl residue at the central carbon of the silylated synthons with a subsequent Et(3)N.3HF-promoted, direct trichloroacetylation across the siloxy system by trichloroacetic anhydride (TCAA), followed by cleavage of the trichloroacetyl group, affords the respective 1,2(2,3)-diacyl- or 1(3)-O-alkyl-2-acyl-sn-glycerols. Alternatively, a reaction sequence involving: (i) attachment of a trichloroacetyl fragment at the stereogenic C2-centre of the monosilylated glycerides; (ii) replacement of the silyl moiety by a short- or long-chain carboxylic acid residue by means of the acylating agent: tetra-n-butylammonium bromide (TBABr)-carboxylic acid anhydride (CAA)-trimethylsilyl bromide (TMSBr); and (iii) removal of the trichloroacetyl replacement, provides pure 1,3-diacyl-sn-glycerols. The TBABr-CAA-TMSBr reagent system allows also a one-step conversion of 1,2-diacylglycerol silyl ethers into homochiral triglycerides with predefined asymmetry and degree of unsaturation. These compounds can also be accessed via a two-step one-pot approach where the trichloroacetyl derivatives of 1,2(2,3)- or 1,3-diacyl-sn-glycerols serve as triester building blocks for establishing the third ester bond at preselected C3(1)- or C2-positions within the glycerol skeleton at the very last synthetic stage. In all instances, the target compounds

Structure Shift of GaN Among Nanowall Network, Nanocolumn, and Compact Film Grown on Si (111) by MBE.

PubMed

Zhong, Aihua; Fan, Ping; Zhong, Yuanting; Zhang, Dongping; Li, Fu; Luo, Jingting; Xie, Yizhu; Hane, Kazuhiro

2018-02-13

Structure shift of GaN nanowall network, nanocolumn, and compact film were successfully obtained on Si (111) by plasma-assisted molecular beam epitaxy (MBE). As is expected, growth of the GaN nanocolumns was observed in N-rich condition on bare Si, and the growth shifted to compact film when the Ga flux was improved. Interestingly, if an aluminum (Al) pre-deposition for 40 s was carried out prior to the GaN growth, GaN grows in the form of the nanowall network. Results show that the pre-deposited Al exits in the form of droplets with typical diameter and height of ~ 80 and ~ 6.7 nm, respectively. A growth model for the nanowall network is proposed and the growth mechanism is discussed. GaN grows in the area without Al droplets while the growth above Al droplets is hindered, resulting in the formation of continuous GaN nanowall network that removes the obstacles of nano-device fabrication.

Structure Shift of GaN Among Nanowall Network, Nanocolumn, and Compact Film Grown on Si (111) by MBE

NASA Astrophysics Data System (ADS)

Zhong, Aihua; Fan, Ping; Zhong, Yuanting; Zhang, Dongping; Li, Fu; Luo, Jingting; Xie, Yizhu; Hane, Kazuhiro

2018-02-01

Structure shift of GaN nanowall network, nanocolumn, and compact film were successfully obtained on Si (111) by plasma-assisted molecular beam epitaxy (MBE). As is expected, growth of the GaN nanocolumns was observed in N-rich condition on bare Si, and the growth shifted to compact film when the Ga flux was improved. Interestingly, if an aluminum (Al) pre-deposition for 40 s was carried out prior to the GaN growth, GaN grows in the form of the nanowall network. Results show that the pre-deposited Al exits in the form of droplets with typical diameter and height of 80 and 6.7 nm, respectively. A growth model for the nanowall network is proposed and the growth mechanism is discussed. GaN grows in the area without Al droplets while the growth above Al droplets is hindered, resulting in the formation of continuous GaN nanowall network that removes the obstacles of nano-device fabrication.

Global Analysis of Apicomplexan Protein S-Acyl Transferases Reveals an Enzyme Essential for Invasion

PubMed Central

Frénal, Karine; Tay, Chwen L; Mueller, Christina; Bushell, Ellen S; Jia, Yonggen; Graindorge, Arnault; Billker, Oliver; Rayner, Julian C; Soldati-Favre, Dominique

2013-01-01

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite’s ability to egress. PMID:23638681

Thyroid receptor ligands. Part 8: Thyromimetics derived from N-acylated-alpha-amino acid derivatives displaying modulated pharmacological selectivity compared with KB-141.

PubMed

Garg, Neeraj; Li, Yi-Lin; Garcia Collazo, Ana Maria; Litten, Chris; Ryono, Denis E; Zhang, Minsheng; Caringal, Yolanda; Brigance, Robert P; Meng, Wei; Washburn, William N; Agback, Peter; Mellström, Karin; Rehnmark, Stefan; Rahimi-Ghadim, Mahmoud; Norin, Thomas; Grynfarb, Marlena; Sandberg, Johnny; Grover, Gary; Malm, Johan

2007-08-01

Based on the scaffold of the pharmacologically selective thyromimetic 2b, structurally a close analog to KB-141 (2a), a number of novel N-acylated-alpha-amino acid derivatives were synthesized and tested in a TR radioligand binding assay as well as in a reporter cell assay. On the basis of TRbeta(1)-isoform selectivity and affinity, as well as affinity to the reporter cell assay, 3d was selected for further studies in the cholesterol-fed rat model. In this model 3d revealed an improved therapeutic window between cholesterol and TSH lowering but decreased margins versus tachycardia compared with 2a.

The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase

PubMed Central

Kim, Myung Hee; Choi, Won-Chan; Kang, Hye Ok; Lee, Jong Suk; Kang, Beom Sik; Kim, Kyung-Jin; Derewenda, Zygmunt S.; Oh, Tae-Kwang; Lee, Choong Hwan; Lee, Jung-Kee

2005-01-01

In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-l-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-Å resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-Å crystal structure of its complex with l-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-β-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases. PMID:16314577

Exploring Cooperative Effects in Oxidative NHC Catalysis: Regioselective Acylation of Carbohydrates.

PubMed

Cramer, David L; Bera, Srikrishna; Studer, Armido

2016-05-23

The utility of oxidative NHC catalysis for both the regioselective and chemoselective functionalization of carbohydrates is explored. Chiral NHCs allow for the highly regioselective oxidative esterification of various carbohydrates using aldehydes as acylation precursors. The transformation was also shown to be amenable to both cis/trans diol isomers, free amino groups, and selective for specific sugar epimers in competition experiments. Efficiency and regioselectivity of the acylation can be improved upon using two different NHC catalysts that act cooperatively. The potential of the method is documented by the regioselective acylation of an amino-linked neodisaccharide. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Saturated amine oxides: Part 8. Hydroacridines: Part 27. Effects of N-oxidation and of N-quaternization on the 15N NMR chemical shifts of N-methylpiperidine-derived mono-, bi-, and tricycloaliphatic tertiary amines.

PubMed

Potmischil, Francisc; Duddeck, Helmut; Nicolescu, Alina; Deleanu, Calin

2007-03-01

The (15)N chemical shifts of 13 N-methylpiperidine-derived mono-, bi- and tricycloaliphatic tertiary amines, their methiodides and their N-epimeric pairs of N-oxides were measured, and the contributions of specific structural parameters to the chemical shifts were determined by multilinear regression analysis. Within the examined compounds, the effects of N-oxidation upon the (15)N chemical shifts of the amines vary from +56 ppm to +90 ppm (deshielding), of which approx. +67.7 ppm is due to the inductive effect of the incoming N(+)--O(-) oxygen atom, whereas the rest is due to the additive shift effects of the various C-alkyl substituents of the piperidine ring. The effects of quaternization vary from -3.1 ppm to +29.3 ppm, of which approx. +8.9 ppm is due to the inductive effect of the incoming N(+)--CH(3) methyl group, and the rest is due to the additive shift effects of the various C-alkyl substituents of the piperidine ring. The shift effects of the C-alkyl substituents in the amines, the N-oxides and the methiodides are discussed. Copyright (c) 2007 John Wiley & Sons, Ltd.

Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

PubMed Central

Winter, E; Brummel, M; Schuch, R; Spener, F

1997-01-01

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

PubMed

Winter, E; Brummel, M; Schuch, R; Spener, F

1997-01-15

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.

Acute aerobic exercise differentially alters acylated ghrelin and perceived fullness in normal-weight and obese individuals.

PubMed

Heden, Timothy D; Liu, Ying; Park, Youngmin; Dellsperger, Kevin C; Kanaley, Jill A

2013-09-01

Adiposity alters acylated ghrelin concentrations, but it is unknown whether adiposity alters the effect of exercise and feeding on acylated ghrelin responses. Therefore, the purpose of this study was to determine whether adiposity [normal-weight (NW) vs. obese (Ob)] influences the effect of exercise and feeding on acylated ghrelin, hunger, and fullness. Fourteen NW and 14 Ob individuals completed two trials in a randomized counterbalanced fashion, including a prior exercise trial (EX) and a no exercise trial (NoEX). During the EX trial, the participants performed 1 h of treadmill walking (55-60% peak O2 uptake) during the evening, 12 h before a 4-h standardized mixed meal test. Frequent blood samples were taken and analyzed for acylated ghrelin, and a visual analog scale was used to assess perceived hunger and fullness. In NW individuals, EX, compared with NoEX, reduced fasting acylated ghrelin concentrations by 18% (P = 0.03), and, in response to feeding, the change in acylated ghrelin (P = 0.02) was attenuated by 39%, but perceived hunger and fullness were unaltered. In Ob individuals, despite no changes in fasting or postprandial acylated ghrelin concentrations with EX, postprandial fullness was attenuated by 46% compared with NoEX (P = 0.05). In summary, exercise performed the night before a meal suppresses acylated ghrelin concentrations in NW individuals without altering perceived hunger or fullness. In Ob individuals, despite no changes in acylated ghrelin concentrations, EX reduced the fullness response to the test meal. Acylated ghrelin and perceived fullness responses are differently altered by acute aerobic exercise in NW and Ob individuals.

Interaction of cholesterol with sphingomyelins and acyl-chain-matched phosphatidylcholines: a comparative study of the effect of the chain length.

PubMed Central

Ramstedt, B; Slotte, J P

1999-01-01

In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells. PMID:9929492

Quantum chemical study of penicillin: Reactions after acylation

NASA Astrophysics Data System (ADS)

Li, Rui; Feng, Dacheng; Zhu, Feng

The density functional theory methods were used on the model molecules of penicillin to determine the possible reactions after their acylation on ?-lactamase, and the results were compared with sulbactam we have studied. The results show that, the acylated-enzyme tetrahedral intermediate can evolves with opening of ?-lactam ring as well as the thiazole ring; the thiazole ring-open products may be formed via ?-lactam ring-open product or from tetrahedral intermediate directly. Those products, in imine or enamine form, can tautomerize via hydrogen migration. In virtue of the water-assisted, their energy barriers are obviously reduced.

Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease.

PubMed

Rockwell, N C; Fuller, R S

2001-10-19

Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.

Monitoring the refinement of crystal structures with {sup 15}N solid-state NMR shift tensor data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalakewich, Keyton; Eloranta, Harriet; Harper, James K.

The {sup 15}N chemical shift tensor is shown to be extremely sensitive to lattice structure and a powerful metric for monitoring density functional theory refinements of crystal structures. These refinements include lattice effects and are applied here to five crystal structures. All structures improve based on a better agreement between experimental and calculated {sup 15}N tensors, with an average improvement of 47.0 ppm. Structural improvement is further indicated by a decrease in forces on the atoms by 2–3 orders of magnitude and a greater similarity in atom positions to neutron diffraction structures. These refinements change bond lengths by more thanmore » the diffraction errors including adjustments to X–Y and X–H bonds (X, Y = C, N, and O) of 0.028 ± 0.002 Å and 0.144 ± 0.036 Å, respectively. The acquisition of {sup 15}N tensors at natural abundance is challenging and this limitation is overcome by improved {sup 1}H decoupling in the FIREMAT method. This decoupling dramatically narrows linewidths, improves signal-to-noise by up to 317%, and significantly improves the accuracy of measured tensors. A total of 39 tensors are measured with shifts distributed over a range of more than 400 ppm. Overall, experimental {sup 15}N tensors are at least 5 times more sensitive to crystal structure than {sup 13}C tensors due to nitrogen’s greater polarizability and larger range of chemical shifts.« less

Metallophosphite-Catalyzed Asymmetric Acylation of α,β -Unsaturated Amides

PubMed Central

Nahm, Mary R.; Potnick, Justin R.; White, Peter S.; Johnson, Jeffrey S.

2007-01-01

The l-menthone-derived TADDOL phosphite 6b catalyzes highly enantioselective conjugate additions of acyl silanes to α,β-unsaturated amides. p-Methoxybenzoyl cyclohexyldimethylsilane adds to a variety of N,N-dimethyl acrylamide derivatives in the presence of the lithium salt of 6b. In many instances the α-silyl-γ-ketoamide product undergoes facile enantioenrichment (to 97–99% ee) upon recrystallization. Desilylation with HF·pyr affords the formal Stetter addition products. Baeyer–Villiger oxidation of the desilylated γ-ketoamides affords useful ester products. An X-ray diffraction study of 6b reveals that the isopropyl group of the menthone ketal influences the position of the syn-pseudoaxial phenyl group in the TADDOL structure. Through a crossover experiment, the silicon migration step in the reaction mechanism is shown to be strictly intramolecular. PMID:16492064

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van De Loo, F.J.; Broun, P.; Turner, S.

1995-07-18

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 andmore » with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.« less

Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

PubMed Central

Hao, Guixia; Burr, Thomas J.

2006-01-01

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N-acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI, ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI. The addition of synthetic AHLs (C16:1 and 3-O-C16:1) complemented grape necrosis in the avsR, avsI, ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR, two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI::lacZ fusion construct. PMID:16513747

Modeling 15N NMR chemical shift changes in protein backbone with pressure

NASA Astrophysics Data System (ADS)

La Penna, Giovanni; Mori, Yoshiharu; Kitahara, Ryo; Akasaka, Kazuyuki; Okamoto, Yuko

2016-08-01

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

PubMed

Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

2007-10-01

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

Toward Green Acylation of (Hetero)arenes: Palladium-Catalyzed Carbonylation of Olefins to Ketones

PubMed Central

2017-01-01

Green Friedel–Crafts acylation reactions belong to the most desired transformations in organic chemistry. The resulting ketones constitute important intermediates, building blocks, and functional molecules in organic synthesis as well as for the chemical industry. Over the past 60 years, advances in this topic have focused on how to make this reaction more economically and environmentally friendly by using green acylating conditions, such as stoichiometric acylations and catalytic homogeneous and heterogeneous acylations. However, currently well-established methodologies for their synthesis either produce significant amounts of waste or proceed under harsh conditions, limiting applications. Here, we present a new protocol for the straightforward and selective introduction of acyl groups into (hetero)arenes without directing groups by using available olefins with inexpensive CO. In the presence of commercial palladium catalysts, inter- and intramolecular carbonylative C–H functionalizations take place with good regio- and chemoselectivity. Compared to classical Friedel–Crafts chemistry, this novel methodology proceeds under mild reaction conditions. The general applicability of this methodology is demonstrated by the direct carbonylation of industrial feedstocks (ethylene and diisobutene) as well as of natural products (eugenol and safrole). Furthermore, synthetic applications to drug molecules are showcased. PMID:29392174

Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

PubMed

Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

2016-02-26

We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate1[OPEN

PubMed Central

Free, Heather C.A.; Smith, Bronwen G.

2018-01-01

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. PMID:29724771

Correlation of proton irradiation induced threshold voltage shifts to deep level traps in AlGaN/GaN heterostructures

NASA Astrophysics Data System (ADS)

Zhang, Z.; Cardwell, D.; Sasikumar, A.; Kyle, E. C. H.; Chen, J.; Zhang, E. X.; Fleetwood, D. M.; Schrimpf, R. D.; Speck, J. S.; Arehart, A. R.; Ringel, S. A.

2016-04-01

The impact of proton irradiation on the threshold voltage (VT) of AlGaN/GaN heterostructures is systematically investigated to enhance the understanding of a primary component of the degradation of irradiated high electron mobility transistors. The value of VT was found to increase monotonically as a function of 1.8 MeV proton fluence in a sub-linear manner reaching 0.63 V at a fluence of 1 × 1014 cm-2. Silvaco Atlas simulations of VT shifts caused by GaN buffer traps using experimentally measured introduction rates, and energy levels closely match the experimental results. Different buffer designs lead to different VT dependences on proton irradiation, confirming that deep, acceptor-like defects in the GaN buffer are primarily responsible for the observed VT shifts. The proton irradiation induced VT shifts are found to depend on the barrier thickness in a linear fashion; thus, scaling the barrier thickness could be an effective way to reduce such degradation.

Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

PubMed

Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

2008-01-01

The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

PubMed Central

Ruscetti, Francis W.; Jacobson, Lewis A.

1972-01-01

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as “complexed” rather than “free” particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of 3H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down. PMID:4591472

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

Protein Structural Information Derived from NMR Chemical Shift with the Neural Network Program TALOS-N

PubMed Central

Shen, Yang; Bax, Ad

2015-01-01

Summary Chemical shifts are obtained at the first stage of any protein structural study by NMR spectroscopy. Chemical shifts are known to be impacted by a wide range of structural factors and the artificial neural network based TALOS-N program has been trained to extract backbone and sidechain torsion angles from 1H, 15N and 13C shifts. The program is quite robust, and typically yields backbone torsion angles for more than 90% of the residues, and sidechain χ1 rotamer information for about half of these, in addition to reliably predicting secondary structure. The use of TALOS-N is illustrated for the protein DinI, and torsion angles obtained by TALOS-N analysis from the measured chemical shifts of its backbone and 13Cβ nuclei are compared to those seen in a prior, experimentally determined structure. The program is also particularly useful for generating torsion angle restraints, which then can be used during standard NMR protein structure calculations. PMID:25502373

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

1998-01-06

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Investigation of acyl migration in mono- and dicaffeoylquinic acids under aqueous basic, aqueous acidic, and dry roasting conditions.

PubMed

Deshpande, Sagar; Jaiswal, Rakesh; Matei, Marius Febi; Kuhnert, Nikolai

2014-09-17

Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties from one quinic acid alcohol group to another, thus interconverting chlorogenic acid regioisomers. It therefore constitutes a special case of transesterification reaction. Acyl migration constitutes an important reaction pathway in both coffee roasting and brewing, altering the structure of chlorogenic acid initially present in the green coffee bean. In this contribution we describe detailed and comprehensive mechanistic studies comparing inter- and intramolecular acyl migration involving the seven most common chlorogenic acids in coffee. We employe aqueous acidic and basic conditions mimicking the brewing of coffee along with dry roasting conditions. We show that under aqueous basic conditions intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl migration. 3-Caffeoylquinic acid was shown to be most labile to basic hydrolysis. We additionally show that the acyl migration process is strongly pH dependent with increased transesterification taking place at basic pH. Under dry roasting conditions acyl migration competes with dehydration to form lactones. We argue that acyl migration precedes lactonization, with 3-caffeoylquinic acid lactone being the predominant product.

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

PubMed

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

PubMed

Veliz-Vallejos, Debora F; van Noorden, Giel E; Yuan, Mengqi; Mathesius, Ulrike

2014-01-01

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

PubMed Central

Akasaka, K.; Li, H.; Yamada, H.; Li, R.; Thoresen, T.; Woodward, C. K.

1999-01-01

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place. PMID:10548039

Raman shifts in electron-irradiated monolayer MoS 2

DOE PAGES

Parkin, William M.; Balan, Adrian; Liang, Liangbo; ...

2016-03-21

Here, we report how the presence of electron-beam-induced sulfur vacancies affects first-order Raman modes and correlate the effects with the evolution of the in situ transmission-electron microscopy (TEM) two-terminal conductivity of monolayer MoS 2 under electron irradiation. We observe a redshift in the E Raman peak and a less pronounced blueshift in the A' 1 peak with increasing electron dose. Using energy-dispersive X-ray spectroscopy (EDS), we show that irradiation causes partial removal of sulfur and correlate the dependence of the Raman peak shifts with S vacancy density (a few %), which is confirmed by first-principles density functional theory calculations. Inmore » situ device current measurements show exponential decrease in channel current upon irradiation. Our analysis demonstrates that the observed frequency shifts are intrinsic properties of the defective systems and that Raman spectroscopy can be used as a quantitative diagnostic tool to characterize MoS 2-based transport channels.« less

Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11.

PubMed

Lagrange, Brice; Benaoudia, Sacha; Wallet, Pierre; Magnotti, Flora; Provost, Angelina; Michal, Fanny; Martin, Amandine; Di Lorenzo, Flaviana; Py, Bénédicte F; Molinaro, Antonio; Henry, Thomas

2018-01-16

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

Preliminary results for a measurement of the n=2 Lamb shift in atomic hydrogen

NASA Astrophysics Data System (ADS)

Bezginov, N.; Valdez, T.; Vutha, A. C.; Kato, K.; Skinner, T. D. G.; Hessels, E. A.

2017-04-01

We perform a measurement of the Lamb shift in atomic hydrogen (n = 2 S1/2 F = 0 to P1/2 F = 1). A beam of protons moving at 0.01 c undergoes charge exchange with hydrogen gas to produce atomic hydrogen in the metastable 2S state. The atoms travel through two microwave regions where we utilize the novel technique of frequency offset separated oscillatory fields (FOSOF). The surviving 2S population is observed using a Lyman-alpha detector. The outcome of this experiment will lead to a measurement of the proton radius, contributing to the resolution of the proton radius puzzle. We present preliminary experimental results, along with systematic studies. This research is funded by NSERC, CRC, CFI and NIST.

Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step.

PubMed

Chen, Chun-Chi; Chen, Teh-Liang; Tsai, Shau-Wei

2006-06-05

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators. Copyright 2006 Wiley Periodicals, Inc.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

1998-01-06

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

Physical characterisation of high amylose maize starch and acylated high amylose maize starches.

PubMed

Lim, Ya-Mei; Hoobin, Pamela; Ying, DanYang; Burgar, Iko; Gooley, Paul R; Augustin, Mary Ann

2015-03-06

The particle size, water sorption properties and molecular mobility of high amylose maize starch (HAMS) and high amylose maize starch acylated with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) were investigated. Acylation increased the mean particle size (D(4,3)) and lowered the specific gravity (G) of the starch granules with an inverse relationship between the length of the fatty acid chain and particle size. Acylation of HAMS with fatty acids lowered the monolayer moisture content with the trend being HAMSBacylated starches and that drying and storage of the starch granules further reduced T2 long. Analysis of the Free Induction Decay (FID) focussing on the short components of T2 (correlated to the solid matrix), indicated that drying and subsequent storage resulted in alterations of starch at 0.33a(w) and that these changes were reduced with acylation. In vitro enzymatic digestibility of heated starch dispersions by bacterial α-amylase was increased by acylation (HAMS

Highly efficient and regioselective acylation of pharmacologically interesting cordycepin catalyzed by lipase in the eco-friendly solvent 2-methyltetrahydrofuran.

PubMed

Chen, Zhi-Gang; Zhang, Dan-Ni; Cao, Lin; Han, Yong-Bin

2013-04-01

A total of nine lipases and three proteases were tested for enzymatic regioselective acylation(s) of cordycepin with vinyl acetate in organic media. The highest conversion with better initial reaction rate was achieved with immobilized Candida antarctica lipase B (Novozym 435). An eco-friendly solvent 2-methyltetrahydrofuran (MeTHF) was thought to be the most suitable reaction medium. Novozym 435 was found to be a useful biocatalyst for the 25-g scale syntheses of cordycepin acetate (96.2% isolated yield), and the biocatalyst displayed excellent regioselectivity and high operational stability during the transformation. The 5'-substituted cordycepin derivative was the sole detectable product from each acylation reaction. Novozym 435 could be recycled for the synthesis of cordycepin derivative on a 25-g scale and 63% of its original activity was maintained after being reused for 7 batches. MeTHF could be considered as an eco-friendly solvent for the large scale use in biotransformation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Lanthanum Tricyanide-Catalyzed Acyl Silane-Ketone Benzoin Additions and Kinetic Resolution of Resultant α-Silyloxyketones

PubMed Central

Tarr, James C.

2010-01-01

We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic α-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant α-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction. PMID:20392127

Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions and kinetic resolution of resultant alpha-silyloxyketones.

PubMed

Tarr, James C; Johnson, Jeffrey S

2010-05-21

We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic alpha-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant alpha-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction.

Modeling {sup 15}N NMR chemical shift changes in protein backbone with pressure

DOE Office of Scientific and Technical Information (OSTI.GOV)

La Penna, Giovanni, E-mail: glapenna@iccom.cnr.it; Mori, Yoshiharu, E-mail: ymori@ims.ac.jp; Kitahara, Ryo, E-mail: ryo@ph.ritsumei.ac.jp

2016-08-28

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change inmore » the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.« less

Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions.

PubMed

Tarr, James C; Johnson, Jeffrey S

2009-09-03

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones.

Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

PubMed

Wang, Nan; Rudolf, Jeffrey D; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2018-06-04

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

Shift scheduling model considering workload and worker’s preference for security department

NASA Astrophysics Data System (ADS)

Herawati, A.; Yuniartha, D. R.; Purnama, I. L. I.; Dewi, LT

2018-04-01

Security department operates for 24 hours and applies shift scheduling to organize its workers as well as in hotel industry. This research has been conducted to develop shift scheduling model considering the workers physical workload using rating of perceived exertion (RPE) Borg’s Scale and workers’ preference to accommodate schedule flexibility. The mathematic model is developed in integer linear programming and results optimal solution for simple problem. Resulting shift schedule of the developed model has equally distribution shift allocation among workers to balance the physical workload and give flexibility for workers in working hours arrangement.

An increase level of acylation stimulating protein is correlated with metabolic risk markers in North Indian obese women.

PubMed

Mishra, Supriya; Gupta, Vani; Mishra, Sameeksha; Gupta, Vandana; Mahdi, Abbas Ali; Sachan, Rekha

2017-12-01

The present study was to investigate the association between serum acylation stimulating protein (ASP) level with metabolic risk factors in North Indian obese women. This is a case control study, total n=322 women aged between 20 and 45 years (n=162 with metabolic syndrome & n=160 without metabolic syndrome) were recruited for the study according to National Cholesterol Education Program Treatment Panel (NCEPATP) guidelines. Serum ASP level were determined by enzyme linked immunosorbent assay. Results indicated that circulating ASP and other metabolic risk factors (waist circumference, triglycerides, fasting plasma glucose etc) were significantly higher in women with metabolic syndrome (WmetS) than in women without syndrome (WometS) (p<0.001). Furthermore circulating ASP was significantly higher possitively correlated with waist circumference (r=0.51, p<0.001), triglyceride (r=0.56, p<0.001), glucose (r=0.70, p<0.001), and negatively correlated with high density lipoprotein(r=-0.56, p<0.001) in women with metabolic syndrome. Conclusively circulating ASP was found to be significantly associated with hyperlipidemia, obesity and obesity related disorders in North Indian obese women. Copyright © 2017 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.

PubMed

Vergnolle, Olivia; Xu, Hua; Blanchard, John S

2013-09-27

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Triazine-Substituted and Acyl Hydrazones: Experiment and Computation Reveal a Stability Inversion at Low pH.

PubMed

Ji, Kun; Lee, Changsuk; Janesko, Benjamin G; Simanek, Eric E

2015-08-03

Condensation of a hydrazine-substituted s-triazine with an aldehyde or ketone yields an equivalent to the widely used, acid-labile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveals that triazine-substituted hydrazones are more labile than acetyl hydrazones at pH>5. The reactivity trends mirror that of the corresponding acetyl hydrazones, with hydrolysis rates increasing along the series (aromatic aldehydeN1 is the critical factor in affecting the reaction rate.

Development of an activity-based probe for acyl-protein thioesterases

PubMed Central

Garland, Megan; Schulze, Christopher J.; Foe, Ian T.; van der Linden, Wouter A.; Child, Matthew A.

2018-01-01

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe. PMID:29364904

Increased hepatic beta-oxidation of docosahexaenoic acid, elongation of eicosapentaenoic acid, and acylation of lysophosphatidate in rats fed a docosahexaenoic acid-enriched diet.

PubMed

Kanazawa, A; Shirota, Y; Fujimoto, K

1997-07-01

Rats were fed a diet supplemented with corn oil (n-3 deficient), soy oil, or a mixture containing 8% 22:6n-3 ethyl ester for 6 wk. The hepatic capacities for the beta-oxidation and synthesis of 22:6n-3, in addition to the acylation of lysophosphatidate, were tested in vitro. In rats that were fed a 22:6n-3-enriched diet, both the beta-oxidation of 22:6n-3 and elongation of 20:5n-3 were enhanced compared to those in rats fed the other diets. Acylation of lysophosphatidate was also enhanced in rats fed a 22:6n-3-enriched diet, while the rate of dephosphorylation of phosphatidate was not changed. The amount of 22:6n-3 in the liver was much less than that consumed in a docosahexaenoic acid-enriched diet. These results suggest that a significant amount of dietary 22:6n-3 was degraded via beta-oxidation, and that a portion of the retroconverted 20:5n-3 was recycled for the synthesis of 22:6n-3. The recycling of 20:5n-3 might contribute to the low level of 22:6n-3 in rats fed an n-3-deficient diet.

Engineering acyl carrier protein to enhance production of shortened fatty acids.

PubMed

Liu, Xueliang; Hicks, Wade M; Silver, Pamela A; Way, Jeffrey C

2016-01-01

The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris. We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

Lanthanum Tricyanide-Catalyzed Acyl Silane-Ketone Benzoin Additions

PubMed Central

Tarr, James C.; Johnson, Jeffrey S.

2009-01-01

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones. PMID:19655731

Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS: "missing" resonances at the dimer interface.

PubMed

Buchko, Garry W; Edwards, Thomas E; Hewitt, Stephen N; Phan, Isabelle Q H; Van Voorhis, Wesley C; Miller, Samuel I; Myler, Peter J

2015-10-01

Using a deuterated sample, all the observable backbone (1)H(N), (15)N, (13)C(a), and (13)C' chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are "missing" in the (1)H-(15)N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (μs-ms) and/or is heterogeneous.

Correlation of proton irradiation induced threshold voltage shifts to deep level traps in AlGaN/GaN heterostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Cardwell, D.; Sasikumar, A.

2016-04-28

The impact of proton irradiation on the threshold voltage (V{sub T}) of AlGaN/GaN heterostructures is systematically investigated to enhance the understanding of a primary component of the degradation of irradiated high electron mobility transistors. The value of V{sub T} was found to increase monotonically as a function of 1.8 MeV proton fluence in a sub-linear manner reaching 0.63 V at a fluence of 1 × 10{sup 14} cm{sup −2}. Silvaco Atlas simulations of V{sub T} shifts caused by GaN buffer traps using experimentally measured introduction rates, and energy levels closely match the experimental results. Different buffer designs lead to different V{sub T} dependences on protonmore » irradiation, confirming that deep, acceptor-like defects in the GaN buffer are primarily responsible for the observed V{sub T} shifts. The proton irradiation induced V{sub T} shifts are found to depend on the barrier thickness in a linear fashion; thus, scaling the barrier thickness could be an effective way to reduce such degradation.« less

Electron spin resonance shifts in S=1 antiferromagnetic chains

NASA Astrophysics Data System (ADS)

Furuya, Shunsuke C.; Maeda, Yoshitaka; Oshikawa, Masaki

2013-03-01

We discuss electron spin resonance (ESR) shifts in spin-1 Heisenberg antiferromagnetic chains with a weak single-ion anisotropy, based on several effective field theories: the O(3) nonlinear sigma model (NLSM) in the Haldane phase, free-fermion theories around the lower and the upper critical fields. In the O(3) NLSM, the single-ion anisotropy corresponds to a composite operator which creates two magnons at the same time and position. Therefore, even inside a parameter range where free magnon approximation is valid for thermodynamics, we have to take interactions among magnons into account in order to include the single-ion anisotropy as a perturbation. Although the O(3) NLSM is only valid in the Haldane phase, an appropriate translation of Faddeev-Zamolodchikov operators of the O(3) NLSM to fermion operators enables one to treat ESR shifts near the lower critical field in a similar manner to discussions in the Haldane phase. Our theory gives quantitative agreements with a numerical evaluation using quantum Monte Carlo simulation, and also with recent ESR experimental results on a spin-1 chain compound Ni(C5H14N2)2N3(PF6).

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Intracerebroventricular urocortin 3 counteracts central acyl ghrelin-induced hyperphagic and gastroprokinetic effects via CRF receptor 2 in rats

PubMed Central

Yeh, Chun; Ting, Ching-Heng; Doong, Ming-Luen; Chi, Chin-Wen; Lee, Shou-Dong; Chen, Chih-Yen

2016-01-01

Purpose Urocortin 3 is a key neuromodulator in the regulation of stress, anxiety, food intake, gut motility, and energy homeostasis, while ghrelin elicits feeding behavior and enhances gastric emptying, adiposity, and positive energy balance. However, the interplays between urocortin 3 and ghrelin on food intake and gastric emptying remain uninvestigated. Methods We examined the differential effects of central O-n-octanoylated ghrelin, des-Gln14-ghrelin, and urocortin 3 on food intake, as well as on charcoal nonnutrient semiliquid gastric emptying in conscious rats that were chronically implanted with intracerebroventricular (ICV) catheters. The functional importance of corticotropin-releasing factor (CRF) receptor 2 in urocortin 3-induced responses was examined by ICV injection of the selective CRF receptor 2 antagonist, astressin2-B. Results ICV infusion of urocortin 3 opposed central acyl ghrelin-elicited hyperphagia via CRF receptor 2 in satiated rats. ICV injection of O-n-octanoylated ghrelin and des-Gln14-ghrelin were equally potent in accelerating gastric emptying in fasted rats, whereas ICV administration of urocortin 3 delayed gastric emptying. In addition, ICV infusion of urocortin 3 counteracted central acyl ghrelin-induced gastroprokinetic effects via CRF receptor 2 pathway. Conclusion ICV-infused urocortin 3 counteracts central acyl ghrelin-induced hyperphagic and gastroprokinetic effects via CRF receptor 2 in rats. Our results clearly showed that enhancing ghrelin and blocking CRF receptor 2 signaling in the brain accelerated gastric emptying, which provided important clues for a new therapeutic avenue in ameliorating anorexia and gastric ileus found in various chronic wasting disorders. PMID:27757017

Acute effect of exercise intensity and duration on acylated ghrelin and hunger in men.

PubMed

Broom, David R; Miyashita, Masashi; Wasse, Lucy K; Pulsford, Richard; King, James A; Thackray, Alice E; Stensel, David J

2017-03-01

Acute exercise transiently suppresses the orexigenic gut hormone acylated ghrelin, but the extent to which exercise intensity and duration determine this response is not fully understood. The effects of manipulating exercise intensity and duration on acylated ghrelin concentrations and hunger were examined in two experiments. In experiment one, nine healthy males completed three, 4-h conditions (control, moderate-intensity running (MOD) and vigorous-intensity running (VIG)), with an energy expenditure of ~2.5 MJ induced in both MOD (55-min running at 52% peak oxygen uptake (V.O 2peak )) and VIG (36-min running at 75% V.O 2peak ). In experiment two, nine healthy males completed three, 9-h conditions (control, 45-min running (EX45) and 90-min running (EX90)). Exercise was performed at 70% V.O 2peak In both experiments, participants consumed standardised meals, and acylated ghrelin concentrations and hunger were quantified at predetermined intervals. In experiment one, delta acylated ghrelin concentrations were lower than control in MOD (ES = 0.44, P = 0.01) and VIG (ES = 0.98, P < 0.001); VIG was lower than MOD (ES = 0.54, P = 0.003). Hunger ratings were similar across the conditions (P = 0.35). In experiment two, delta acylated ghrelin concentrations were lower than control in EX45 (ES = 0.77, P < 0.001) and EX90 (ES = 0.68, P < 0.001); EX45 and EX90 were similar (ES = 0.09, P = 0.55). Hunger ratings were lower than control in EX45 (ES = 0.20, P = 0.01) and EX90 (ES = 0.27, P = 0.001); EX45 and EX90 were similar (ES = 0.07, P = 0.34). Hunger and delta acylated ghrelin concentrations remained suppressed at 1.5 h in EX90 but not EX45. In conclusion, exercise intensity, and to a lesser extent duration, are determinants of the acylated ghrelin response to acute exercise. © 2017 Society for Endocrinology.

Chemical shifts of diamagnetic azafullerenes: (C 59N) 2 and C 59HN

NASA Astrophysics Data System (ADS)

Bühl, Michael; Curioni, Alessandro; Andreoni, Wanda

1997-08-01

13C and 15N chemical shifts have been calculated for the azafullerenes (C 59N) 2 and C 59HN using the GIAO (gauge including atomic orbitals)-SCF method based on the geometry obtained with the density functional theory BLYP scheme Our results are in good agreement with experimental data, in particular, for the "anomalous" shift of the saturated carbon. Combined with previous calculations of the structural stability and electronic as well as vibrational properties, the present findings confirm the calculated structures for both molecules and establish the [6,6]-closed configuration for the dimer.

40 CFR 721.10175 - 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18 and C18-unsatd...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1-Propanaminium, N-(3-aminopropyl)-2... 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18 and C18-unsatd. acyl... chemical substance identified as 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12...

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

PubMed Central

Gupta, Chhitar M.; Costello, Catherine E.; Khorana, H. Gobind

1979-01-01

Sonicated vesicles of 1-fatty acyl-2-ω-(2-diazo-3,3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphoryl-cholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C—H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms. Images PMID:16592675

Tropical Colonial Ports: Shifting concepts, 1500s-1800s

PubMed

Reid, Brian

2016-12-01

Based on a study of the history of exploration and settlement in North Australia in the 18th and 19th centuries, I became particularly interested in the concept of a 'good port' in the tropics and how in time this concept shifted. The threat of fevers played a significant part in these shifts. In this overview, I examine how similar shifts in the concept of a good port occurred in the maritime silk and spice routes of South and Shout East Asia.

Agent-based model of diffusion of N-acyl homoserine lactones in a multicellular environment of Pseudomonas aeruginosa and Candida albicans.

PubMed

Pérez-Rodríguez, Gael; Dias, Sónia; Pérez-Pérez, Martín; Fdez-Riverola, Florentino; Azevedo, Nuno F; Lourenço, Anália

2018-03-08

Experimental incapacity to track microbe-microbe interactions in structures like biofilms, and the complexity inherent to the mathematical modelling of those interactions, raises the need for feasible, alternative modelling approaches. This work proposes an agent-based representation of the diffusion of N-acyl homoserine lactones (AHL) in a multicellular environment formed by Pseudomonas aeruginosa and Candida albicans. Depending on the spatial location, C. albicans cells were variably exposed to AHLs, an observation that might help explain why phenotypic switching of individual cells in biofilms occurred at different time points. The simulation and algebraic results were similar for simpler scenarios, although some statistical differences could be observed (p < 0.05). The model was also successfully applied to a more complex scenario representing a small multicellular environment containing C. albicans and P. aeruginosa cells encased in a 3-D matrix. Further development of this model may help create a predictive tool to depict biofilm heterogeneity at the single-cell level.

Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension

PubMed Central

1994-01-01

In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side- chain of tunicamycin, in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons), including potent inhibitors of glycosylation, are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for

Conformational transition of membrane-associated terminally-acylated HIV-1 Nef

PubMed Central

Akgun, Bulent; Satija, Sushil; Nanda, Hirsh; Pirrone, Gregory F.; Shi, Xiaomeng; Engen, John R.; Kent, Michael S.

2013-01-01

Many proteins are post-translationally modified by acylation targetting them to lipid membranes. While methods such as X-ray crystallography and NMR are available to determine the structure of folded proteins in solution, the precise position of folded domains relative to a membrane remains largely unknown. We used neutron and X-ray reflection methods to measure the displacement of the core domain of HIV Nef from lipid membranes upon insertion of the N-terminal myristate group. Nef is one of several HIV-1 accessory proteins and an essential factor in AIDS progression. Upon insertion of the myristate and residues from the N-terminal arm, Nef transitions from a closed to open conformation that positions the core domain 70 Å from the lipid headgroups. This work rules out speculation that the Nef core remains closely associated with the membrane to optimize interactions with the cytoplasmic domain of MHC-1. PMID:24035710

Among Metabolic Factors, Significance of Fasting and Postprandial Increases in Acyl and Desacyl Ghrelin and the Acyl/Desacyl Ratio in Obstructive Sleep Apnea before and after Treatment

PubMed Central

Chihara, Yuichi; Akamizu, Takashi; Azuma, Masanori; Murase, Kimihiko; Harada, Yuka; Tanizawa, Kiminobu; Handa, Tomohiro; Oga, Toru; Mishima, Michiaki; Chin, Kazuo

2015-01-01

Study Objectives: There are reports suggesting that obstructive sleep apnea (OSA) may itself cause weight gain. However, recent reports showed increases in body mass index (BMI) following continuous positive airway pressure (CPAP) treatments. When considering weight changes, changes in humoral factors that have significant effects on appetite such as acyl (AG) and desacyl ghrelin (DAG), leptin, insulin, and glucose and their interactions, examples of which are AG/DAG and AG/insulin, are important. The aim of this study was to test the hypothesis that some appetite-related factors had a specific profile before and after CPAP treatment. Methods: Metabolic parameters were measured cross-sectionally while fasting and 30, 60, 90, and 120 min following breakfast in no or mild OSA (apnea-hypopnea index < 15, n = 15) and moderate-to-severe OSA (apnea-hypopnea index ≥ 15, n = 39) participants in a single institute. There were no differences in age, sex, BMI, or visceral fat accumulation between the two groups. Twenty-one patients with moderate-to-severe OSA who received CPAP treatment also prospectively underwent the same testing following 3 months of CPAP treatment. Results: Although fasting and postprandial glucose, insulin, and leptin levels did not differ between no or mild OSA and moderate-to-severe OSA participants, AG and DAG, including AG/DAG and AG/insulin, under fasting and postprandial conditions were significantly increased in the moderate-to-severe OSA patients (p < 0.01). After 3 months of CPAP treatment in 21 of the moderate-to-severe OSA participants, AG/DAG did not change significantly, but other ghrelin-related parameters including AG/insulin significantly decreased compared with values before treatment but remained higher than in no or mild OSA. Conclusions: Among several important metabolic factors, ghrelin-related factors had the strongest associations with moderate-to-severe OSA. These results indicate that continuous changes in ghrelin secretion in

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

PubMed Central

Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

2015-01-01

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

New parasite inhibitors encompassing novel conformationally-locked 5'-acyl sulfamoyl adenosines.

PubMed

Dixit, Shailesh S; Upadhayaya, Ram Shankar; Chattopadhyaya, Jyoti

2012-08-14

We describe the design, synthesis and biological evaluation of conformationally-locked 5'-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3'-endo, North-type) nucleosides have been synthesized by covalently attaching a 4'-CH(2)-O-2' bridge () across C2'-C4' of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5'-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC(50) = 0.25-0.51 μM. In particular, the potent 5'-pentanyl acyl sulfamoyl adenosine derivative 17 (IC(50) = 0.25 μM) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC(50) >4 μM) and in diploid human fibroblasts MRC-5 cell lines (CC(50) 4 μM). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5' can modulate the toxicity of 5'-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

The Brainstem Switch for Gaze Shifts in Humans

DTIC Science & Technology

2001-10-25

Page 1 of 4 THE BRAINSTEM SWITCH FOR GAZE SHIFTS IN HUMANS A. N. Kumar1, R. J. Leigh1,2, S. Ramat3 Department of 1Biomedical Engineering, Case...omnipause neurons during gaze shifts. Using the scleral search coil technique, eye movements were measured in seven normal subjects, as they made...voluntary, disjunctive gaze shifts comprising saccades and vergence movements. Conjugate oscillations of small amplitude and high frequency were identified

Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates.

PubMed

Hengge, Alvan C; Stein, Ross L

2004-01-27

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.

A study of the red-shift of a neutral donor bound exciton in GaN nanorods by hydrogenation

NASA Astrophysics Data System (ADS)

Park, Byung-Guon; Lee, Sang-Tae; Reddeppa, Maddaka; Kim, Moon-Deock; Oh, Jae-Eung; Lee, Sang-Kwon

2017-09-01

In this paper we account for the physics behind the exciton peak shift in GaN nanorods (NRs) due to hydrogenation. GaN NRs were selectively grown on a patterned Ti/Si(111) substrate using plasma-assisted molecular beam epitaxy, and the effect of hydrogenation on their optical properties was investigated in detail using low-temperature photoluminescence measurements. Due to hydrogenation, the emissions corresponding to the donor-acceptor pair and yellow luminescence in GaN NRs were strongly suppressed, while the emission corresponding to the neutral to donor bound exciton (D0X) exhibited red-shift. Thermal annealing of hydrogenated GaN NRs demonstrated the recovery of the D0X and deep level emission. To determine the nature of the D0X peak shift due to hydrogenation, comparative studies were carried out on various diameters of GaN NRs, which can be controlled by different growth conditions and wet-etching times. Our experimental results reveal that the D0X shift depends on the diameter of the GaN NRs after hydrogenation. The results clearly demonstrate that the hydrogenation leads to band bending of GaN NRs as compensated by hydrogen ions, which causes a red-shift in the D0X emission.

A study of the red-shift of a neutral donor bound exciton in GaN nanorods by hydrogenation.

PubMed

Park, Byung-Guon; Lee, Sang-Tae; Reddeppa, Maddaka; Kim, Moon-Deock; Oh, Jae-Eung; Lee, Sang-Kwon

2017-09-08

In this paper we account for the physics behind the exciton peak shift in GaN nanorods (NRs) due to hydrogenation. GaN NRs were selectively grown on a patterned Ti/Si(111) substrate using plasma-assisted molecular beam epitaxy, and the effect of hydrogenation on their optical properties was investigated in detail using low-temperature photoluminescence measurements. Due to hydrogenation, the emissions corresponding to the donor-acceptor pair and yellow luminescence in GaN NRs were strongly suppressed, while the emission corresponding to the neutral to donor bound exciton (D 0 X) exhibited red-shift. Thermal annealing of hydrogenated GaN NRs demonstrated the recovery of the D 0 X and deep level emission. To determine the nature of the D 0 X peak shift due to hydrogenation, comparative studies were carried out on various diameters of GaN NRs, which can be controlled by different growth conditions and wet-etching times. Our experimental results reveal that the D 0 X shift depends on the diameter of the GaN NRs after hydrogenation. The results clearly demonstrate that the hydrogenation leads to band bending of GaN NRs as compensated by hydrogen ions, which causes a red-shift in the D 0 X emission.

Density functional theory studies on the nano-scaled composites consisted of graphene and acyl hydrazone molecules

NASA Astrophysics Data System (ADS)

Ren, J. L.; Zhou, L.; Lv, Z. C.; Ding, C. H.; Wu, Y. H.; Bai, H. C.

2016-07-01

Graphene, which is the first obtained single atomic layer 2D materials, has drawn a great of concern in nano biotechnology due to the unique property. On one hand, acyl hydrazone compounds belonging to the Schif bases have aroused considerable attention in medicine, pharmacy, and analytical reagent. However, few understanding about the interaction between graphene and acyl hydrazone molecules is now available. And such investigations are much crucial for the applications of these new nano-scaled composites. The current work revealed theoretical investigations on the nano-scaled composites built by acyl hydrazone molecules loaded on the surface of graphene. The relative energy, electronic property and the interaction between the counterparts of graphene/acyl hydrazone composites are investigated based on the density functional theory calculations. According to the obtained adsorption energy, the formation of the nano-scaled composite from the isolated graphene and acyl hydrazone molecule is exothermic, and thus it is energetically favorable to form these nano composites in viewpoint of total energy change. The frontier molecular orbital for the nano composite is mainly distributed at the graphene part, leading to that the energy levels of the frontier molecular orbital of the nano composites are very close to that of isolated graphene. Moreover, the counterpart interaction for the graphene/acyl hydrazone composites is also explored based on the discussions of orbital hybridization, charge redistribution and Van der Waals interaction.

Acyl carrier protein structural classification and normal mode analysis

PubMed Central

Cantu, David C; Forrester, Michael J; Charov, Katherine; Reilly, Peter J

2012-01-01

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well. PMID:22374859

Dihydropyranone Formation by Ipso C–H Activation in a Glucal 3-Carbamate-Derived Rhodium Acyl Nitrenoid

PubMed Central

Hurlocker, Brisa; Abascal, Nadia C.; Repka, Lindsay M.; Santizo-Deleon, Elsy; Smenton, Abigail L.; Baranov, Victoria; Gupta, Ritu; Bernard, Sarah E.; Chowdhury, Shenjuti; Rojas, Christian M.

2011-01-01

By using (N-tosyloxy)-3-O-carbamoyl-D-glucal 10, which removes the need for a hypervalent iodine(III) oxidant, we provide evidence for rhodium nitrenoid-mediated ipso C–H activation as the origin of a C3-oxidized dihydropyranone product 3. This system may be especially susceptible to such a pathway due to the ease of forming a cation upon hydride transfer to the rhodium-complexed acyl nitrene. PMID:21381715

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling | Center for Cancer Research

Cancer.gov

The cover image illuminates the non-enzymatic “ghost writers” of lysine acylation. Meier et al. detail the development of a chemoproteomic strategy that harnesses thioester reactivity to discover candidate cellular targets of non-enzymatic acylation. Application of this approach reveals that glycolytic enzymes can be strongly inhibited by reactive thioesters, including the

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko

2014-03-07

Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipidmore » methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.« less

Further acylated flavonol bisdesmosides from Sinocrassula indica.

PubMed

Xie, Hai-Hui; Yoshikawa, Masayuki

2013-01-01

Further investigation on the whole herbs of Sinocrassula indica (Crassulaceae) led to the isolation of four new acylated flavonol bisdesmosides, sinocrassosides A₁₃, B₆, B₇, and D₄, together with kaempferol 3-O-β-D-(6-O-acetyl)glucopyranosyl-7-O-α-L-rhamnopyranoside. Their structures were established by spectral and chemical methods.

Enzymatic acylation of flavonoid glycosides by a carbohydrate esterase of family 16.

PubMed

Biely, Peter; Cziszárová, Mária; Wong, Ken K Y; Fernyhough, Alan

2014-11-01

The acetyl esterase of Trichoderma reesei belonging to carbohydrate esterase (CE) family 16 catalyzes transacylations to carbohydrate moieties of flavonoid glycosides, esculin and rutin. The enzyme recognizes as acyl donors vinyl esters of short carboxylic acids. Esculin was acylated at position 3 of the glucosyl residue in aqueous solutions saturated with vinyl acetate and vinyl propionate. The yields of esculin monoacetate and monopropionate of esculin in aqueous medium (esculin 40 mM, enzyme 40 µg/ml, 40 °C, 3 days) were 67 and 55 %, respectively. Replacement of water by 2-propanol was required for a similar acylation of rutin at 4 mM concentration. The yields of rutin monoacetate and propionate were 60 and 30 %, respectively. The results indicate that the enzyme could be used for an easy modification of solubility and hydrophobicity of glycosylated compounds, including drugs and functional food additives.

Anti-cancer agents based on N-acyl-2, 3-dihydro-1H-pyrrolo[2,3-b] quinoline derivatives and a method of making

DOEpatents

Gakh, Andrei; Krasavin, Mikhail; Karapetian, Ruben; Rufanov, Konstantin A; Konstantinov, Igor; Godovykh, Elena; Soldatkina, Olga; Sosnov, Andrey V

2013-04-16

The present disclosure relates to novel compounds that can be used as anti-cancer agents in the prostate cancer therapy. In particular, the invention relates to N-acyl derivatives of 2,3-dihydro-1H-pyrrolo[2,3-b]quinolines having the structural Formula (I), ##STR00001## stereoisomers, tautomers, racemics, prodrugs, metabolites thereof, or pharmaceutically acceptable salt and/or solvate thereof. The meaning of R1 is independently selected from H; C1-C6 Alkyl, cyclo-Alkyl or iso-Alkyl substituents; R2 is selected from C1-C6 Alkyl, cyclo-Alkyl or iso-Alkyl; substituted or non-substituted, fused or non-fused to substituted or non-substituted aromatic ring, aryl or heteroaryl groups. The invention also relates to methods for preparing said compounds, and to pharmaceutical compositions comprising said compounds.

Serum Levels of Acyl-Carnitines along the Continuum from Normal to Alzheimer's Dementia.

PubMed

Cristofano, Adriana; Sapere, Nadia; La Marca, Giancarlo; Angiolillo, Antonella; Vitale, Michela; Corbi, Graziamaria; Scapagnini, Giovanni; Intrieri, Mariano; Russo, Claudio; Corso, Gaetano; Di Costanzo, Alfonso

2016-01-01

This study aimed to determine the serum levels of free L-carnitine, acetyl-L-carnitine and 34 acyl-L-carnitine in healthy subjects and in patients with or at risk of Alzheimer's disease. Twenty-nine patients with probable Alzheimer's disease, 18 with mild cognitive impairment of the amnestic type, 24 with subjective memory complaint and 46 healthy subjects were enrolled in the study, and the levels of carnitine and acyl-carnitines were measured by tandem mass spectrometry. The concentrations of acetyl-L-carnitine progressively decreased passing from healthy subjects group (mean±SD, 5.6±1.3 μmol/L) to subjective memory complaint (4.3±0.9 μmol/L), mild cognitive impairment (4.0±0.53 μmol/L), up to Alzheimer's disease (3.5±0.6 μmol/L) group (p<0.001). The differences were significant for the comparisons: healthy subjects vs. subjective memory complaint, mild cognitive impairment or Alzheimer's disease group; and subjective memory complaint vs. Alzheimer's disease group. Other acyl-carnitines, such as malonyl-, 3-hydroxyisovaleryl-, hexenoyl-, decanoyl-, dodecanoyl-, dodecenoyl-, myristoyl-, tetradecenoyl-, hexadecenoyl-, stearoyl-, oleyl- and linoleyl-L-carnitine, showed a similar decreasing trend, passing from healthy subjects to patients at risk of or with Alzheimer's disease. These results suggest that serum acetyl-L-carnitine and other acyl-L-carnitine levels decrease along the continuum from healthy subjects to subjective memory complaint and mild cognitive impairment subjects, up to patients with Alzheimer's disease, and that the metabolism of some acyl-carnitines is finely connected among them. These findings also suggest that the serum levels of acetyl-L-carnitine and other acyl-L-carnitines could help to identify the patients before the phenotype conversion to Alzheimer's disease and the patients who would benefit from the treatment with acetyl-L-carnitine. However, further validation on a larger number of samples in a longitudinal study is needed

Orientation-dependent surface core-level shifts and chemical shifts on clean and H 2S-covered GaAs

NASA Astrophysics Data System (ADS)

Ranke, W.; Finster, J.; Kuhr, H. J.

1987-08-01

Photoelectron spectra of the As 3d and Ga 3d core levels were studied in situ on a cylindrically shaped GaAs single crystal for the six inequivalent orientations (001), (113), (111), (110), (11¯1) and (11¯3). On the clean surface, prepared by molecular beam epitaxy (MBE), surface core levels are shifted by 0.25 to 0.55 eV towards smaller binding energy (BE) for As 3d and -0.25 to -0.35 eV towards higher BE for Ga, depending on orientation. Additional As causes As 3d contributions shifted between -0.45 and -0.7 eV towards higher BE. The position and intensity of them is influenced by H 2S adsorption. At 150 K, H 2S adsorbs preferentially on As sites. As chemical shifts appear at -0.6 to -0.9 eV towards higher BE. Simultaneously, As accumulation occurs on all orientations with the exception of (110). High temperature adsorption (550 K, 720 K) influences mainly the Ga 3d peaks. Two peaks shifted by about -0.45 and -0.8 eV towards higher Be were found which are attributed to Ga atoms with one or two sulfur ligands, respectively. At 720 K, also As depletion is observed. The compatibility of surface core-level positions and intensities with recent structural models for the (111) and (11¯1) surfaces is discussed.

Synthesis of 2-acyl-1,4-diketones via the diacylation of {alpha},{beta}-unsaturated ketones

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, N.S.; Yu, S.; Kabalka, G.W.

1998-08-17

The first example of a diacylation of the carbon-carbon double bond in {alpha},{beta}-unsaturated ketones is described. The reaction of acylcyanocuprate reagents with {alpha},{beta}-unsaturated ketones, followed by C-acylation, produces 2-acyl-1,4-diketones in good yields (50--89%). The 1,4-addition of organocuprate reagents to conjugated enones, followed by trapping of the enolate intermediates with various electrophiles, is one of the most useful synthetic reactions. However, to the best of the authors` knowledge, 1,4-acylation followed by trapping of the enolate intermediates with acid chloride has not been reported.

Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group.

PubMed

Schikora, Adam; Schenk, Sebastian T; Hartmann, Anton

2016-04-01

Bacterial quorum sensing (QS) mechanisms play a crucial role in the proper performance and ecological fitness of bacterial populations. Many key physiological processes are regulated in a QS-dependent manner by auto-inducers, like the N-acyl homoserine lactones (AHLs) in numerous Gram-negative bacteria. In addition, also the interaction between bacteria and eukaryotic hosts can be regulated by AHLs. Those mechanisms gained much attention, because of the positive effects of different AHL molecules on plants. This positive impact ranges from growth promotion to induced resistance and is quite contrasting to the rather negative effects observed in the interactions between bacterial AHL molecules and animals. Only very recently, we began to understand the molecular mechanisms underpinning plant responses to AHL molecules. In this review, we gathered the latest information in this research field. The first part gives an overview of the bacterial aspects of quorum sensing. Later we focus on the impact of AHLs on plant growth and AHL-priming, as one of the most understood phenomena in respect to the inter-kingdom interactions based on AHL-quorum sensing molecules. Finally, we discuss the potential benefits of the understanding of bacteria-plant interaction for the future agricultural applications.

Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants.

PubMed

Götz, Christine; Fekete, Agnes; Gebefuegi, Istvan; Forczek, Sándor T; Fuksová, Kvetoslava; Li, Xiaojing; Englmann, Matthias; Gryndler, Milan; Hartmann, Anton; Matucha, Miroslav; Schmitt-Kopplin, Philippe; Schröder, Peter

2007-11-01

Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.

Doping dependent blue shift and linewidth broadening of intersubband absorption in non-polar m-plane AlGaN/GaN multiple quantum wells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kotani, Teruhisa, E-mail: tkotani@iis.u-tokyo.ac.jp; Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505; Advanced Technology Research Laboratories, Sharp Corporation, 2613-1 Ichinomoto-cho, Tenri, Nara 632-8567

2015-09-14

Blue shift and broadening of the absorption spectra of mid-infrared intersubband transition in non-polar m-plane AlGaN/GaN 10 quantum wells were observed with increasing doping density. As the doping density was increased from 6.6 × 10{sup 11} to 6.0 × 10{sup 12 }cm{sup −2} per a quantum well, the intersubband absorption peak energy shifted from 274.0 meV to 302.9 meV, and the full width at half maximum increased from 56.4 meV to 112.4 meV. Theoretical calculations reveal that the blue shift is due to many body effects, and the intersubband linewidth in doped AlGaN/GaN QW is mainly determined by scattering due to interface roughness, LO phonons, and ionized impurities.

GluN2B in corticostriatal circuits governs choice learning and choice shifting

PubMed Central

Brigman, Jonathan L.; Daut, Rachel; Wright, Tara; Gunduz-Cinar, Ozge; Graybeal, Carolyn; Davis, Margaret I.; Jiang, Zhihong; Saksida, Lisa; Jinde, Seiichiro; Pease, Matthew; Bussey, Timothy J.; Lovinger, David M.; Nakazawa, Kazu; Holmes, Andrew

2013-01-01

A choice that reliably produces a preferred outcome can be automated to liberate cognitive resources for other tasks. Should an outcome become less desirable, behavior must adapt in parallel or become perseverative. Corticostriatal systems are known to mediate choice learning and flexibility, but the molecular mechanisms subserving the instantiation of these processes are not well understood. We integrated mouse behavioral, immunocytochemical, in vivo electrophysiological, genetic, and pharmacological approaches to study choice. We found that the dorsal striatum (DS) was increasingly activated with choice learning, whereas reversal of learned choice engaged prefrontal regions. In vivo, DS neurons showed activity associated with reward anticipation and receipt that emerged with learning and relearning. Corticostriatal or striatal GluN2B gene deletion, or DS-restricted GluN2B antagonism, impaired choice learning, whereas cortical GluN2B deletion or OFC GluN2B antagonism impaired shifting. Our convergent data demonstrate how corticostriatal GluN2B circuits govern the ability to learn and shift choice behavior. PMID:23831965

Evaluation of anti-quorum-sensing activity of edible plants and fruits through inhibition of the N-acyl-homoserine lactone system in Chromobacterium violaceum and Pseudomonas aeruginosa.

PubMed

Musthafa, K Syed; Ravi, A Veera; Annapoorani, A; Packiavathy, I Sybiya Vasantha; Pandian, S Karutha

2010-01-01

To find out an alternative strategy to antibiotic usage against bacterial infection. The purpose of this study is to describe the quorum-sensing (QS) inhibitory activity of edible plants and fruits against N-acyl-homoserine lactone (AHL)-mediated violacein production in Chromobacterium violaceum and virulence factor expression in Pseudomonas aeruginosa PAO1. Aqueous extracts of Ananas comosus (Bromeliaceae), Musa paradiciaca (Musaceae), Manilkara zapota (Sapotaceae) and Ocimum sanctum (Lamiaceae) were prepared and anti-QS activity of each extract was tested against AHL-mediated phenotypic expressions of C. violaceum and PAO1. Most of these extracts showed significant reduction in AHL-mediated violacein production in C. violaceum as well as pyocyanin pigment, staphylolytic protease, elastase production and biofilm formation in PAO1. However, these extracts were not inhibitory to bacterial growth, revealing that the QS inhibition by the extracts is not related to static or killing effects on the bacteria. The present study identified the anti-QS activity of A. comosus, M. paradiciaca, M. zapota and O. sanctum. An AHL-inactivating compound from these plant sources can be used as an alternative to antibiotic compounds to prevent AHL-mediated bacterial infection in higher organisms. Copyright © 2010 S. Karger AG, Basel.

Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance

2011-07-01

Bacterial acyl carrier protein synthase plays an essential role in the synthesis of fatty acids, nonribosomal peptides and polyketides. In Mycobacterium tuberculosis, AcpS or group I phosphopentatheine transferase exhibits two different structural conformations depending upon the pH. The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS–ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix andmore » in the conformation of the α3–α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4–6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS–ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS–ADP adopt different conformations depending upon the pH conditions of the crystallization solution.« less

Mycobacterial glycolipids di-O-acylated trehalose and tri-O-acylated trehalose downregulate inducible nitric oxide synthase and nitric oxide production in macrophages.

PubMed

Espinosa-Cueto, Patricia; Escalera-Zamudio, Marina; Magallanes-Puebla, Alejandro; López-Marín, Luz María; Segura-Salinas, Erika; Mancilla, Raúl

2015-06-23

Tuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines. In this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ). These findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.

Benfotiamine, a synthetic S-acyl thiamine derivative, has different mechanisms of action and a different pharmacological profile than lipid-soluble thiamine disulfide derivatives

PubMed Central

Volvert, Marie-Laure; Seyen, Sandrine; Piette, Marie; Evrard, Brigitte; Gangolf, Marjorie; Plumier, Jean-Christophe; Bettendorff, Lucien

2008-01-01

Background Lipid-soluble thiamine precursors have a much higher bioavailability than genuine thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylthiamine O-monophosphate), an amphiphilic S-acyl thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of thiamine diphosphate and so enhancing transketolase activity. As the brain is particularly sensitive to thiamine deficiency, we wanted to test whether intracellular thiamine and thiamine phosphate levels are increased in the brain after oral benfotiamine administration. Results Benfotiamine that is practically insoluble in water, organic solvents or oil was solubilized in 200 mM hydroxypropyl-β-cyclodextrin and the mice received a single oral administration of 100 mg/kg. Though thiamine levels rapidly increased in blood and liver to reach a maximum after one or two hours, no significant increase was observed in the brain. When mice received a daily oral administration of benfotiamine for 14 days, thiamine derivatives were increased significantly in the liver but not in the brain, compared to control mice. In addition, incubation of cultured neuroblastoma cells with 10 μM benfotiamine did not lead to increased intracellular thiamine levels. Moreover, in thiamine-depleted neuroblastoma cells, intracellular thiamine contents increased more rapidly after addition of thiamine to the culture medium than after addition of benfotiamine for which a lag period was observed. Conclusion Our results show that, though benfotiamine strongly increases thiamine levels in blood and liver, it has no significant effect in the brain. This would explain why beneficial effects of benfotiamine have only been observed in peripheral tissues, while sulbutiamine, a lipid-soluble thiamine disulfide derivative, that increases thiamine derivatives in the brain as well as in cultured cells, acts as a central nervous system drug. We propose that

Benfotiamine, a synthetic S-acyl thiamine derivative, has different mechanisms of action and a different pharmacological profile than lipid-soluble thiamine disulfide derivatives.

PubMed

Volvert, Marie-Laure; Seyen, Sandrine; Piette, Marie; Evrard, Brigitte; Gangolf, Marjorie; Plumier, Jean-Christophe; Bettendorff, Lucien

2008-06-12

Lipid-soluble thiamine precursors have a much higher bioavailability than genuine thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylthiamine O-monophosphate), an amphiphilic S-acyl thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of thiamine diphosphate and so enhancing transketolase activity. As the brain is particularly sensitive to thiamine deficiency, we wanted to test whether intracellular thiamine and thiamine phosphate levels are increased in the brain after oral benfotiamine administration. Benfotiamine that is practically insoluble in water, organic solvents or oil was solubilized in 200 mM hydroxypropyl-beta-cyclodextrin and the mice received a single oral administration of 100 mg/kg. Though thiamine levels rapidly increased in blood and liver to reach a maximum after one or two hours, no significant increase was observed in the brain. When mice received a daily oral administration of benfotiamine for 14 days, thiamine derivatives were increased significantly in the liver but not in the brain, compared to control mice. In addition, incubation of cultured neuroblastoma cells with 10 muM benfotiamine did not lead to increased intracellular thiamine levels. Moreover, in thiamine-depleted neuroblastoma cells, intracellular thiamine contents increased more rapidly after addition of thiamine to the culture medium than after addition of benfotiamine for which a lag period was observed. Our results show that, though benfotiamine strongly increases thiamine levels in blood and liver, it has no significant effect in the brain. This would explain why beneficial effects of benfotiamine have only been observed in peripheral tissues, while sulbutiamine, a lipid-soluble thiamine disulfide derivative, that increases thiamine derivatives in the brain as well as in cultured cells, acts as a central nervous system drug. We propose that benfotiamine only penetrates the cells

Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions

USDA-ARS?s Scientific Manuscript database

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles inacyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for se...

N-acyl Taurines and Acylcarnitines Cause an Imbalance in Insulin Synthesis and Secretion Provoking β Cell Dysfunction in Type 2 Diabetes.

PubMed

Aichler, Michaela; Borgmann, Daniela; Krumsiek, Jan; Buck, Achim; MacDonald, Patrick E; Fox, Jocelyn E Manning; Lyon, James; Light, Peter E; Keipert, Susanne; Jastroch, Martin; Feuchtinger, Annette; Mueller, Nikola S; Sun, Na; Palmer, Andrew; Alexandrov, Theodore; Hrabe de Angelis, Martin; Neschen, Susanne; Tschöp, Matthias H; Walch, Axel

2017-06-06

The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives.

PubMed

Zhou, Xiaohua; Tai, Akihiro; Yamamoto, Itaru

2003-03-01

It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.

Standardized pivot shift test improves measurement accuracy.

PubMed

Hoshino, Yuichi; Araujo, Paulo; Ahlden, Mattias; Moore, Charity G; Kuroda, Ryosuke; Zaffagnini, Stefano; Karlsson, Jon; Fu, Freddie H; Musahl, Volker

2012-04-01

The variability of the pivot shift test techniques greatly interferes with achieving a quantitative and generally comparable measurement. The purpose of this study was to compare the variation of the quantitative pivot shift measurements with different surgeons' preferred techniques to a standardized technique. The hypothesis was that standardizing the pivot shift test would improve consistency in the quantitative evaluation when compared with surgeon-specific techniques. A whole lower body cadaveric specimen was prepared to have a low-grade pivot shift on one side and high-grade pivot shift on the other side. Twelve expert surgeons performed the pivot shift test using (1) their preferred technique and (2) a standardized technique. Electromagnetic tracking was utilized to measure anterior tibial translation and acceleration of the reduction during the pivot shift test. The variation of the measurement was compared between the surgeons' preferred technique and the standardized technique. The anterior tibial translation during pivot shift test was similar between using surgeons' preferred technique (left 24.0 ± 4.3 mm; right 15.5 ± 3.8 mm) and using standardized technique (left 25.1 ± 3.2 mm; right 15.6 ± 4.0 mm; n.s.). However, the variation in acceleration was significantly smaller with the standardized technique (left 3.0 ± 1.3 mm/s(2); right 2.5 ± 0.7 mm/s(2)) compared with the surgeons' preferred technique (left 4.3 ± 3.3 mm/s(2); right 3.4 ± 2.3 mm/s(2); both P < 0.01). Standardizing the pivot shift test maneuver provides a more consistent quantitative evaluation and may be helpful in designing future multicenter clinical outcome trials. Diagnostic study, Level I.

Night shift work and incident diabetes among U.S. black women

PubMed Central

Vimalananda, Varsha G.; Palmer, Julie R.; Gerlovin, Hanna; Wise, Lauren A.; Rosenzweig, James L.; Rosenberg, Lynn; Narváez, Edward A. Ruiz

2015-01-01

Aims To assess shift work in relation to incident type 2 diabetes among African American women. Methods In the Black Women's Health Study (BWHS), an ongoing prospective cohort study, we followed 28,041 participants for incident diabetes during 2005-2013. They answered questions in 2005 about having worked the night shift. We estimated hazard ratios (HR) and 95% confidence intervals (CI) for incident diabetes using Cox proportional hazards models. The basic multivariable model included age, time period, family history of diabetes, education, and neighborhood SES. In further models, we controlled for lifestyle factors and body mass index (BMI). Results Over the 8 years of follow-up, there were 1,786 incident diabetes cases. Relative to never having worked the night shift, HRs (95% CI) of diabetes were 1.17 (1.04, 1.31) for 1-2 years of night shift work, 1.23 (1.06, 1.41) for 3-9 years, and 1.42 (1.19, 1.70) for ≥ 10 years (P-trend < 0.0001). The monotonic positive association between night shift work and type 2 diabetes remained after multivariable adjustment (P-trend = 0.02). The association did not vary by obesity status, but was stronger in women aged < 50 years. Conclusions Long durations of shift work were associated with an increased risk of type 2 diabetes. The association was only partially explained by lifestyle factors and BMI. A better understanding of the mechanisms by which shift work may affect risk of diabetes is needed in view of the high prevalence of shift work among U.S. workers. PMID:25586362

Low acylation stimulating protein levels are associated with cardiometabolic disorders–secondary to autoimmune activation?

PubMed Central

Onat, Altan; Altay, Servet; Yüksel, Murat; Karadeniz, Yusuf; Can, Günay; Yüksel, Hüsniye; Ademoğlu, Evin

2017-01-01

Objective: We investigated the possible association of serum acylation stimulating protein (ASP) with cardiometabolic disorders and the evidence of autoimmune activation. Methods: Population-based randomly selected 1024 participants were cross-sectionally and prospectively analyzed. ASP concentrations were measured with a validated ELISA kit. Correlations were sought separately in subjects with no cardiometabolic disorders (n=427) designated as “healthy.” Results: ASP was positively correlated with total testosterone and inversely correlated with platelet activating factor (PAF), PAF-acetylhydrolase (AH), in each gender, and positively correlated in “healthy” men with lipoprotein [Lp](a) and apolipoprotein B. Correlations of ASP with PAF values ≥22 nmol/L were abolished, contrasted to a strongly inverse one in subjects with PAF <22 nmol/L. In linear regression analyses in the whole sample, ASP was inversely associated independently with PAF and PAF-AH and, in men, positively with Lp(a) and sex hormone-binding globulin. Prevalent and (at 2.0 years’ follow-up) incident metabolic syndrome (MetS, n=393), diabetes (n=154), and coronary heart disease (CHD, n=171) were analyzed by sex-, age-, and Lp(a)-adjusted logistic regression, using tertiles of ASP and PAF. The lower two (<42 nmol/L) ASP tertiles were a risk factor in combined sexes for MetS and diabetes. In women, incident CHD was predicted by either reduced or elevated ASP tertiles. Conclusion: Findings can be explained by the notion of operation of immune responses against both ASP and oxidized PAF-like lipids of Lp(a) to yield for “reduced” values and increased likelihood of cardiometabolic disorders. PMID:27599666

Preparation and enhanced daylight-induced photocatalytic activity of C,N,S-tridoped titanium dioxide powders.

PubMed

Zhou, Minghua; Yu, Jiaguo

2008-04-15

A simple method for preparing highly daylight-induced photoactive nanocrystalline C,N,S-tridoped TiO2 powders was developed by a solid-phase reaction. The as-prepared TiO2 powders were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV-vis diffuse reflectance spectra, N2 adsorption-desorption measurements and transmission electron microscopy (TEM). The photocatalytic activity was evaluated by the photocatalytic oxidation of formaldehyde under daylight irradiation in air. The results show that daylight-induced photocatalytic activities of the as-prepared TiO2 powders were improved by C,N,S-tridoping. The C,N,S-tridoped TiO2 powders exhibited stronger absorption in the near UV and visible-light region with red shift in the band-gap transition. When the molar ratio of CS(NH2)2 to xerogel TiO2 powders (prepared by hydrolysis of Ti(OC4H9)4 in distilled water) (R) was kept in 3, the daylight-induced photocatalytic activities of the as-prepared C,N,S-tridoped TiO2 powders were about more than six times greater than that of Degussa P25 and un-doped TiO2 powders. The high activities of the C,N,S-tridoped TiO2 can be attributed to the results of the synergetic effects of strong absorption in the near UV and visible-light region, red shift in adsorption edge and two phase structures of un-doped TiO2 and C,N,S-tridoped TiO2.

Long-range interactions of hydrogen atoms in excited states. III. n S -1 S interactions for n ≥3

NASA Astrophysics Data System (ADS)

Adhikari, C. M.; Debierre, V.; Jentschura, U. D.

2017-09-01

The long-range interaction of excited neutral atoms has a number of interesting and surprising properties such as the prevalence of long-range oscillatory tails and the emergence of numerically large van der Waals C6 coefficients. Furthermore, the energetically quasidegenerate n P states require special attention and lead to mathematical subtleties. Here we analyze the interaction of excited hydrogen atoms in n S states (3 ≤n ≤12 ) with ground-state hydrogen atoms and find that the C6 coefficients roughly grow with the fourth power of the principal quantum number and can reach values in excess of 240 000 (in atomic units) for states with n =12 . The nonretarded van der Waals result is relevant to the distance range R ≪a0/α , where a0 is the Bohr radius and α is the fine-structure constant. The Casimir-Polder range encompasses the interatomic distance range a0/α ≪R ≪ℏ c /L , where L is the Lamb shift energy. In this range, the contribution of quasidegenerate excited n P states remains nonretarded and competes with the 1 /R2 and 1 /R4 tails of the pole terms, which are generated by lower-lying m P states with 2 ≤m ≤n -1 , due to virtual resonant emission. The dominant pole terms are also analyzed in the Lamb shift range R ≫ℏ c /L . The familiar 1 /R7 asymptotics from the usual Casimir-Polder theory is found to be completely irrelevant for the analysis of excited-state interactions. The calculations are carried out to high precision using computer algebra in order to handle a large number of terms in intermediate steps of the calculation for highly excited states.

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

PubMed

Paturiaux-Hanocq, F; Hanocq-Quertier, J; de Almeida, M L; Nolan, D P; Pays, A; Vanhamme, L; Van den Abbeele, J; Wasunna, C L; Carrington, M; Pays, E

2000-04-21

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.

Measurement of the Lamb shift in the hydrogen atom (n = 2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokolov, Y.L.; Yakovlev, V.P.

1982-07-01

A principle is proposed for the observation of the stationary interference pattern of two phase-shifted components of the 2p (or 2s) state of the hydrogen atom (Yu. L. Sokolov, Sov. Phys. JETP 36, 243 (1973)); (Proc. 6-th Internat. Conf. on Atomic Phys., Riga, 1978, p. 207). An atomic interferometer, a device analogous in principle to a two-channel optical (such as Michelson's) interferometer, is used to measure the frequency of the (2s/sub 1/2/, F = 0)--(2p/sub 1/2/, F = 0) transition in the hydrogen atom, which is found to equal 909.9014 +- 0.0019 MHz. The corresponding Lamb shift is delta(H, nmore » = 2) = 1057.8594 +- 0.0019 MHz.« less

A new acylated isoflavone glucoside from Pterocarpus santalinus.

PubMed

Krishnaveni, K S; Srinivasa Rao, J V

2000-09-01

Phytochemical investigation on the constituents of heartwood of Pterocarpus santalinus resulted in the isolation of a new acylated isoflavone glucoside. The structure of the new compound was elucidated on the basis of spectral studies as 4',5-dihydroxy-7-O-methyl isoflavone 3'-O-D-(3''-E-cinnamoyl)glucoside.

Distinct zooplankton regime shift patterns across ecoregions of the U.S. Northeast continental shelf Large Marine Ecosystem

NASA Astrophysics Data System (ADS)

Morse, R. E.; Friedland, K. D.; Tommasi, D.; Stock, C.; Nye, J.

2017-01-01

We investigated regime shifts in seasonal zooplankton communities of the Northeast continental shelf Large Marine Ecosystem (NES) and its subcomponent ecoregions over a multi-decadal period (1977-2013). Our cross ecoregion analysis shows that regime shifts in different ecoregions often exhibited very distinct characteristics, emphasizing more granular fluctuations in NES plankton communities relative to previous work. Shifts early in the time series generally reflected an increase in abundance levels. The response of zooplankton abundance within fall communities was more similar among ecoregions than for spring communities. The Gulf of Maine exhibited highly distinct patterns from other ecoregions, with regime shifts identified in the early 1980s, early 2000s, and mid-2000s for spring communities. Regime shifts were identified in the early to mid-1990s for the NES, Georges Bank, and the Mid-Atlantic Bight ecoregions, while the fall communities experienced shifts in the early 1990s and late 1980s for the NES and Georges Bank, but in the late 1990s in the Mid-Atlantic Bight. A constrained correspondence analysis of zooplankton community against local and basin-scale climatological indices suggests that water temperature, stratification, and the Atlantic multidecadal oscillation (AMO) were the predominant factors in driving the zooplankton community composition.

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains

PubMed Central

Kassan, Adam; Herms, Albert; Fernández-Vidal, Andrea; Bosch, Marta; Schieber, Nicole L.; Reddy, Babu J.N.; Fajardo, Alba; Gelabert-Baldrich, Mariona; Tebar, Francesc; Enrich, Carlos; Gross, Steven P.

2013-01-01

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of “emerging” LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage. PMID:24368806

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

PubMed Central

Casabon, Israël; Swain, Kendra; Crowe, Adam M.

2014-01-01

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids.

PubMed

Huo, Tianyao; Ferruzzi, Mario G; Schwartz, Steven J; Failla, Mark L

2007-10-31

A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P < 0.05) by addition of TG (2.5% v/w) to the meal and was dependent on fatty acyl chain length in structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These

Genetics Home Reference: short-chain acyl-CoA dehydrogenase deficiency

MedlinePlus

... Orphanet: Short chain acyl-CoA dehydrogenase deficiency Screening, Technology and Research in Genetics Patient Support and Advocacy Resources (5 links) Children Living with Inherited Metabolic Disease (CLIMB) Children's Mitochondrial ...

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

PubMed

Mostyn, Shannon N; Carland, Jane E; Shimmon, Susan; Ryan, Renae M; Rawling, Tristan; Vandenberg, Robert J

2017-09-20

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC 50 of 9 μM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 μM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

N-Allyl-N-Sulfonyl Ynamides as Synthetic Precursors to Amidines and Vinylogous Amidines. An Unexpected N-to-C 1,3-Sulfonyl Shift in Nitrile Synthesis

PubMed Central

DeKorver, Kyle A.; Johnson, Whitney L.; Zhang, Yu; Hsung, Richard P.; Dai, Huifang; Deng, Jun; Lohse, Andrew G.; Zhang, Yan-Shi

2011-01-01

A detailed study of amidine synthesis from N-allyl-N-sulfonyl ynamides is described here. Mechanistically, this is a fascinating reaction consisting of diverging pathways that could lead to deallylation or allyl transfer depending upon the oxidation state of palladium catalysts, the nucleophilicity of amines, and the nature of the ligands. It essentially constitutes a Pd(0)-catalyzed aza-Claisen rearrangement of N-allyl ynamides, which can also be accomplished thermally. An observation of N-to-C 1,3-sulfonyl shift was made when examining these aza-Claisen rearrangements thermally. This represents a useful approach to nitrile synthesis. While attempts to render this 1,3-sulfonyl shift stereoselective failed, we uncovered another set of tandem sigmatropic rearrangements, leading to vinyl imidate formation. Collectively, this work showcases the rich array of chemistry one can discover using these ynamides. PMID:21563776

C-H carbonylation: In situ acyl triflates ace it

NASA Astrophysics Data System (ADS)

Lee, Yong Ho; Morandi, Bill

2018-02-01

A simple palladium catalyst has mediated the facile formation of aroyl triflates -- an extremely reactive class of electrophiles. These intermediates, generated in situ, enable the Friedel-Crafts acylation of traditionally unreactive arenes, addressing a significant gap in C-H carbonylation methodology.

Identification, quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis).

PubMed

Chen, Chu; Xu, Xue-Min; Chen, Yang; Yu, Meng-Yao; Wen, Fei-Yan; Zhang, Hao

2013-12-01

A novel acylated flavonol glycoside: isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (1), together with two known acylated flavonol glycosides: quercetin (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (2) and kaempferol (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (3) were isolated from the n-butanol fraction of sea buckthorn (Hippophae rhamnoides ssp. sinensis) berries for the first time by chromatographic methods, and their structures were elucidated using UV, MS, (1)H and (13)C NMR, and 2D NMR. Compounds 1-3 showed good scavenging activities, with respective IC50 values of 8.91, 4.26 and 30.90 μM toward the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical; respective Trolox equivalent antioxidant capacities of 2.89, 4.04 and 2.44 μM μM(-1) toward 2,2'-azino-bis-3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical. The quantitative analysis of the isolated acylated flavonol glycosides was performed by HPLC-DAD method. The contents of compounds 1-3 were in the range of 12.2-31.4, 4.0-25.3, 7.5-59.7 mg/100 g dried berries and 9.1-34.5, 75.1-182.1, 29.2-113.4 mg/100 g dried leaves, respectively. Copyright © 2013. Published by Elsevier Ltd.

The orexigenic hormone acyl-ghrelin increases adult hippocampal neurogenesis and enhances pattern separation

PubMed Central

Kent, Brianne A.; Beynon, Amy L.; Hornsby, Amanda K.E.; Bekinschtein, Pedro; Bussey, Timothy J.; Davies, Jeffrey S.; Saksida, Lisa M.

2015-01-01

Summary An important link exists between intact metabolic processes and normal cognitive functioning; however, the underlying mechanisms remain unknown. There is accumulating evidence that the gut hormone ghrelin, an orexigenic peptide that is elevated during calorie restriction (CR) and known primarily for stimulating growth hormone release, has important extra-hypothalamic functions, such as enhancing synaptic plasticity and hippocampal neurogenesis. The present study was designed to evaluate the long-term effects of elevating acyl-ghrelin levels, albeit within the physiological range, on the number of new adult born neurons in the dentate gyrus (DG) and performance on the Spontaneous Location Recognition (SLR) task, previously shown to be DG-dependent and sensitive to manipulations of plasticity mechanisms and cell proliferation. The results revealed that peripheral treatment of rats with acyl-ghrelin enhanced both adult hippocampal neurogenesis and performance on SLR when measured 8–10 days after the end of acyl-ghrelin treatment. Our data show that systemic administration of physiological levels of acyl-ghrelin can produce long-lasting improvements in spatial memory that persist following the end of treatment. As ghrelin is potentially involved in regulating the relationship between metabolic and cognitive dysfunction in ageing and neurodegenerative disease, elucidating the underlying mechanisms holds promise for identifying novel therapeutic targets and modifiable lifestyle factors that may have beneficial effects on the brain. PMID:25462915

Franck Condon shift assessment in 2D MoS2

NASA Astrophysics Data System (ADS)

Gupta, Sunny; Shirodkar, Sharmila N.; Kaplan, Daniel; Swaminathan, Venkataraman; Yakobson, Boris I.

2018-03-01

Optical spectroscopy (OS) techniques are often coupled with first-principles density functional theoretical (DFT) calculations for determining the precise influence of defects on the electronic and structural properties of two-dimensional (2D) transition metal dichalcogenides. Such calculations are carried out presuming there is little or no effect of vibrational transitions on the observed electronic spectrum. However, if the effect of change in vibrational energy (Franck Condon (FC) shift) associated with such a transition is large, it could possibly lead to a different origin for the observed peak. One such instance is the attribution of the 0.75 eV cathodoluminescence peak by Fabbri et al (2016 Nat. Commun. 7 13044) to an optical transition from an S vacancy level in the band gap, under the assumption that the FC shift is negligible. Here, by first principles constrained DFT calculations using hybrid HSE06 functional we show that this combined prediction of OS and DFT calculations is valid for 2D MoS2 since the FC shift associated with electronic transitions from a sulfur vacancy is indeed small ~28 meV. Based on our calculations we conclude that it is reasonable to make a direct connection between DFT calculations and optical spectroscopy techniques in this material, hence, establishing a one to one relation between defect related emission bands and electronic transitions from the defect levels.

U.S. sulfur dioxide emission reductions: Shifting factors and a carbon dioxide penalty

DOE PAGES

Brown, Marilyn Ann; Li, Yufei; Massetti, Emanuele; ...

2017-01-18

For more than 20 years, the large-scale application of flue gas desulfurization technology has been a dominant cause of SO 2 emission reductions. From 1994–2004, electricity generation from coal increased, but the shift to low-sulfur coal eclipsed this. From 2004–2014, electricity generation from coal decreased, but a shift to higher-sulfur subbituminous and lignite coal overshadowed this. Here, the shift in coal quality has also created a CO 2 emissions penalty, representing 2% of the sector’s total emissions in 2014.

Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry.

PubMed

Sachon, Emmanuelle; Nielsen, Per Franklin; Jensen, Ole Nørregaard

2007-06-01

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.

A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

PubMed Central

2009-01-01

Background The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria. Results An aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity. Conclusion This is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum

Expression of Cyanobacterial Acyl-ACP Reductase Elevates the Triacylglycerol Level in the Red Alga Cyanidioschyzon merolae.

PubMed

Sumiya, Nobuko; Kawase, Yasuko; Hayakawa, Jumpei; Matsuda, Mami; Nakamura, Mami; Era, Atsuko; Tanaka, Kan; Kondo, Akihiko; Hasunuma, Tomohisa; Imamura, Sousuke; Miyagishima, Shin-ya

2015-10-01

Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family

PubMed Central

Gan, Han Ming; Dailey, Lucas K.; Halliday, Nigel; Williams, Paul; Hudson, André O.

2016-01-01

Background Members of the genus Novosphingobium have been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated. Methods In this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by the N. subterraneum DSM 12447 strain. Results Three predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference. Discussion This study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae. PMID:27635318

Acyl Coenzyme A Thioesterase 7 Regulates Neuronal Fatty Acid Metabolism To Prevent Neurotoxicity

PubMed Central

Ellis, Jessica M.; Wong, G. William

2013-01-01

Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7N−/−, revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7N−/− mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7N−/− mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity. PMID:23459938

Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer.

PubMed

Siddiqui, Muhammad Faisal; Sakinah, Mimi; Singh, Lakhveer; Zularisam, A W

2012-10-31

Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling. Copyright © 2012 Elsevier B.V. All rights reserved.

Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity.

PubMed

Ryabtsova, Oxana; Jansen, Koen; Van Goethem, Sebastiaan; Joossens, Jurgen; Cheng, Jonathan D; Lambeir, Anne-Marie; De Meester, Ingrid; Augustyns, Koen; Van der Veken, Pieter

2012-05-15

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery. Copyright © 2012 Elsevier Ltd. All rights reserved.

Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.

PubMed

Fehling, E; Lessire, R; Cassagne, C; Mukherjee, K D

1992-06-05

All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.

Configurational and constitutional information storage: multiple dynamics in systems based on pyridyl and acyl hydrazones.

PubMed

Chaur, Manuel N; Collado, Daniel; Lehn, Jean-Marie

2011-01-03

The C=N group of hydrazones can undergo E/Z isomerization both photochemically and thermally, allowing the generation of a closed process that can be tuned by either of these two physical stimuli. On the other hand, hydrazine-exchange reactions enable a constitutional change in a given hydrazone. The two classes of processes: 1) configurational (physically stimulated) and 2) constitutional (chemically stimulated) give access to short-term and long-term information storage, respectively. Such transformations are reported herein for two hydrazones (bis-pyridyl hydrazone and 2-pyridinecarboxaldehyde phenylhydrazone) that undergo a closed, chemically or physically driven process, and, in addition, can be locked or unlocked at will by metal-ion coordination or removal. These features also extend to acyl hydrazones derived from 2-pyridinecarboxaldehyde. Similarly to the terpydine-like hydrazones, such acyl hydrazones can undergo both constitutional and configurational changes, as well as metal-ion coordination. All these types of hydrazones represent dynamic systems capable of acting as multiple state molecular devices, in which the presence of coordination sites furthermore allows the metal ion-controlled locking and unlocking of the interconversion of the different states. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

S-Matrix to potential inversion of low-energy α-12C phase shifts

NASA Astrophysics Data System (ADS)

Cooper, S. G.; Mackintosh, R. S.

1990-10-01

The IP S-matrix to potential inversion procedure is applied to phase shifts for selected partial waves over a range of energies below the inelastic threshold for α-12C scattering. The phase shifts were determined by Plaga et al. Potentials found by Buck and Rubio to fit the low-energy alpha cluster resonances need only an increased attraction in the surface to accurately reproduce the phase-shift behaviour. Substantial differences between the potentials for odd and even partial waves are necessary. The surface tail of the potential is postulated to be a threshold effect.

Plasma fatty acyl-carnitines during 8 weeks of overfeeding: relation to diet energy expenditure and body composition: the PROOF study.

PubMed

Bray, George A; Redman, Leanne M; de Jonge, Lilian; Rood, Jennifer; Sutton, Elizabeth F; Smith, Steven R

2018-06-01

Overfeeding is a strategy for evaluating the effects of excess energy intake. In this secondary analysis we tested the possibility that different levels of dietary protein might differentially modify the response of fatty acyl-carnitines to overfeeding. Twenty-three healthy adult men and women were overfed by 40% for 8 weeks while in-patients with diets containing 5% (LPD), 15% (NPD) or 25% (HPD) protein. Plasma fatty acyl-carnitines were measured by gas chromatography/mass spectrometry (GC/MS) at baseline and after 8 weeks of overfeeding. Measurements included: body composition by DXA, energy expenditure by ventilated hood and doubly-labeled water, fat cell size from subcutaneous fat biopsies, and fat distribution by CT scan. Analysis was done on 5 groups of fatty acyl-carnitines identified by principal components analysis and 6 individual short-chain fatty acyl carnitines. Higher protein intake was associated with significantly lower 8 week levels of medium chain fatty acids and C2, C4-OH and C 6:1, but higher values of C3 and C5:1 acyl-carnitines derived from essential amino acids. In contrast energy and fat intake were only weakly related to changes in fatty acyl-carnitines. A decease or smaller rise in 8 week medium chain acyl-carnitines was associated with an increase in sleeping energy expenditure (P = 0.0004), and fat free mass (P < 0.0001) and a decrease in free fatty acid concentrations (FFA) (P = 0.0067). In contrast changes in short-chain fatty acyl-carnitines were related to changes in resting energy expenditure (P = 0.0026), and fat free mass (P = 0.0007), and C4-OH was positively related to FFA (P = 0006). Protein intake was the major factor influencing changes in fatty acyl carnitines during overfeeding with higher values of most acyl-fatty acids on the low protein diet. The association of dietary protein and fat intake may explain the changes in energy expenditure and metabolic variables resulting in the observed

Interaction between rhein acyl glucuronide and methotrexate based on human organic anion transporters.

PubMed

Yuan, Yuan; Yang, Hua; Kong, Linghua; Li, Yuan; Li, Ping; Zhang, Hongjian; Ruan, Jianqing

2017-11-01

Rhein, a major bioactive compound of many medicinal herbs and the prodrug of diacerein, is often used with low dose of methotrexate as drug combination to treat rheumatoid arthritis. In this study, potential drug-drug interaction between methotrexate and rhein was investigated based on organic anion transporters (OAT). Our study demonstrated that rhein acyl glucuronide (RAG), the major metabolite of rhein in the human blood circulation, significantly inhibited the uptake of p-aminohippurate in hOAT1 transfected cells with IC 50 value of 691 nM and estrone sulfate uptake in hOAT3 transfected cells with IC 50 value of 78.5 nM. As the substrate of both hOAT1 and hOAT3, the methotrexate transport was significantly inhibited by RAG in hOAT1 transfected cells at 50 μM and hOAT3 transfected cells at 1 μM by 69% and 87%, respectively. Further in vivo study showed that after co-administrated with RAG in rats the AUC 0-24 values of methotrexate increased from 3109 to 5370 ng/mL*hr and the t 1/2 was prolonged by 40.5% (from 7.4 to 10.4 h), demonstrating the inhibitory effect of RAG on methotrexate excretion. In conclusion, rhein acyl glucuronide could significantly decrease the transport of methotrexate by both hOAT1 and hOAT3. The combination use of rhein, diacerein or other rhein-containing herbs with methotrexate may cause obvious drug-drug interaction and require close monitoring for potential drug interaction in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

An insight on acyl migration in solvent-free ethanolysis of model triglycerides using Novozym 435.

PubMed

Sánchez, Daniel Alberto; Tonetto, Gabriela Marta; Ferreira, María Luján

2016-02-20

In this work, the ethanolysis of triglycerides catalyzed by immobilized lipase was studied, focusing on the secondary reaction of acyl migration. The catalytic tests were performed in a solvent-free reaction medium using Novozym 435 as biocatalyst. The selected experimental variables were biocatalyst loading (5-20mg), reaction time (30-90min), and chain length of the fatty acids in triglycerides with and without unsaturation (short (triacetin), medium (tricaprylin) and long (tripalmitin/triolein)). The formation of 2-monoglyceride by ethanolysis of triglycerides was favored by long reaction times and large biocatalyst loading with saturated short- to medium-chain triglycerides. In the case of long-chain triglycerides, the formation of this monoglyceride was widely limited by acyl migration. In turn, acyl migration increased the yield of ethyl esters and minimized the content of monoglycerides and diglycerides. Thus, the enzymatic synthesis of biodiesel was favored by long-chain triglycerides (which favor the acyl migration), long reaction times and large biocatalyst loading. The conversion of acylglycerides made from long-chain fatty acids with unsaturation was relatively low due to limitations in their access to the active site of the lipase. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct membrane properties are differentially influenced by cardiolipin content and acyl chain composition in biomimetic membranes.

PubMed

Pennington, Edward Ross; Fix, Amy; Sullivan, E Madison; Brown, David A; Kennedy, Anthony; Shaikh, Saame Raza

2017-02-01

Cardiolipin (CL) has a critical role in maintaining mitochondrial inner membrane structure. In several conditions such as heart failure and aging, there is loss of CL content and remodeling of CL acyl chains, which are hypothesized to impair mitochondrial inner membrane biophysical organization. Therefore, this study discriminated how CL content and acyl chain composition influenced select properties of simple and complex mitochondrial mimicking model membranes. We focused on monolayer excess area/molecule (a measure of lipid miscibility), bilayer phase transitions, and microdomain organization. In monolayer compression studies, loss of tetralinoleoyl [(18:2) 4 ] CL content decreased the excess area/molecule. Replacement of (18:2) 4 CL acyl chains with tetraoleoyl [(18:1) 4 ] CL or tetradocosahexaenoyl [(22:6) 4 ] CL generally had little influence on monolayer excess area/molecule; in contrast, replacement of (18:2) 4 CL acyl chains with tetramyristoyl [(14:0) 4 ] CL increased monolayer excess area/molecule. In bilayers, calorimetric studies showed that substitution of (18:2) 4 CL with (18:1) 4 CL or (22:6) 4 CL lowered the phase transition temperature of phosphatidylcholine vesicles whereas (14:0) 4 CL had no effect. Finally, quantitative imaging of giant unilamellar vesicles revealed differential effects of CL content and acyl chain composition on microdomain organization, visualized with the fluorescent probe Texas Red DHPE. Notably, microdomain areas were decreased by differing magnitudes upon lowering of (18:2) 4 CL content and substitution of (18:2) 4 CL with (14:0) 4 CL or (22:6) 4 CL. Conversely, exchanging (18:2) 4 CL with (18:1) 4 CL increased microdomain area. Altogether, these data demonstrate that CL content and fatty acyl composition differentially target membrane physical properties, which has implications for understanding how CL regulates mitochondrial activity and the design of CL-specific therapeutics. Copyright © 2016 Elsevier B.V. All rights

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE PAGES

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

2015-09-18

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Design, synthesis and activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-oleoylethanolamine analogues.

PubMed

Grijalvo, Santiago; Bedia, Carmen; Triola, Gemma; Casas, Josefina; Llebaria, Amadeu; Teixidó, Jordi; Rabal, Obdulia; Levade, Thierry; Delgado, Antonio; Fabriàs, Gemma

2006-10-01

The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or thioether moieties. It appears that, within the 2-oxooctanamide family, the C3-OH group of the sphingosine molecule is required for inhibition both in vitro and in cultured cells. Furthermore, although the (E)-4 double bond is not essential for inhibitory activity, the (E) configuration is required, since the analogue with a (Z)-4 unsaturation was not inhibitory. None of the oleamides inhibited the aCDase in vitro. Conversely, with the exception of N-oleoylethanolamine and its analogs with S-decyl and S-hexadecyl substituents, all the synthesized oleamides inhibited the aCDase in cultured cells, although with a relatively low potency. We conclude that novel aCDase inhibitors can evolve from N-acylation of sphingoid bases with electron deficient-acyl groups. In contrast, chemical modification of the N-oleoylsphingosine backbone does not seem to offer an appropriate strategy to obtain aCDase inhibitors.

Acylated iridoids from the roots of Valeriana officinalis var. latifolia.

PubMed

Han, Zhu-zhen; Yan, Zhao-hui; Liu, Qing-xin; Hu, Xian-qing; Ye, Ji; Li, Hui-liang; Zhang, Wei-dong

2012-10-01

Phytochemical investigation of the roots of Valeriana officinalis var. latifolia resulted in the isolation and characterization of six new acylated iridoids, (5S,7S,8S,9S)-7-hydroxy-8-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (1), (5S,7S,8S,9S)-7-hydroxy-10-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (2), (5S,8S,9S)-10-isovaleroyloxy-Δ⁴,¹¹-dihyronepetalactone (3), (5S,6S,8S,9R)-6-isovaleroyloxy-Δ⁴,¹¹-1,3-diol (4), (5S,6S,8S,9R)-1,3-isovaleroxy-Δ4,11-1,3-diol (5), and (5S,6S,8S,9R)-3-isovaleroxy-6-isovaleroyloxy-Δ⁴,¹¹-1,3-diol (6). Their structures were determined mainly by 1D and 2D NMR spectroscopic techniques. We also report herein for the first time the single crystal X-ray structure of compound 1. In addition, the cytotoxic activities of compounds 1-6 were evaluated against A549 (human lung adenocarcinoma), HCT116 (human colon carcinoma), SK-BR-3 (human breast carcinoma), and HepG2 (human hepatoma) cell lines. Compound 6 showed weak cell growth inhibition of A549, HCT116, SK-BR-3, and HepG2 cells. Georg Thieme Verlag KG Stuttgart · New York.

The Effects of Exercise on Food Intake and Hunger: Relationship with Acylated Ghrelin and Leptin

PubMed Central

Vatansever-Ozen, Serife; Tiryaki-Sonmez, Gul; Bugdayci, Guler; Ozen, Guclu

2011-01-01

This study investigated the effects of a long bout of aerobic exercise on hunger and energy intake and circulating levels of leptin and acylated ghrelin. Ten healthy male subjects undertook two, 4 h trials in a randomized crossover design. In the exercise trial subjects ran for 105 min at 50% of maximal oxygen uptake and the last 15 min at 70% of maximal oxygen uptake followed by a 120 min rest period. In the control trial, subjects rested for 4 h. Subjects consumed a buffet test meal at 180 min during each trial. Hunger ratings, acylated ghrelin, leptin, glucose and insulin concentrations were measured at 0, 1, 2, 3 and 4 h. No differences were found at baseline values for hunger, acylated ghrelin, leptin, insulin and glucose for both trials (p > 0.05). The estimated energy expenditure of the exercise trial was 1550 ± 136 kcal. Exercise did not change subsequent absolute energy intake, but produced a significant decrease (p < 0.05) in relative energy intake. A two-way ANOVA revealed a significant (p < 0. 05) interaction effect for hunger and acylated ghrelin. In conclusion, this exercise regimen had a positive effect on reducing appetite which is related to reduced acylated ghrelin responses over time. This finding lends support for a role of exercise in weight management. Key points Physical exercise is a strategy used to counteract obesity, since it lowers the energetic balance by increasing energy expenditure. However, because any energy expended in exercise elevates the intensity of hunger and drives food consumption, it is pertinent to ask how effective exercise could be in helping people to lose weight or to prevent weight gain. The effects of exercise on hunger sensations and food intake are fairly controversial and depend on the intensity and duration of exercise. 120 min prolonged treadmill exercise with mix intensity, temporarily decreased hunger sensations, acylated ghrelin and relative energy intake. Variations in exercise intensity should

Acyl ghrelin improves cognition, synaptic plasticity deficits and neuroinflammation following amyloid β (Aβ1-40) administration in mice.

PubMed

Santos, V V; Stark, R; Rial, D; Silva, H B; Bayliss, J A; Lemus, M B; Davies, J S; Cunha, R A; Prediger, R D; Andrews, Z B

2017-05-01

Ghrelin is a metabolic hormone that has neuroprotective actions in a number of neurological conditions, including Parkinson's disease (PD), stroke and traumatic brain injury. Acyl ghrelin treatment in vivo and in vitro also shows protective capacity in Alzheimer's disease (AD). In the present study, we used ghrelin knockout (KO) and their wild-type littermates to test whether or not endogenous ghrelin is protective in a mouse model of AD, in which human amyloid β peptide 1-40 (Aβ 1-40 ) was injected into the lateral ventricles i.c.v. Recognition memory, using the novel object recognition task, was significantly impaired in ghrelin KO mice and after i.c.v. Aβ 1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Spatial orientation, as assessed by the Y-maze task, was also significantly impaired in ghrelin KO mice and after i.c.v. Aβ 1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Ghrelin KO mice had deficits in olfactory discrimination; however, neither i.c.v. Aβ 1-40 treatment, nor acyl ghrelin injections affected olfactory discrimination. We used stereology to show that ghrelin KO and Aβ 1-40 increased the total number of glial fibrillary acidic protein expressing astrocytes and ionised calcium-binding adapter expressing microglial in the rostral hippocampus. Finally, Aβ 1-40 blocked long-term potentiation induced by high-frequency stimulation and this effect could be acutely blocked with co-administration of acyl ghrelin. Collectively, our studies demonstrate that ghrelin deletion affects memory performance and also that acyl ghrelin treatment may delay the onset of early events of AD. This supports the idea that acyl ghrelin treatment may be therapeutically beneficial with respect to restricting disease progression in AD. © 2017 British Society for Neuroendocrinology.

Des-acyl ghrelin inhibits the capacity of macrophages to stimulate the expression of aromatase in breast adipose stromal cells.

PubMed

Au, CheukMan C; Docanto, Maria M; Zahid, Heba; Raffaelli, Francesca-Maria; Ferrero, Richard L; Furness, John B; Brown, Kristy A

2017-06-01

Des-acyl ghrelin is the unacylated form of the well-characterized appetite-stimulating hormone ghrelin. It affects a number of physiological processes, including increasing adipose lipid accumulation and inhibiting adipose tissue inflammation. Breast adipose tissue inflammation in obesity is associated with an increase in the expression of the estrogen biosynthetic enzyme, aromatase, and is hypothesized to create a hormonal milieu conducive to tumor growth. We previously reported that des-acyl ghrelin inhibits the expression and activity of aromatase in isolated human adipose stromal cells (ASCs), the main site of aromatase expression in the adipose tissue. The current study aimed to examine the effect of des-acyl ghrelin on the capacity of mouse macrophages (RAW264.7 cells) and human adipose tissue macrophages (ATMs) to stimulate aromatase expression in primary human breast ASCs. RAW264.7 cells were treated with 0, 10 and 100pM des-acyl ghrelin following activation with phorbol 12-myristate 13-acetate, and cells and conditioned media were collected after 6 and 24h. The effect of des-acyl ghrelin on macrophage polarization was examined by assessing mRNA expression of pro-inflammatory M1-specific marker Cd11c and anti-inflammatory M2-specific marker Cd206, as well as expression of Tnf and Ptgs2, known mediators of the macrophage-dependent stimulation of aromatase. TNF protein in conditioned media was assessed by ELISA. The effect of RAW264.7 and ATM-conditioned media on aromatase expression in ASCs was assessed after 6h. Results demonstrate des-acyl ghrelin significantly increases the expression of Cd206 and suppresses the expression of Cd11c, Tnf and Ptgs2 in activated RAW264.7 cells. Treatment of RAW264.7 and ATMs with des-acyl ghrelin also significantly reduces the capacity of these cells to stimulate aromatase transcript expression in human breast ASCs. Overall, these findings suggest that in addition to direct effects on aromatase in ASCs, des-acyl ghrelin also

Role for phospholipid acyl chains and cholesterol in pulmonary infections and inflammation

PubMed Central

Shaikh, Saame Raza; Fessler, Michael B.

2016-01-01

Bacterial and viral respiratory tract infections result in millions of deaths worldwide and are currently the leading cause of death from infection. Acute inflammation is an essential element of host defense against infection, but can be damaging to the host when left unchecked. Effective host defense requires multiple lipid mediators, which collectively have proinflammatory and/or proresolving effects on the lung. During pulmonary infections, phospholipid acyl chains and cholesterol can be chemically and enzymatically oxidized, as well as truncated and modified, producing complex mixtures of bioactive lipids. We review recent evidence that phospholipids and cholesterol and their derivatives regulate pulmonary innate and adaptive immunity during infection. We first highlight data that oxidized phospholipids generated in the lung during infection stimulate pattern recognition receptors, such as TLRs and scavenger receptors, thereby amplifying the pulmonary inflammatory response. Next, we discuss evidence that oxidation of endogenous pools of cholesterol during pulmonary infections produces oxysterols that also modify the function of both innate and adaptive immune cells. Last, we conclude with data that n-3 polyunsaturated fatty acids, both in the form of phospholipid acyl chains and through enzymatic processing into endogenous proresolving lipid mediators, aid in the resolution of lung inflammation through distinct mechanisms. Unraveling the complex mechanisms of induction and function of distinct classes of bioactive lipids, both native and modified, may hold promise for developing new therapeutic strategies for improving pulmonary outcomes in response to infection. PMID:27286794

Compartmentalized acyl-CoA metabolism in skeletal muscle regulates systemic glucose homeostasis.

PubMed

Li, Lei O; Grevengoed, Trisha J; Paul, David S; Ilkayeva, Olga; Koves, Timothy R; Pascual, Florencia; Newgard, Christopher B; Muoio, Deborah M; Coleman, Rosalind A

2015-01-01

The impaired capacity of skeletal muscle to switch between the oxidation of fatty acid (FA) and glucose is linked to disordered metabolic homeostasis. To understand how muscle FA oxidation affects systemic glucose, we studied mice with a skeletal muscle-specific deficiency of long-chain acyl-CoA synthetase (ACSL)1. ACSL1 deficiency caused a 91% loss of ACSL-specific activity and a 60-85% decrease in muscle FA oxidation. Acsl1(M-/-) mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1(M-/-) mice ran only 48% as far as controls. At the time that Acsl1(M-/-) mice were exhausted but control mice continued to run, liver and muscle glycogen and triacylglycerol stores were similar in both genotypes; however, plasma glucose concentrations in Acsl1(M-/-) mice were ∼40 mg/dL, whereas glucose concentrations in controls were ∼90 mg/dL. Excess use of glucose and the likely use of amino acids for fuel within muscle depleted glucose reserves and diminished substrate availability for hepatic gluconeogenesis. Surprisingly, the content of muscle acyl-CoA at exhaustion was markedly elevated, indicating that acyl-CoAs synthesized by other ACSL isoforms were not available for β-oxidation. This compartmentalization of acyl-CoAs resulted in both an excessive glucose requirement and severely compromised systemic glucose homeostasis. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

CF₃CF₂N=S(F)CF₃: vibrational spectra and conformational properties.

PubMed

Robles, Norma L; Oberhammer, Heinz; Mews, Rüdiger; Cutin, Edgardo H

2014-05-05

The structural, conformational, and configurational properties of 1,1,1-trifluoro-N-(1,1,2,2,2-pentafluoroethyl) methanesulfinimidoyl fluoride, CF3CF2N=S(F)CF3 have been studied by vibrational spectroscopy [IR (vapor) and Raman (liquid)] and quantum chemical calculations [B3LYP, MP2 and B3PW91 levels of theory (using the 6-311+G(d) and 6-311+G(2df) basis sets). According to these theoretical approximations, CF3CF2N=S(F)CF3 might be found in the gas phase as a mixture of a favoured anticlinal form (C-N bond anticlinal with respect to the C-S-F bisector angle) and a less abundant syn conformer showing C1 symmetry as well (ΔG°≈1.5 kcal mol(-1)). However, corresponding vibrational modes for these conformers show only small shifts which would not allow confidently detecting the rather small contribution of this second form in the experimental spectra. Copyright © 2014 Elsevier B.V. All rights reserved.

13C and (15)N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine.

PubMed

Stueber, Dirk; Grant, David M

2002-09-04

The (13)C and (15)N chemical shift tensor principal values for adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine are measured on natural abundance samples. Additionally, the (13)C and (15)N chemical shielding tensor principal values in these four nucleosides are calculated utilizing various theoretical approaches. Embedded ion method (EIM) calculations improve significantly the precision with which the experimental principal values are reproduced over calculations on the corresponding isolated molecules with proton-optimized geometries. The (13)C and (15)N chemical shift tensor orientations are reliably assigned in the molecular frames of the nucleosides based upon chemical shielding tensor calculations employing the EIM. The differences between principal values obtained in EIM calculations and in calculations on isolated molecules with proton positions optimized inside a point charge array are used to estimate the contributions to chemical shielding arising from intermolecular interactions. Moreover, the (13)C and (15)N chemical shift tensor orientations and principal values correlate with the molecular structure and the crystallographic environment for the nucleosides and agree with data obtained previously for related compounds. The effects of variations in certain EIM parameters on the accuracy of the shielding tensor calculations are investigated.

Acyl coenzyme a preference of diacylglycerol acyltransferase from the maturing seeds of cuphea, maize, rapeseed, and canola.

PubMed

Cao, Y Z; Huang, A H

1987-07-01

In their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations from maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of triacylglycerols from the acyl groups at 24, 32, and 40 degrees C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24 degrees C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40 degrees C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology.

Shifts in leaf N:P stoichiometry during rehabilitation in highly alkaline bauxite processing residue sand

PubMed Central

Goloran, Johnvie B.; Chen, Chengrong; Phillips, Ian R.; Elser, James J.

2015-01-01

Large quantities of sodic and alkaline bauxite residue are produced globally as a by-product from alumina refineries. Ecological stoichiometry of key elements [nitrogen (N) and phosphorus (P)] plays a critical role in establishing vegetation cover in bauxite residue sand (BRS). Here we examined how changes in soil chemical properties over time in rehabilitated sodic and alkaline BRS affected leaf N to P stoichiometry of native species used for rehabilitation. Both Ca and soil pH influenced the shifts in leaf N:P ratios of the study species as supported by consistently significant positive relationships (P < 0.001) between these soil indices and leaf N:P ratios. Shifts from N to P limitation were evident for N-fixing species, while N limitation was consistently experienced by non-N-fixing plant species. In older rehabilitated BRS embankments, soil and plant indices (Ca, Na, pH, EC, ESP and leaf N:P ratios) tended to align with those of the natural ecosystem, suggesting improved rehabilitation performance. These findings highlight that leaf N:P stoichiometry can effectively provide a meaningful assessment on understanding nutrient limitation and productivity of native species used for vegetating highly sodic and alkaline BRS, and is a crucial indicator for assessing ecological rehabilitation performance. PMID:26443331

Shifts in leaf N:P stoichiometry during rehabilitation in highly alkaline bauxite processing residue sand.

PubMed

Goloran, Johnvie B; Chen, Chengrong; Phillips, Ian R; Elser, James J

2015-10-07

Large quantities of sodic and alkaline bauxite residue are produced globally as a by-product from alumina refineries. Ecological stoichiometry of key elements [nitrogen (N) and phosphorus (P)] plays a critical role in establishing vegetation cover in bauxite residue sand (BRS). Here we examined how changes in soil chemical properties over time in rehabilitated sodic and alkaline BRS affected leaf N to P stoichiometry of native species used for rehabilitation. Both Ca and soil pH influenced the shifts in leaf N:P ratios of the study species as supported by consistently significant positive relationships (P < 0.001) between these soil indices and leaf N:P ratios. Shifts from N to P limitation were evident for N-fixing species, while N limitation was consistently experienced by non-N-fixing plant species. In older rehabilitated BRS embankments, soil and plant indices (Ca, Na, pH, EC, ESP and leaf N:P ratios) tended to align with those of the natural ecosystem, suggesting improved rehabilitation performance. These findings highlight that leaf N:P stoichiometry can effectively provide a meaningful assessment on understanding nutrient limitation and productivity of native species used for vegetating highly sodic and alkaline BRS, and is a crucial indicator for assessing ecological rehabilitation performance.

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine.

PubMed Central

Savage, T. J.; Hristova, M. K.; Croteau, R.

1996-01-01

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev.& Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane. PMID:12226360

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

PubMed Central

Kawashima, Y; Hirose, A; Kozuka, H

1986-01-01

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed. PMID:2874791

Downregulation of carnitine acyl-carnitine translocase by miRNAs 132 and 212 amplifies glucose-stimulated insulin secretion.

PubMed

Soni, Mufaddal S; Rabaglia, Mary E; Bhatnagar, Sushant; Shang, Jin; Ilkayeva, Olga; Mynatt, Randall; Zhou, Yun-Ping; Schadt, Eric E; Thornberry, Nancy A; Muoio, Deborah M; Keller, Mark P; Attie, Alan D

2014-11-01

We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. Small interfering RNA-mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma β-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine-mediated regulation of IS. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

PubMed

Kumar, Ish; Pratt, R F

2005-08-02

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

[Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

PubMed

Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

2010-01-01

Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

Shifting Foliar N:P Ratios with Experimental Soil Warming in Tussock Tundra

NASA Astrophysics Data System (ADS)

Jasinski, B.; Mack, M. C.; Schuur, E.; Mauritz, M.; Walker, X. J.

2017-12-01

Warming temperatures in the Arctic and boreal ecosystems are currently driving widespread permafrost thaw. Thermokarst is one form of thaw, in which a deepening active soil layer and associated hydrologic changes can lead to increased nutrient availability and shifts in plant community composition. Individual plant species often differ in their ability to access nutrients and adapt to new environmental conditions. While nitrogen (N) is often the nutrient most limiting to Arctic plant communities, the extent to which plant available phosphorus (P) from previously frozen mineral soil may increase as the active layer deepens is still uncertain. To understand the changing relationship between species' uptake of N and P in a thermokarst environment, we assessed foliar N:P ratios from 2015 in two species, a tussock sedge (Eriophorum vaginatum) and a dwarf shrub (Rubus chamaemorus), at a moist acidic tussock tundra experimental passive soil warming site. The passive soil warming treatment increased active layer depth in warmed plots by 35.4 cm (+/- 1.1 cm), an 80% increase over the control plots. E.vaginatum demonstrated a 16.9% decrease (p=0.012, 95% CI [-27.99%, -5.94%]) in foliar N:P ratios in warmed plots, driven mostly by an increase in foliar phosphorus. Foliar N:P ratios of R.chamaemorus showed no significant change. However, foliar samples of R.chamaemorus were significantly enriched in the isotope 15N in soil warming plots (9.9% increase (p=0.002, 95% CI [4.45%, 15.39%])), while the sedge E.vaginatum was slightly depleted. These results suggest that (1) in environments with thawing mineral soil plant available phosphorus may increase more quickly than nitrogen, and (2) that species' uptake strategies and responses to increasing N and P will vary, which has implications for future ecological shifts in thawing ecosystems.

An Unambiguous Nomenclature for the Acyl-quinic Acids Commonly Known as Chlorogenic Acids.

PubMed

Abrankó, László; Clifford, Michael N

2017-05-10

The history of the acyl-quinic acids is briefly reviewed, the merits and limitations of the various nomenclature systems applicable are critically compared, and their limitations are highlighted, in particular their inability to provide an unambiguous description of all quinic acid enantiomers and diastereoisomers and associated acyl-quinic acids. Recommendations are made for a nomenclature system that in combination with IUPAC numbering achieves this objective. A comprehensive set of structures for the quinic acid enantiomers and diastereoisomers is presented. The Supporting Information provides an explanation of trivial names and a decision tree to determine which quinic acid isomer a structure represents.

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases.

PubMed

Westin, Maria A K; Alexson, Stefan E H; Hunt, Mary C

2004-05-21

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.

Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression.

PubMed

Ziesack, Marika; Rollins, Nathan; Shah, Aashna; Dusel, Brendon; Webster, Gordon; Silver, Pamela A; Way, Jeffrey C

2018-05-15

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( Cp FatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify Cp FatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from Cp FatB1 into Cp FatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE PAGES

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton; ...

2016-10-24

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Regioselective lipase-catalyzed synthesis of 3-o-acyl derivatives of resveratrol and study of their antioxidant properties.

PubMed

Torres, Pamela; Poveda, Ana; Jimenez-Barbero, Jesús; Ballesteros, Antonio; Plou, Francisco J

2010-01-27

One of the approaches to increasing the bioavailability of resveratrol is to protect its 3-OH phenolic group. In this work, regioselective acylation of resveratrol at 3-OH was achieved by transesterification with vinyl acetate catalyzed by immobilized lipase from Alcaligenes sp. (lipase QLG). The maximum yield of 3-O-acetylresveratrol was approximately 75%, as the lipase also catalyzes its further acetylation affording the diester 3,4'-di-O-acetylresveratrol and finally the peracetylated derivative. Long saturated and unsaturated fatty acid vinyl esters were also effective as acyl donors with similar regioselectivity. In contrast, lipase B from Candida antarctica catalyzes the acylation of the phenolic group 4'-OH with 80% yield and negligible formation of higher esters. The analysis of the antioxidant properties showed that the Trolox equivalent antioxidant capability (TEAC) values for the acetyl and stearoyl derivatives at 3-OH were, respectively, 40% and 25% referred to resveratrol. The addition of an acyl chain in the 3-OH position caused a higher loss of activity compared with that at the 4'-OH.

Use of 13Cα Chemical-Shifts in Protein Structure Determination

PubMed Central

Vila, Jorge A.; Ripoll, Daniel R.; Scheraga, Harold A.

2008-01-01

A physics-based method, aimed at determining protein structures by using NOE-derived distances together with observed and computed 13C chemical shifts, is proposed. The approach makes use of 13Cα chemical shifts, computed at the density functional level of theory, to obtain torsional constraints for all backbone and side-chain torsional angles without making a priori use of the occupancy of any region of the Ramachandran map by the amino acid residues. The torsional constraints are not fixed but are changed dynamically in each step of the procedure, following an iterative self-consistent approach intended to identify a set of conformations for which the computed 13Cα chemical shifts match the experimental ones. A test is carried out on a 76-amino acid all-α-helical protein, namely the B. Subtilis acyl carrier protein. It is shown that, starting from randomly generated conformations, the final protein models are more accurate than an existing NMR-derived structure model of this protein, in terms of both the agreement between predicted and observed 13Cα chemical shifts and some stereochemical quality indicators, and of similar accuracy as one of the protein models solved at a high level of resolution. The results provide evidence that this methodology can be used not only for structure determination but also for additional protein structure refinement of NMR-derived models deposited in the Protein Data Bank. PMID:17516673

Clarification of the Mechanism of Acylation Reaction and Origin of Substrate Specificity of the Serine-Carboxyl Peptidase Sedolisin through QM/MM Free Energy Simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qin; Yao, Jianzhuang; Wiodawer, Alexander

2011-01-01

Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzyme-catalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers asmore » well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N H of P1 -Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton

Detecting Fermi-level shifts by Auger electron spectroscopy in Si and GaAs

NASA Astrophysics Data System (ADS)

Debehets, J.; Homm, P.; Menghini, M.; Chambers, S. A.; Marchiori, C.; Heyns, M.; Locquet, J. P.; Seo, J. W.

2018-05-01

In this paper, changes in surface Fermi-level of Si and GaAs, caused by doping and cleaning, are investigated by Auger electron spectroscopy. Based on the Auger voltage contrast, we compared the Auger transition peak energy but with higher accuracy by using a more accurate analyzer and an improved peak position determination method. For silicon, a peak shift as large as 0.46 eV was detected when comparing a cleaned p-type and n-type wafer, which corresponds rather well with the theoretical difference in Fermi-levels. If no cleaning was applied, the peak position did not differ significantly for both wafer types, indicating Fermi-level pinning in the band gap. For GaAs, peak shifts were detected after cleaning with HF and (NH4)2S-solutions in an inert atmosphere (N2-gas). Although the (NH4)2S-cleaning in N2 is very efficient in removing the oxygen from the surface, the observed Ga- and As-peak shifts are smaller than those obtained after the HF-cleaning. It is shown that the magnitude of the shift is related to the surface composition. After Si-deposition on the (NH4)2S-cleaned surface, the Fermi-level shifts back to a similar position as observed for an as-received wafer, indicating that this combination is not successful in unpinning the Fermi-level of GaAs.

Precise Determination of the 1s Lamb Shift in Hydrogen-Like Lead and Gold Using Microcalorimeters

NASA Technical Reports Server (NTRS)

Kraft-Bermuth, S.; Andrianov, V.; Bleile, A.; Echler, A.; Egelhof, P.; Grabitz, P.; Ilieva, S.; Kiselev, O.; Kilbourne, C.; McCammon, D.;

Shifting balances in U.S. metropolitan and nonmetropolitan area growth.

PubMed

Garnick, D H

1984-12-01

"This paper assesses some of the recent attempts to explain the perceived growth reversal between metropolitan and nonmetropolitan areas in the United States during the 1970s. The paper argues that the reversal in population trends was not a one-time, radical shift in settlement trends, but rather the result of more continuous underlying industrial trends. Indeed, since 1979, population growth has again become faster in metropolitan than nonmetropolitan areas." The paper includes three sections. Regional and area population and industrial earnings growth patterns are first summarized for the 1960s, 1970s, and 1980s. Theories of polarization and polar reversal are then evaluated and found to be inadequate. Finally, a reconstruction of the neoclassical model is proposed. excerpt

Genetics Home Reference: very long-chain acyl-CoA dehydrogenase deficiency

MedlinePlus

... Very long chain acyl-CoA dehydrogenase deficiency Screening, Technology, and Research in Genetics Virginia Department of Health (PDF) Patient Support and Advocacy Resources (4 links) Children's Mitochondrial Disease Network (UK) FOD (Fatty Oxidation Disorders) ...

The acylation state of mycobacterial lipomannans modulates innate immunity response through toll-like receptor 2.

PubMed

Gilleron, Martine; Nigou, Jérôme; Nicolle, Delphine; Quesniaux, Valérie; Puzo, Germain

2006-01-01

Detection of Mycobacterium tuberculosis antigens by professional phagocytes via toll-like receptors (TLR) contributes to controlling chronic M. tuberculosis infection. Lipomannans (LM), which are major lipoglycans of the mycobacterial envelope, were recently described as agonists of TLR2 with potent activity on proinflammatory cytokine regulation. LM correspond to a heterogeneous population of acyl- and glyco-forms. We report here the purification and the complete structural characterization of four LM acyl-forms from Mycobacterium bovis BCG using MALDI MS and 2D (1)H-(31)P NMR analyses. All this biochemical work provided the tools to investigate the implication of LM acylation degree on its proinflammatory activity. The latter was ascribed to the triacylated LM form, essentially an agonist of TLR2, using TLR2/TLR1 heterodimers for signaling. Altogether, these findings shed more light on the molecular basis of LM recognition by TLR.

Preservation of polyunsaturated fatty acyl glycerides via intramolecular antioxidant coupling

USDA-ARS?s Scientific Manuscript database

Ferulic acid and its esters are known to be effective antioxidants. Feruloyl di-gamma-linolenoylglycerol was assessed for its ability to serve as an antioxidant for preventing the oxidation of its gamma-linolenoyl polyunsaturated fatty acyl groups in model membrane phospholipid vesicles. The molec...

Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

PubMed Central

Gram, Lone; Christensen, Allan Beck; Ravn, Lars; Molin, Søren; Givskov, Michael

1999-01-01

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 106 CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N2 atmosphere when mean cell densities increased to 106 CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 106 CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity. PMID:10427034

Acyl Coenzyme A Preference of Diacylglycerol Acyltransferase from the Maturing Seeds of Cuphea, Maize, Rapeseed, and Canola 1

PubMed Central

Cao, Yi-Zhi; Huang, Anthony H. C.

1987-01-01

In their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations from maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of triacylglycerols from the acyl groups at 24, 32, and 40°C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24°C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40°C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology. PMID:16665518

A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis.

PubMed

Wang, Kun; Froehlich, John E; Zienkiewicz, Agnieszka; Hersh, Hope Lynn; Benning, Christoph

2017-07-01

The lipid composition of thylakoid membranes inside chloroplasts is conserved from leaves to developing embryos. A finely tuned lipid assembly machinery is required to build these membranes during Arabidopsis thaliana development. Contrary to thylakoid lipid biosynthetic enzymes, the functions of most predicted chloroplast lipid-degrading enzymes remain to be elucidated. Here, we explore the biochemistry and physiological function of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A 1 In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific phosphatidylglycerol that contains 16:1 Δ3trans as its second acyl group. Thus far, a specific function of this 16:1 Δ3trans -containing phosphatidylglycerol in chloroplasts has remained elusive. The PLIP1 gene is highly expressed in seeds, and plip1 mutant seeds contain less oil and exhibit delayed germination compared with the wild type. Acyl groups released by PLIP1 are exported from the chloroplast, reincorporated into phosphatidylcholine, and ultimately enter seed triacylglycerol. Thus, 16:1 Δ3trans uniquely labels a small but biochemically active plastid phosphatidylglycerol pool in developing Arabidopsis embryos, which is subject to PLIP1 activity, thereby contributing a small fraction of the polyunsaturated fatty acids present in seed oil. We propose that acyl exchange involving thylakoid lipids functions in acyl export from plastids and seed oil biosynthesis. © 2017 American Society of Plant Biologists. All rights reserved.

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

PubMed Central

Slocombe, S P; Piffanelli, P; Fairbairn, D; Bowra, S; Hatzopoulos, P; Tsiantis, M; Murphy, D J

1994-01-01

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo. PMID:8016261

Enhanced separation and analysis procedure reveals production of tri-acylated mannosylerythritol lipids by Pseudozyma aphidis.

PubMed

Goossens, Eliane; Wijnants, Marc; Packet, Dirk; Lemière, Filip

2016-11-01

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants because of their high fermentation yields (>100 g l -1 ) and during the last two decades they have gained a lot of attention due to their interesting self-assembling properties and biological activities. In this study, MELs were produced by fed-batch bioreactor fermentation of rapeseed oil with Pseudozyma aphidis MUCL 27852. This high-level MEL-producing yeast secretes four conventional MEL structures, -A, -B, -C and -D, which differ in their degree of acetylation. During our research, unknown compounds synthesized by P. aphidis were detected by thin-layer chromatography. The unknown compounds were separated by flash chromatography and identified as tri-acylated MELs by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The third fatty acid chain on the tri-acylated MELs was positioned on the primary alcohol of the erythritol moiety and comprised long-chain acids, mainly oleic and linoleic acid, which are not found in conventional di-acylated MELs. Furthermore, the LC-MS analysis time of conventional MELs was reduced to almost one-third by switching from HPLC-MS/MS to ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Provided optimization of the fermentation yield, P. aphidis could be an interesting novel producer of tri-acylated MELs and, thereby expand the supply and applicability of biosurfactants.

Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?

PubMed

Özcan, Behiye; Neggers, Sebastian J C M M; Miller, Anne Reifel; Yang, Hsiu-Chiung; Lucaites, Virginia; Abribat, Thierry; Allas, Soraya; Huisman, Martin; Visser, Jenny A; Themmen, Axel P N; Sijbrands, Eric J G; Delhanty, Patric J D; van der Lely, Aart Jan

2014-06-01

The objective of this study was to assess the effects of a continuous overnight infusion of des-acyl ghrelin (DAG) on acylated ghrelin (AG) levels and glucose and insulin responses to a standard breakfast meal (SBM) in eight overweight patients with type 2 diabetes. Furthermore, in the same patients and two additional subjects, the effects of DAG infusion on AG concentrations and insulin sensitivity during a hyperinsulinemic-euglycemic clamp (HEC) were assessed. A double-blind, placebo-controlled cross-over study design was implemented, using overnight continuous infusions of 3 and 10  μg DAG/kg per h and placebo to study the effects on a SBM. During a HEC, we studied the insulin sensitivity. We observed that, compared with placebo, overnight DAG administration significantly decreased postprandial glucose levels, both during continuous glucose monitoring and at peak serum glucose levels. The degree of improvement in glycemia was correlated with baseline plasma AG concentrations. Concurrently, DAG infusion significantly decreased fasting and postprandial AG levels. During the HEC, 2.5  h of DAG infusion markedly decreased AG levels, and the M-index, a measure of insulin sensitivity, was significantly improved in the six subjects in whom we were able to attain steady-state euglycemia. DAG administration was not accompanied by many side effects when compared with placebo. DAG administration improves glycemic control in obese subjects with type 2 diabetes through the suppression of AG levels. DAG is a good candidate for the development of compounds in the treatment of metabolic disorders or other conditions with a disturbed AG:DAG ratio, such as type 2 diabetes mellitus or Prader-Willi syndrome. © 2014 European Society of Endocrinology.

Total and cause-specific mortality of U.S. nurses working rotating night shifts.

PubMed

Gu, Fangyi; Han, Jiali; Laden, Francine; Pan, An; Caporaso, Neil E; Stampfer, Meir J; Kawachi, Ichiro; Rexrode, Kathryn M; Willett, Walter C; Hankinson, Susan E; Speizer, Frank E; Schernhammer, Eva S

2015-03-01

Rotating night shift work imposes circadian strain and is linked to the risk of several chronic diseases. To examine associations between rotating night shift work and all-cause; cardiovascular disease (CVD); and cancer mortality in a prospective cohort study of 74,862 registered U.S. nurses from the Nurses' Health Study. Lifetime rotating night shift work (defined as ≥3 nights/month) information was collected in 1988. During 22 years (1988-2010) of follow-up, 14,181 deaths were documented, including 3,062 CVD and 5,413 cancer deaths. Cox proportional hazards models estimated multivariable-adjusted hazard ratios (HRs) and 95% CIs. All-cause and CVD mortality were significantly increased among women with ≥5 years of rotating night shift work, compared to women who never worked night shifts. Specifically, for women with 6-14 and ≥15 years of rotating night shift work, the HRs were 1.11 (95% CI=1.06, 1.17) and 1.11 (95% CI=1.05, 1.18) for all-cause mortality and 1.19 (95% CI=1.07, 1.33) and 1.23 (95% CI=1.09, 1.38) for CVD mortality. There was no significant association between rotating night shift work and all-cancer mortality (HR≥15years=1.08, 95% CI=0.98, 1.19) or mortality of any individual cancer, with the exception of lung cancer (HR≥15years=1.25, 95% CI=1.04, 1.51). Women working rotating night shifts for ≥5 years have a modest increase in all-cause and CVD mortality; those working ≥15 years of rotating night shift work have a modest increase in lung cancer mortality. These results add to prior evidence of a potentially detrimental effect of rotating night shift work on health and longevity. Copyright © 2015 American Journal of Preventive Medicine. All rights reserved.

Total and Cause-Specific Mortality of U.S. Nurses Working Rotating Night Shifts

PubMed Central

Gu, Fangyi; Han, Jiali; Laden, Francine; Pan, An; Caporaso, Neil E.; Stampfer, Meir J.; Kawachi, Ichiro; Rexrode, Kathryn M.; Willett, Walter C.; Hankinson, Susan E.; Speizer, Frank; Schernhammer, Eva S.

2014-01-01

Background Rotating night shift work imposes circadian strain and is linked to the risk of several chronic diseases. Purpose To examine associations between rotating night shift work and all-cause, cardiovascular disease (CVD), and cancer mortality in a prospective cohort study of 74,862 registered U.S. nurses from the Nurses’ Health Study. Methods Lifetime rotating night shift work (defined as ≥3 nights/month) information was collected in 1988. During 22 years (1988–2010) of follow-up, 14,181 deaths were documented, including 3,062 CVD and 5,413 cancer deaths. Cox proportional hazards models (2013) estimated multivariable-adjusted hazard ratios (HRs) and 95% CIs. Results All-cause and CVD mortality were significantly increased among women with ≥5 years of rotating night shift work, compared to women who never worked night shifts. Specifically, for women with 6–14 and ≥15 years of rotating night shift work, the HRs were 1.11 (95% CI=1.06, 1.17) and 1.11 (95% CI=1.05, 1.18) for all-cause mortality and 1.19 (95% CI=1.07, 1.33) and 1.23 (95% CI=1.09, 1.38) for CVD mortality. There was no association between rotating night shift work and all-cancer mortality (HR≥15years=1.08, 95% CI=0.89, 1.19) or any other cancer, with the exception of lung cancer (HR≥15years=1.25, 95% CI=1.04, 1.51). Conclusions Women working rotating night shifts for ≥5 five years have a modest increase in all-cause and CVD mortality; those working ≥15 years of rotating night shift work have a modest increase in lung cancer mortality. These results add to prior evidence of a potentially detrimental effect of rotating night shift work on health and longevity. PMID:25576495

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

PubMed

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration

PubMed Central

Banks, Courtney J.; Rodriguez, Nathan W.; Gashler, Kyle R.; Pandya, Rushika R.; Mortenson, Jeffrey B.; Whited, Matthew D.; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur; Reddi, Amit R.; Tessem, Jeffery S.; Torres, Matthew P.; Bikman, Benjamin T.

2017-01-01

ABSTRACT In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival. PMID:28739857

Impact of N-plasma and Ga-irradiation on MoS2 layer in molecular beam epitaxy