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...

Validation of a method for the quantitation of ghrelin and unacylated ghrelin by HPLC.

PubMed

Staes, Edith; Rozet, Eric; Ucakar, Bernard; Hubert, Philippe; Préat, Véronique

2010-02-05

An HPLC/UV method was first optimized for the separation and quantitation of human acylated and unacylated (or des-acyl) ghrelin from aqueous solutions. This method was validated by an original approach using accuracy profiles based on tolerance intervals for the total error measurement. The concentration range that achieved adequate accuracy extended from 1.85 to 59.30microM and 1.93 to 61.60microM for acylated and unacylated ghrelin, respectively. Then, optimal temperature, pH and buffer for sample storage were determined. Unacylated ghrelin was found to be stable in all conditions tested. At 37 degrees C acylated ghrelin was stable at pH 4 but unstable at pH 7.4, the main degradation product was unacylated ghrelin. Finally, this validated HPLC/UV method was used to evaluate the binding of acylated and unacylated ghrelin to liposomes.

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.

Mechanism of lipid mobilization by the small intestine after transport blockade

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

Halpern, J.; Tso, P.; Mansbach, C.M. II

1988-07-01

The nonionic detergent, Pluronic L-81 (L-81) has been shown to block the transport of intestinal mucosal triacylglycerol (TG) in chylomicrons. This results in large lipid masses within the enterocyte that are greater in diameter than chylomicrons. On removal of L-81, mucosal TG is rapidly mobilized and appears in the lymph. We questioned whether the blocked TG requires partial or complete hydrolysis before its transport. Rats were infused intraduodenally with (3H)glyceryl, (14C)oleoyl trioleate (TO) and 0.5 mg L-81/h for 8 h, followed by 120 mumol/h linoleate for 18 h. Mesenteric lymph was collected and analyzed for TG content and radioactivity. Anmore » HPLC method was developed to separate TG on the basis of its acyl group species. The assumed acyl group composition was confirmed by gas liquid chromatography analysis. TG lymphatic output was low for the first 8 h but increased to 52 mumol/h at the 11th h of infusion (3 h after stopping L-81). 38% of the infused TO was retained in the mucosa after the 8-h infusion. 95% of mucosal TG was TO, 92% of the radioactivity was in TG, and 2.4% of the 14C disintegrations per minute was in fatty acid. HPLC analysis of lymph at 6, 10, 12, and 14.5 h of infusion showed a progressive rise in TG composed of one linoleate and two oleates, to 39%; and in TG composed of two linoleates and one oleate to 20% at 14.5 h of infusion. On a mass basis, however, 80% of the TG acyl groups were oleate. 3H/14C ratios in the various TG acyl group species reflected the decrease in oleate. We conclude that first, unlike liver, most mucosal TG is not hydrolyzed before transport. The mechanism of how the large lipid masses present in mucosal cells after L-81 infusion are converted to the much smaller chylomicrons is unknown. Second, the concomitant infusion of linoleate did not impair lymph TG delivery after L-81 blockade.« less

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, likely because of a decrease in the acylation process. Desacyl ghrelin increases in gestational diabetes, possibly reflecting resistance to the inhibitory effect of insulin on ghrelin secretion.

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 because of a decrease in the acylation process. Desacyl ghrelin increases in gestational diabetes, possibly reflecting resistance to the inhibitory effect of insulin on ghrelin secretion. PMID:19240252

Comprehensive two-dimensional liquid chromatography tandem diode array detector (DAD) and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa.

PubMed

Li, Duxin; Schmitz, Oliver J

2015-01-01

The analysis of chemical constituents in Chinese herbal medicines (CHMs) is a challenge because of numerous compounds with various polarities and functional groups. Liquid chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry (LC/MS) is of particular interest in the analysis of herbal components. One of the main attributes of QTOF that makes it an attractive analytical technique is its accurate mass measurement for both precursor and product ions. For the separation of CHMs, comprehensive two-dimensional chromatography (LCxLC) provides much higher resolving power than traditional one-dimensional separation. Therefore, a LCxLC-QTOF-MS system was developed and applied to the analysis of flavonoids and iridoid glycosides in aqueous extracts of Hedyotis diffusa (Rubiaceae). Shift gradient was applied in the two-dimensional separation in the LCxLC system to increase the orthogonality and effective peak distribution area of the analysis. Tentative identification of compounds was done by accurate mass interpretation and validation by UV spectrum. A clear classification of flavonol glycosides (FGs), acylated FGs, and iridoid glycosides (IGs) was shown in different regions of the LCxLC contour plot. In total, five FGs, four acylated FGs, and three IGs were tentatively identified. In addition, several novel flavonoids were found, which demonstrates that LCxLC-QTOF-MS detection also has great potential in herbal medicine analysis.

Synthesis and Characterization of AlCl3 Impregnated Molybdenum Oxide as Heterogeneous Nano-Catalyst for the Friedel-Crafts Acylation Reaction in Ambient Condition.

PubMed

Jadhav, Arvind H; Chinnappan, Amutha; Hiremath, Vishwanath; Seo, Jeong Gil

2015-10-01

Aluminum trichloride (AlCl3) impregnated molybdenum oxide heterogeneous nano-catalyst was prepared by using simple impregnation method. The prepared heterogeneous catalyst was characterized by powder X-ray diffraction, FT-IR spectroscopy, solid-state NMR spectroscopy, SEM imaging, and EDX mapping. The catalytic activity of this protocol was evaluated as heterogeneous catalyst for the Friedel-Crafts acylation reaction at room temperature. The impregnated MoO4(AlCl2)2 catalyst showed tremendous catalytic activity in Friedel-Crafts acylation reaction under solvent-free and mild reaction condition. As a result, 84.0% yield of acyl product with 100% consumption of reactants in 18 h reaction time at room temperature was achieved. The effects of different solvents system with MoO4(AlCl2)2 catalyst in acylation reaction was also investigated. By using optimized reaction condition various acylated derivatives were prepared. In addition, the catalyst was separated by simple filtration process after the reaction and reused several times. Therefore, heterogeneous MoO4(AlCl2)2 catalyst was found environmentally benign catalyst, very convenient, high yielding, and clean method for the Friedel-Crafts acylation reaction under solvent-free and ambient reaction condition.

Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B.

PubMed

Wolfson, Adi; Atyya, Aviad; Dlugy, Christina; Tavor, Dorith

2010-03-01

Glycerol triacetate was successfully used as a green solvent and as the acyl donor in the transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized Candida antarctica lipase B. Immobilized lipase was more catalytically active than free lipase and could be easily separated from the reaction mixture by filtration. In addition, it was found that increasing either the reaction temperature or the enzyme to substrate ratio increased the conversion of isoamyl alcohol. Using triacetin as the solvent also enabled the separation of product by simple extraction with petroleum ether and catalyst recycling.

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.

Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2[C][W

PubMed Central

Sanjaya; Miller, Rachel; Durrett, Timothy P.; Kosma, Dylan K.; Lydic, Todd A.; Muthan, Bagyalakshmi; Koo, Abraham J.K.; Bukhman, Yury V.; Reid, Gavin E.; Howe, Gregg A.; Ohlrogge, John; Benning, Christoph

2013-01-01

Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves. PMID:23417035

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

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

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

PubMed

Yevglevskis, Maksims; Lee, Guat L; Nathubhai, Amit; Petrova, Yoana D; James, Tony D; Threadgill, Michael D; Woodman, Timothy J; Lloyd, Matthew D

2018-04-30

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC 50  = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation. Copyright © 2018 Elsevier Inc. All rights reserved.

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…

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.

Exogenous fatty acid metabolism in bacteria.

PubMed

Yao, Jiangwei; Rock, Charles O

2017-10-01

Bacterial type II fatty acid synthesis (FASII) is a target for novel antibiotic development. All bacteria encode for mechanisms to incorporate exogenous fatty acids, and some bacteria can use exogenous fatty acids to bypass FASII inhibition. Bacteria encode three different mechanisms for activating exogenous fatty acids for incorporation into phospholipid synthesis. Exogenous fatty acids are converted into acyl-CoA in Gammaproteobacteria such as E. coli. Acyl-CoA molecules constitute a separate pool from endogenously synthesized acyl-ACP. Acyl-CoA can be used for phospholipid synthesis or broken down by β-oxidation, but cannot be used for lipopolysaccharide synthesis. Exogenous fatty acids are converted into acyl-ACP in some Gram-negative bacteria. The resulting acyl-ACP undergoes the same fates as endogenously synthesized acyl-ACP. Exogenous fatty acids are converted into acyl-phosphates in Gram-positive bacteria, and can be used for phospholipid synthesis or become acyl-ACP. Only the order Lactobacillales can use exogenous fatty acids to bypass FASII inhibition. FASII shuts down completely in presence of exogenous fatty acids in Lactobacillales, allowing Lactobacillales to synthesize phospholipids entirely from exogenous fatty acids. Inhibition of FASII cannot be bypassed in other bacteria because FASII is only partially down-regulated in presence of exogenous fatty acid or FASII is required to synthesize essential metabolites such as β-hydroxyacyl-ACP. Certain selective pressures such as FASII inhibition or growth in biofilms can select for naturally occurring one step mutations that attenuate endogenous fatty acid synthesis. Although attempts have been made to estimate the natural prevalence of these mutants, culture-independent metagenomic methods would provide a better estimate. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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.

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

Enzymatic synthesis of structured lipids.

PubMed

Iwasaki, Yugo; Yamane, Tsuneo

2004-01-01

Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2- position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A2-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

Incorporation of Extracellular Fatty Acids by a Fatty Acid Kinase-Dependent Pathway in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Frank, Matthew W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Summary Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids. PMID:24673884

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

PubMed

Garver, W S; Kemp, J D; Kuehn, G D

1992-12-01

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

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.

3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis.

PubMed

Wilson, Michael B; Pawlus, Alison D; Brinkman, Doug; Gardner, Gary; Hegeman, Adrian D; Spivak, Marla; Cohen, Jerry D

2017-06-01

Honey bees, Apis mellifera, collect antimicrobial plant resins from the environment and deposit them in their nests as propolis. This behavior is of practical concern to beekeepers since the presence of propolis in the hive has a variety of benefits, including the suppression of disease symptoms. To connect the benefits that bees derive from propolis with particular resinous plants, we determined the identity and botanical origin of propolis compounds active against bee pathogens using bioassay-guided fractionation against the bacterium Paenibacillus larvae, the causative agent of American foulbrood. Eleven dihydroflavonols were isolated from propolis collected in Fallon, NV, including pinobanksin-3-octanoate. This hitherto unknown derivative and five other 3-acyl-dihydroflavonols showed inhibitory activity against both P. larvae (IC 50  = 17-68 μM) and Ascosphaera apis (IC 50  = 8-23 μM), the fungal agent of chalkbrood. A structure-activity relationship between acyl group size and antimicrobial activity was found, with longer acyl groups increasing activity against P. larvae and shorter acyl groups increasing activity against A. apis. Finally, it was determined that the isolated 3-acyl-dihydroflavonols originated from Populus fremontii, and further analysis showed these compounds can also be found in other North American Populus spp. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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

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

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.

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.

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

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.

Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry.

PubMed

Jerz, Gerold; Wybraniec, Sławomir; Gebers, Nadine; Winterhalter, Peter

2010-07-02

In this study, preparative ion-pair high-speed countercurrent chromatography was directly coupled to an electrospray ionization mass-spectrometry device (IP-HSCCC/ESI-MS-MS) for target-guided fractionation of high molecular weight acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra (Nyctaginaceae). The direct identification of six principal acyl-oligosaccharide linked betacyanins in the mass range between m/z 859 and m/z 1359 was achieved by positive ESI-MS ionization and gave access to the genuine pigment profile already during the proceeding of the preparative separation. Inclusively, all MS/MS-fragmentation data were provided during the chromatographic run for a complete analysis of substitution pattern. On-line purity evaluation of the recovered fractions is of high value in target-guided screening procedures and for immediate decisions about suitable fractions used for further structural analysis. The applied preparative hyphenation was shown to be a versatile screening method for on-line monitoring of countercurrent chromatographic separations of polar crude pigment extracts and also traced some minor concentrated compounds. For the separation of 760mg crude pigment extract the biphasic solvent system tert.-butylmethylether/n-butanol/acetonitrile/water 2:2:1:5 (v/v/v/v) was used with addition of ion-pair forming reagent trifluoroacetic acid. The preparative HSCCC-eluate had to be modified by post-column addition of a make-up solvent stream containing formic acid to reduce ion-suppression caused by trifluoroacetic acid and later significantly maximized response of ESI-MS/MS detection of target substances. A variable low-pressure split-unit guided a micro-eluate to the ESI-MS-interface for sensitive and direct on-line detection, and the major volume of the effluent stream was directed to the fraction collector for preparative sample recovery. The applied make-up solvent mixture significantly improved smoothness of the continuously measured IP-HSCCC-ESI-MS base peak ion trace in the experimental range of m/z 50-2200 by masking stationary phase bleeding and generating a stable single solvent phase for ESI-MS/MS detection. Immediate structural data were retrieved throughout the countercurrent chromatography run containing complete MS/MS-fragmentation pattern of the separated acyl-substituted betanidin oligoglycosides. Single ion monitoring indicated clearly the base-line separation of higher concentrated acylated betacyanin components. Copyright 2010 Elsevier B.V. All rights reserved.

Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry.

PubMed

Zhang, Jie; Wang, Liang-Sheng; Gao, Jin-Ming; Xu, Yan-Jun; Li, Lian-Fang; Li, Chong-Hui

2012-01-01

Anthocyanins are important plant secondary metabolites. They show strong antioxidant activities and have potential as anti-cancer agents. Viola yedoensis and V. prionantha are traditional Chinese medicines and ornamental plants. However, the anthocyanin compositions of these two species are still unresolved. To develop a rapid and reliable high-performance liquid chromatography (HPLC) method for the separation and identification of anthocyanins from V. yedoensis and V. prionantha. Samples were extracted in methanol-water-formic acid-TFA (70:27:2:1, v/v). HPLC analysis was done on a C(18) column (TSK-GEL ODS-80Ts: 150 × 4.6 mm i.d.). Four solvent systems were tested to optimise the separation of anthocyanins using different gradient separation systems. HPLC-photodiode array detection (DAD) coupled to electrospray ionisation mass spectrometry (ESI-MS) was used to carry out the comprehensive characterisation of anthocyanins. Fourteen anthocyanins were characterised within 40 min with satisfactory peak resolution by a gradient composed of 10% aqueous formic acid and formic acid-acetonitrile-water (10:40:50, v/v). The calibration curve showed an excellent linear regression (r(2)  = 0.9995) and low intra- and inter-day variations (RSD < 3.67%). The detected anthocyanins derived from Dp, Cy, Pt, Mv and Pn, could be divided into three groups: non-acylated glycosides, acetylglycosides and coumaroylglycosides. Anthocyanins distribution exhibited remarkable differences in aglycone levels and acylation patterns. The optimised method was successfully applied for the analysis of 14 anthocyanins from V. yedoensis and V. prionantha. The identification of anthocyanin constitutions is valuable for breeding and will open up new prospects for their medicinal application. Copyright © 2011 John Wiley & Sons, Ltd.

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.

Assessment of the levels of serum Hsp 70 and ghrelin in children with simple febrile convulsions.

PubMed

Kilic, Mehmet; Gündüzalp, Mehmet; Taskin, Erdal; Aydin, Süleyman; Serin, Hepsen M

2016-04-01

In this study we aimed to evaluate the serum levels of Heat-shock protein (Hsp) 70 and acylated and desacylated ghrelin in patients suffering from a simple febrile convulsion. This cross-sectional study included patients who were diagnosed with a simple febrile convulsion, afebrile tonic-clonic epileptic seizure and upper respiratory tract infection when admitted to our hospital. All patients were aged between six months and 60 months. Patients enrolled in this study were divided into five groups. Group I: patients with a simple febrile convulsion and body temperature of 38º C to 39° C; group II: patients with a simple febrile convulsion and body temperature of 39.1° C to 41° C; group III: patients with primary generalised tonic-clonic seizure and normal body temperature; group IV: patients with upper respiratory infection without convulsion and a body temperature of 38° C to 39° C; and group V: patients with upper respiratory infection without convulsion and a body temperature of 39.1° C to 41° C. The control group included healthy children who were followed up in the healthy children polyclinic. Serum levels of Hsp70 and acylated and des-acylated ghrelin were studied from the blood samples collected from the patients and control group. Serum levels of Hsp70 were higher in the febrile convulsion (groups I, II) and epileptic convulsion and infection (groups IV, V) groups than in the controls (P<0.0001). Moreover, serum levels of acylated and desacylated ghrelin were higher in the simple febrile convulsion (groups I and II) and epileptic convulsion and infection (groups IV and V) groups than in the control (P<0.05). We demonstrated that serum levels of Hsp70 and acylated and desacylated ghrelin increased in patients with a simple febrile convulsion.

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

Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry.

PubMed

Bansal, Sunil; Durrett, Timothy P

2016-09-01

Acetyl-triacylglycerols (acetyl-TAG) possess an sn-3 acetate group, which confers useful chemical and physical properties to these unusual triacylglycerols (TAG). Current methods for quantification of acetyl-TAG are time consuming and do not provide any information on the molecular species profile. Electrospray ionization mass spectrometry (ESI-MS)-based methods can overcome these drawbacks. However, the ESI-MS signal intensity for TAG depends on the aliphatic chain length and unsaturation index of the molecule. Therefore response factors for different molecular species need to be determined before any quantification. The effects of the chain length and the number of double-bonds of the sn-1/2 acyl groups on the signal intensity for the neutral loss of short chain length sn-3 groups were quantified using a series of synthesized sn-3 specific structured TAG. The signal intensity for the neutral loss of the sn-3 acyl group was found to negatively correlated with the aliphatic chain length and unsaturation index of the sn-1/2 acyl groups. The signal intensity of the neutral loss of the sn-3 acyl group was also negatively correlated with the size of that chain. Further, the position of the group undergoing neutral loss was also important, with the signal from an sn-2 acyl group much lower than that from one located at sn-3. Response factors obtained from these analyses were used to develop a method for the absolute quantification of acetyl-TAG. The increased sensitivity of this ESI-MS-based approach allowed successful quantification of acetyl-TAG in various biological settings, including the products of in vitro enzyme activity assays.

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 before application to clinical practice.

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 study is needed before application to clinical practice. PMID:27196316

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.

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

Lipid-protein interaction in the phosphatidylcholine exchange protein.

PubMed Central

Devaux, P F; Moonen, P; Bienvenue, A; Wirtz, K W

1977-01-01

Incorporation of 2-acyl spin-labeled lecithin into the phosphatidylcholine protein from bovine liver results in an immobilization of the spin-label at the methyl and the carboxyl terminal end of the acyl chain. The nitroxide group on the protein-bound lecithin molecule is not accessible to ascorbate. This suggests that lecithin is buried in a pocket on the protein, which effectively shields the acyl chains from the medium. PMID:194240

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.

Gatekeeping versus Promiscuity in the Early Stages of the Andrimid Biosynthetic Assembly Line

PubMed Central

Magarvey, Nathan A.; Fortin, Pascal D.; Thomas, Paul M.; Kelleher, Neil L.; Walsh, Christopher T.

2009-01-01

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal pep-tide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-β-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-β-Phe, which in turn is activated by AdmJ and ATP to form (S)-β-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-β-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or β-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA. PMID:18652473

Methods for the synthesis of deuterated vinyl pyridine monomers

DOEpatents

Hong, Kunlun; Yang, Jun; Bonnesen, Peter V

2014-02-25

Methods for synthesizing deuterated vinylpyridine compounds of the Formula (1), wherein the method includes: (i) deuterating an acyl pyridine of the Formula (2) in the presence of a metal catalyst and D.sub.2O, wherein the metal catalyst is active for hydrogen exchange in water, to produce a deuterated acyl compound of Formula (3); (ii) reducing the compound of Formula (3) with a deuterated reducing agent to convert the acyl group to an alcohol group, and (iii) dehydrating the compound produced in step (ii) with a dehydrating agent to afford the vinylpyridine compound of Formula (1). The resulting deuterated vinylpyridine compounds are also described.

Methods for the synthesis of deuterated vinyl pyridine monomers

DOEpatents

Hong, Kunlun; Yang, Jun; Bonnesen, Peter V

2015-01-13

Methods for synthesizing deuterated vinylpyridine compounds of the Formula (1), wherein the method includes: (i) deuterating an acyl pyridine of the Formula (2) in the presence of a metal catalyst and D.sub.2O, wherein the metal catalyst is active for hydrogen exchange in water, to produce a deuterated acyl compound of Formula (3); (ii) reducing the compound of Formula (3) with a deuterated reducing agent to convert the acyl group to an alcohol group, and (iii) dehydrating the compound produced in step (ii) with a dehydrating agent to afford the vinylpyridine compound of Formula (1). The resulting deuterated vinylpyridine compounds are also described.

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

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.

Gauging a Hydrocarbon Ruler by an Intrinsic Exciton Probe†

PubMed Central

Khan, M. Adil; Neale, Chris; Michaux, Catherine; Pomés, Régis; Privé, Gilbert G.; Woody, Robert W.; Bishop, Russell E.

2016-01-01

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of Gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid: lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane β-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26–Trp66 phenol–indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton’s expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler–exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment. PMID:17375935

Catalytic Kinetic Resolution of Saturated N-Heterocycles by Enantioselective Amidation with Chiral Hydroxamic Acids.

PubMed

Kreituss, Imants; Bode, Jeffrey W

2016-12-20

The preparation of enantioenriched chiral compounds by kinetic resolution dates back to the laboratories of Louis Pasteur in the middle of the 19th century. Unlike asymmetric synthesis, this process can always deliver enantiopure material (ee > 99%) if the reactions are allowed to proceed to sufficient conversion and the selectivity of the process is not unity (s > 1). One of the most appealing and practical variants is acylative kinetic resolution, which affords easily separable reaction products, and several highly efficient enzymatic and small molecule catalysts are available. Unfortunately, this method is applicable to limited substrate classes such as alcohols and primary benzylamines. This Account focuses on our work in catalytic acylative kinetic resolution of saturated N-heterocycles, a class of molecules that has been notoriously difficult to access via asymmetric synthesis. We document the development of hydroxamic acids as suitable catalysts for enantioselective acylation of amines through relay catalysis. Alongside catalyst optimization and reaction development, we present mechanistic studies and theoretical calculation accounting for the origins of selectivity and revealing the concerted nature of many amide-bond forming reactions. Immobilization of the hydroxamic acid to form a polymer supported reagent allows simplification of the experimental setup, improvement in product purification, and extension of the substrate scope. The kinetic resolutions are operationally straight forward: reactions proceed at room temperature and open to air conditions, without generation of difficult-to-remove side products. This was utilized to achieve decagram scale resolution of antimalarial drug mefloquine to prepare more than 50 g of (+)-erythro-meflqouine (er > 99:1) from the racemate. The immobilized quasienantiomeric acyl hydroxamic acid reagents were also exploited for a rare practical implementation of parallel kinetic resolution that affords both enantiomers of the amine products in high enantiopurity. The success of this process relied on identification of two cleavable acyl groups alongside implementation of flow-chemistry techniques to ensure reusability of the resolving agents. The work discussed in this Account has laid foundations for new catalyst design as well as development of desymmetrization and dynamic kinetic resolution processes. In the meantime, as all the requisite reagents are commercially available, we hope that hydroxamic acid promoted acylative kinetic resolution will become a method of choice for preparation of saturated N-heterocycles in enantiopure form.

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

Multicomponent Approach to the Synthesis of Oxidized Amides through Nitrile Hydrozirconation

PubMed Central

Wan, Shuangyi; Green, Michael E.; Park, Jung-Hyun; Floreancig, Paul E.

2008-01-01

“Oxidized” amides, as represented by acyl aminals and acyl hemiaminals, are integral subunits of several natural products that exhibit useful biological activity. In this manuscript a multicomponent approach to these groups from acylimine intermediates is demonstrated. The acylimines are accessed through a sequence of nitrile hydrozirconation and acylation, making this highly versatile amide synthesis useful for a range of range of applications in target- and diversity-oriented synthesis. PMID:18020344

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

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

Regulation of membrane proteins by dietary lipids: effects of cholesterol and docosahexaenoic acid acyl chain-containing phospholipids on rhodopsin stability and function.

PubMed

Bennett, Michael P; Mitchell, Drake C

2008-08-01

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (T(m)) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (E(a)) was determined from DSC thermograms by four separate methods. Both T(m) and E(a) varied with bilayer composition. Cholesterol increased the T(m) both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E(a) in the absence of DHA, but raised E(a) in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T(m) for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (E(a)) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Shifting Native Chemical Ligation into Reverse through N→S Acyl Transfer

PubMed Central

Macmillan, Derek; Adams, Anna; Premdjee, Bhavesh

2011-01-01

Peptide thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for further development and discovery. PMID:22347724

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.

Pharmaceutical analysis of synthetic lipid A-based vaccine adjuvants in poly (D,L-lactic-co-glycolic acid) nanoparticle formulations.

PubMed

Hamdy, Samar; Haddadi, Azita; Somayaji, Vishwa; Ruan, David; Samuel, John

2007-08-15

The present study had two main objectives. First, was to compare the immune stimulatory effect of two synthetic lipid A analogues (7-acyl lipid A and pentaerythritol-based lipid A (PET lipid A)) on maturation/stimulation of bone marrow derived dendritic cells (DCs). Our second objective was to develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of lipid A-based vaccine adjuvants. Treatment of immature DCs with 7-acyl lipid A and PET lipid A up regulated the surface expression of CD86 and CD40 molecules, and also induced similar profile of pro-inflammatory cytokine secretion. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, coupled to a Waters 2795 separations module with an autosampler. Calibration curves with R(2)>0.999 were constructed over the concentration range of 1.25-20 microg/ml for the solution of 7-acyl lipid A and PET lipid A. The method was tested in a 3 day validation protocol. The accuracy of the assay at different concentrations tested ranged from 89 to 108% and from 92 to 107% for 7-acyl lipid A and PET lipid A, respectively. The limit of quantification for both 7-acyl lipid A and PET lipid A was 1.25 microg/ml (signal/noise (S/N)) ratio >15:1. The sensitivity of the method (the limit of detection) was 0.35 and 0.15 ng for 7-acyl lipid A and PET lipid A, respectively (S/N ratio between 4:1 or 3:1). As a preliminary application, this method has been successfully applied to the determination of 7-acyl lipid A and PET lipid A content in poly (D,L-lactic-co-glycolic acid) nanoparticles (PLGA-NP).

Highly efficient and regioselective synthesis of dihydromyricetin esters by immobilized lipase.

PubMed

Li, Wei; Wu, Huan; Liu, Benguo; Hou, Xuedan; Wan, Duanji; Lou, Wenyong; Zhao, Jian

2015-04-10

Dihydromyricetin is the principle component of the Chinese herbal tea Teng-cha and a promising ingredient for functional food and nutraceuticals, but its low solubility limits its application potentials. This study explored enzymatic acylation of dihydromyricetin to improve its solubility in lipid systems. Acylation was achieved with several lipases with the synthesis of a major (>86%) product and a minor product. Isolation and purification of the products by preparative HPLC followed by LC-MS, (13)C NMR, (1)H NMR and 2 D-HSQC NMR analyses showed that the major product was a dihydromyricetin monoester with the acylation site at the 3-OH group of C ring. Quantum chemical calculations revealed that the 3-OH had the lowest antioxidant activity, and therefore acylation at this site was expected to have minimum impact on the antioxidant activity. Several factors, including solvent, acyl donor, enzyme origin, molar ratio of substrates and reaction temperature and time, exhibited significant effects on the initial rate, conversion yield and regioselectivity of the reaction. Acylation occurred only with vinyl acetate as the acyl donor, and highest conversion yields were achieved with immobilized Penicillium expansum lipase and Novozyme 435 with DMSO and acetonitrile being the best solvents. In general, the acylation results were found to be superior to previous reports on acylation of aglycone flavonoids with respects to conversion yield and regioselectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

Abnormal relationships between the neural response to high- and low-calorie foods and endogenous acylated ghrelin in women with active and weight-recovered anorexia nervosa

PubMed Central

Holsen, Laura M.; Lawson, Elizabeth A.; Christensen, Kara; Klibanski, Anne; Goldstein, Jill M.

2014-01-01

Evidence contributing to the understanding of neurobiological mechanisms underlying appetite dysregulation in anorexia nervosa draws heavily on separate lines of research into neuroendocrine and neural circuitry functioning. In particular, studies consistently cite elevated ghrelin and abnormal activation patterns in homeostatic (hypothalamus) and hedonic (striatum, amygdala, insula) regions governing appetite. The current preliminary study examined the interaction of these systems, based on research demonstrating associations between circulating ghrelin levels and activity in these regions in healthy individuals. In a cross-sectional design, we studied 13 women with active anorexia nervosa (AN), 9 women weight-recovered from AN (AN-WR), and 12 healthy-weight control women using a food cue functional magnetic resonance imaging paradigm, with assessment of fasting levels of acylated ghrelin. Healthy-weight control women exhibited significant positive associations between fasting acylated ghrelin and activity in the right amygdala, hippocampus, insula, and orbitofrontal cortex in response to high-calorie foods, associations which were absent in the AN and AN-WR groups. Women with AN-WR demonstrated a negative relationship between ghrelin and activity in the left hippocampus in response to high-calorie foods, while women with AN showed a positive association between ghrelin and activity in the right orbitofrontal cortex in response to low-calorie foods. Findings suggest a breakdown in the interaction between ghrelin signaling and neural activity in relation to reward responsivity in AN, a phenomenon that may be further characterized using pharmacogenetic studies. PMID:24862390

Separation and characterization of potato lipid acylhydrolases

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

Hasson, E.P.; Laties, G.G.

1976-02-01

Three distinct potato (Solanum tuberosum) lipid acyl-hydrolases have been isolated and characterized. Nonfluorescent esters of the fluorescent alcohols, N-methylindoxyl and N-methylumbelliferone, have been used as convenient substrates for lipid acyl-hydrolase estimation. Enzyme I has been shown to be a neutral lipase which favors glyceryl triolein over the di- and monoolein, which shows no activity with phospho- and galactolipids and which favors long chain fatty acid esters of N-methylindoxyl over the butyrate ester. Enzyme II, while attacking glyceryl mono- and diolein, as well as favoring the butyrate ester of N-methylindoxyl over the myristate ester, is basically a phospholipid and galactolipid acyl-hydrolase.more » Enzyme III may reasonably be considered an esterase, since it hydrolyzes glyceryl monoolein exclusively among the neutral lipids, shows minimal activity on phospho- and galactolipids, and hydrolyzes N-methylindoxylbutyrate exclusively compared with N-methylindoxylmyristate.« less

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

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.

A biotin-conjugated pyridine-based isatoic anhydride, a selective room temperature RNA-acylating agent for the nucleic acid separation.

PubMed

Ursuegui, S; Yougnia, R; Moutin, S; Burr, A; Fossey, C; Cailly, T; Laayoun, A; Laurent, A; Fabis, F

2015-03-28

Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic acid separation. 2'-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-coated magnetic beads and disulfide chemical cleavage led to isolation of highly enriched RNA samples from an initial 9/1 DNA-RNA mixture. Starting from the parent compound N-methylisatoic anhydride A which was used at 65 °C, we improved the extraction process by designing a new generation of isatoic anhydrides that are able to react under smoother conditions. Among them, a pyridine-based isatoic anhydride derivative 15f was found to be reactive at room temperature, leading to enhance the efficiency and selectivity of the extraction process by significantly reducing DNA side extraction. The extracted and purified RNAs can then be detected by RT-PCR.

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

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...

Rapid and Green Separation of Mono- and Diesters of Monochloropropanediols by Ultrahigh Performance Supercritical Fluid Chromatography-Mass Spectrometry Using Neat Carbon Dioxide as a Mobile Phase.

PubMed

Jumaah, Firas; Jędrkiewicz, Renata; Gromadzka, Justyna; Namieśnik, Jacek; Essén, Sofia; Turner, Charlotta; Sandahl, Margareta

2017-09-20

This study demonstrates the effect of column selectivity and density of supercritical carbon dioxide (SC-CO 2 ) on the separation of monochloropropanediol (MCPD) esters, known as food toxicants, using SC-CO 2 without addition of cosolvent in ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS). This study shows that over 20 2-monochloropropanediol (2-MCPD) and 3-monochloropropanediol (3-MCPD) mono- and diesters are separated on a 2-picolylamine column in less than 12 min. The presence and position of a hydroxyl group in the structure, the number of unsaturated bonds, and the acyl chain length play a significant role in the separation of MCPD esters. The flow rate, backpressure, and column oven temperature, which affect the density of the mobile phase, were shown to have a substantial impact on retention, efficiency, and selectivity. The developed method was successfully applied for the determination of MCPD esters in refined oils and showed a close to excellent green analysis score using the Analytical Eco-Scale.

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.

Abnormal relationships between the neural response to high- and low-calorie foods and endogenous acylated ghrelin in women with active and weight-recovered anorexia nervosa.

PubMed

Holsen, Laura M; Lawson, Elizabeth A; Christensen, Kara; Klibanski, Anne; Goldstein, Jill M

2014-08-30

Evidence contributing to the understanding of neurobiological mechanisms underlying appetite dysregulation in anorexia nervosa draws heavily on separate lines of research into neuroendocrine and neural circuitry functioning. In particular, studies consistently cite elevated ghrelin and abnormal activation patterns in homeostatic (hypothalamus) and hedonic (striatum, amygdala, insula) regions governing appetite. The current preliminary study examined the interaction of these systems, based on research demonstrating associations between circulating ghrelin levels and activity in these regions in healthy individuals. In a cross-sectional design, we studied 13 women with active anorexia nervosa (AN), 9 women weight-recovered from AN (AN-WR), and 12 healthy-weight control women using a food cue functional magnetic resonance imaging paradigm, with assessment of fasting levels of acylated ghrelin. Healthy-weight control women exhibited significant positive associations between fasting acylated ghrelin and activity in the right amygdala, hippocampus, insula, and orbitofrontal cortex in response to high-calorie foods, associations which were absent in the AN and AN-WR groups. Women with AN-WR demonstrated a negative relationship between ghrelin and activity in the left hippocampus in response to high-calorie foods, while women with AN showed a positive association between ghrelin and activity in the right orbitofrontal cortex in response to low-calorie foods. Findings suggest a breakdown in the interaction between ghrelin signaling and neural activity in relation to reward responsivity in AN, a phenomenon that may be further characterized using pharmacogenetic studies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

“Prokaryotic Pathway” Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis

PubMed Central

Awai, Koichiro

2017-01-01

Abstract Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called “prokaryotic pathway,” which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not “prokaryotic” in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. PMID:29145606

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

PubMed Central

Sobek, H; Görisch, H

1989-01-01

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported. PMID:2508625

Structural effect of quaternary ammonium chitin derivatives on their bactericidal activity and specificity.

PubMed

Morkaew, Tirut; Pinyakong, Onruthai; Tachaboonyakiat, Wanpen

2017-08-01

The effect of the quaternary ammonium chitin structure on the bactericidal activity and specificity against Escherichia coli and Staphylococcus aureus was investigated. Quaternary ammonium chitins were synthesized by the separate acylation of chitin (CT) with carboxymethyl trimethylammonium chloride (CMA), 3-carboxypropyl trimethylammonium chloride (CPA) and N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB). The successful acylation was confirmed by newly formed ester linkage. All three derivatives had a higher surface charge than chitin due to the additional positively charged quaternary ammonium groups. The N-short alkyl substituent (methyl) of CTCMA and CTCPA increased the hydrophilicity whilst the N-long alkyl substituent (dodecyl) of CTDDMAB increased the hydrophobicity compared to chitin. Chitin did not exhibit any bactericidal activity, while CTCMA and CTCPA completely killed E. coli and S. aureus in 30 and 60min, respectively, and CTDDMAB completely killed S. aureus in 10min but did not kill E. coli after a 2-h exposure. Therefore, the N-short alkyl substituent was more effective for killing E. coli and the N-long alkyl substituent conferred specific bactericidal activity against S. aureus. Copyright © 2017 Elsevier B.V. All rights reserved.

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen

PubMed Central

Bowalgaha, Kushari; Elliot, David J; Mackenzie, Peter I; Knights, Kathleen M; Swedmark, Stellan; Miners, John O

2005-01-01

Aims To characterize the kinetics of S-naproxen (‘naproxen’) acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. Methods Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. Results Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent Km values (±SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 ± 13 µm (16, 43) and 473 ± 108 µm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent Km (72 µm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective ‘probe’ fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis–Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. Conclusion UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug. PMID:16187975

Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

PubMed

McCormack, M; Brecher, P

1987-06-15

Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes.

Identification of tetraacylglycerols in lesquerella oil by electrospray ionization mass spectrometry of the lithium adducts

USDA-ARS?s Scientific Manuscript database

Tetraacylglycerol (an acylglycerol estolide) contains an acyl chain attached to the hydroxyl group of another acyl chain attached to the glycerol backbone. Lequerolic acid (Ls, OH1420:111) is the main fatty acid in lequerella oil and can be used in industry. We have used electrospray ionization mass...

Biogenesis of ER subdomains containing DGAT2, an enzyme involved in industrial oil biosynthesis

USDA-ARS?s Scientific Manuscript database

Diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the committed step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl group from the acyl-CoA pool to the sn-3 position of diacylglycerol. The substrate specificity and overall activity of these enzymes play a key role...

BAHD or SCPL acyltransferase? What a dilemma for acylation in the world of plant phenolic compounds.

PubMed

Bontpart, Thibaut; Cheynier, Véronique; Ageorges, Agnès; Terrier, Nancy

2015-11-01

Phenolic compounds are secondary metabolites involved in several plant growth and development processes, including resistance to biotic and abiotic stresses. The biosynthetic pathways leading to the vast diversity of plant phenolic products often include an acylation step, with phenolic compounds being the donor or acceptor molecules. To date, two acyltransferase families using phenolic compounds as acceptor or donor molecules have been described, with each using a different 'energy-rich' acyl donor. BAHD-acyltransferases, named after the first four biochemically characterized enzymes of the group, use acyl-CoA thioesters as donor molecules, whereas SCPL (Serine CarboxyPeptidase Like)-acyltransferases use 1-O-β-glucose esters. Here, common and divergent specifications found in the literature for both enzyme families were analyzed to answer the following questions. Are both acyltransferases involved in the synthesis of the same molecule (or same group of molecules)? Are both acyltransferases recruited in the same plant? How does the subcellular localization of these enzymes impact metabolite trafficking in plant cells? © 2015 INRA. New Phytologist © 2015 New Phytologist Trust.

Gastrointestinal symptoms in idiopathic pulmonary fibrosis patients treated with pirfenidone and herbal medicine.

PubMed

Shimizu, Y; Shimoyama, Y; Kawada, A; Kusano, M; Hosomi, Y; Sekiguchi, M; Kawata, T; Horie, T; Ishii, Y; Yamada, M; Dobashi, K; Takise, A

2014-01-01

Pirfenidone is an antifibrotic agent for patients with pulmonary fibrosis, but this drug has adverse gastrointestinal (GI) effects. The first aim of this study was to assess GI symptoms due to pirfenidone by using a new questionnaire for reflux symptoms and dismotility symptoms. Whether adding herbal medicine of rikkunshi-to improved GI symptoms due to pirfenidone therapy was also investigated. This was a randomized controlled trial performed on 17 IPF patients. The patients were assigned to two groups, and the study period was 8 weeks. The pirfenidone group received pirfenidone therapy for 8 weeks with add-on rikkunshi-to from 4 weeks, while the control group did not receive either of these agents. To assess the effects of RK, plasma levels of acyl-ghrelin and des-acyl-ghrelin, serum KL-6 and surfactant protein-D, and pulmonary function tests were monitored. GI symptoms were most severe during the initial 2 weeks of pirfenidone therapy at a dose of 600 mg/day. Both reflux symptoms and dismotility symptoms deteriorated. Rikkunshi-to improved GI symptoms to the level prior to pirfenidone therapy. Plasma levels of des-acyl-ghrelin and acyl-/des-acyl-ghrelin ratio changed significantly at 8 weeks compared to 2 weeks. GI adverse events due to PFD were most severe in the first 2 weeks of treatment at a dose of 600 mg/day, and both reflux and dismotility symptoms deteriorated, but the drug was well tolerated at 1200 mg/day. Rikkunshi-to contributed to improvement of GI symptoms, but plasma ghrelin levels did not reflect the improvement of GI symptoms.

Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli

PubMed Central

Cole, M.

1969-01-01

1. The penicillin acylase of Eschericha coli N.C.I.B. 8743 is a reversible enzyme. Reaction rates for the two directions have been determined. 2. Measurements of the rates of enzymic synthesis of penicillins from 6-aminopenicillanic acid and various carboxylic acids revealed that p-hydroxyphenylacetic acid was the best substrate, followed by phenylacetic, 2-thienylacetic, substituted phenylacetic, 3-hexenoic and n-hexanoic acids. 3. The rate of synthesis of penicillin improved when amides or N-acylglycines were used; α-aminobenzylpenicillin and phenoxymethylpenicillin were only synthesized when using these more energy-rich compounds. 4. Phenyl-acetylglycine was the best substrate for the synthesis of benzylpenicillin compared with other derivatives of phenylacetic acid. 5. The enzyme was specific for acyl-l-amino acids, benzylpenicillin being synthesized from phenylacetyl-l-α-aminophenylacetic acid but not from phenylacetyl-d-α-aminophenylacetic acid. 6. α-Phenoxyethylpenicillin was synthesized from 6-aminopenicillanic acid and α-phenoxypropionylthioglycollic acid non-enzymically, but the rate was faster in the presence of the enzyme. 7. The E. coli acylase catalysed the acylation of hydroxylamine by acids or amides to give hydroxamic acids, the phenylacetyl group being the most suitable acyl group. The enzyme also catalysed other acyl-group transfers. PMID:4982418

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 patterns of fatty acyl carnitines. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

Investigation of Resonant AC-DC Magnetic Field Effects.

DTIC Science & Technology

1987-07-31

phosphatidylethanolamine with smaller amounts of phosphatidylinosital and phosphatidic acid . The fluidity of the acyl chain region of these lipids at room temperature...fusion and lipid lateral separation in phosphatidylcholine- phosphatidic acid vesicles. Biochem 25:6978-6987. Liboff AR (1985): Cyclotron resonance in

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.

Central acylated ghrelin improves memory function and hippocampal AMPK activation and partly reverses the impairment of energy and glucose metabolism in rats infused with β-amyloid.

PubMed

Kang, Suna; Moon, Na Rang; Kim, Da Sol; Kim, Sung Hoon; Park, Sunmin

2015-09-01

Ghrelin is a gastric hormone released during the fasting state that targets the hypothalamus where it induces hunger; however, emerging evidence suggests it may also affect memory function. We examined the effect of central acylated-ghrelin and DES-acetylated ghrelin (native ghrelin) on memory function and glucose metabolism in an experimentally induced Alzheimer's disease (AD) rat model. AD rats were divided into 3 groups and Non-AD rats were used as a normal-control group. Each rat in the AD groups had intracerebroventricular (ICV) infusion of β-amyloid (25-35; 16.8nmol/day) into the lateral ventricle for 3 days, and then the pumps were changed to infuse either acylated-ghrelin (0.2nmol/h; AD-G), DES-acylated ghrelin (0.2nmol/h; AD-DES-G), or saline (control; AD-C) for 3 weeks. The Non-AD group had ICV infusion of β-amyloid (35-25) which does not deposit in the hippocampus. During the next 3 weeks memory function, food intake, body weight gain, body fat composition, and glucose metabolism were measured. AD-C exhibited greater β-amyloid deposition compared to Non-AD-C, and AD-G suppressed the increased β-amyloid deposition and potentiated the phosphorylation AMPK. In addition, AD-G increased the phosphorylation GSK and decreased the phosphorylation of Tau in comparison to AD-C and AD-DES-G. Cognitive function, measured by passive avoidance and water maze tests, was much lower in AD-C than Non-AD-C whereas AD-G but not AD-DES-G prevented the decrease (p<0.021). Body weight gain was lower in AD-C group than Non-AD-C group without changing epididymal fat mass. AD-G reversed the decrease in body weight which was due to increased energy intake and decreased energy expenditure. The AD-G group exhibited a decrease in the second part of serum glucose levels during an oral glucose tolerance test (OGTT) compared to the AD-C and AD-DES-G group (p<0.009). However, area under the curve of insulin during the first part of OGTT was higher in AD-DES-G than other groups, whereas during the second part it was suppressed in AD-G as much as Non-AD. In conclusion, central acylated ghrelin in rats prevented the deterioration of memory function, and energy and glucose metabolisms were partially improved, possibly due to less β-amyloid accumulation. This research suggests that interventions such as intermittent fasting to facilitate sustained elevations of acyl-ghrelin should be investigated for cognitive and metabolic benefits, especially in person with early symptoms of memory impairment. Copyright © 2015 Elsevier Inc. All rights reserved.

Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds.

PubMed

Banaś, Walentyna; Sanchez Garcia, Alicia; Banaś, Antoni; Stymne, Sten

2013-06-01

The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.

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...

Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae1[OPEN

PubMed Central

Uebler, Joseph B.; Liu, Xiaoxiao

2017-01-01

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato (Solanum lycopersicum), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3ʹ, and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petunia axillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris, tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris, PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3ʹ position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3ʹ position in tomato, is absent in P. axillaris. Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis. PMID:28701351

Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae.

PubMed

Nadakuduti, Satya Swathi; Uebler, Joseph B; Liu, Xiaoxiao; Jones, A Daniel; Barry, Cornelius S

2017-09-01

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato ( Solanum lycopersicum ), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3', and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petunia axillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris , tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris , PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3' position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3' position in tomato, is absent in P. axillaris Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis*

PubMed Central

Goblirsch, Brandon R.; Jensen, Matthew R.; Mohamed, Fatuma A.; Wackett, Lawrence P.; Wilmot, Carrie M.

2016-01-01

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety—unusual for a thiolase—are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation. PMID:27815501

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

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

Goblirsch, Brandon R.; Jensen, Matthew R.; Mohamed, Fatuma A.

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry aremore » precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety—unusual for a thiolase—are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.« less

Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques.

PubMed

He, Jingren; Santos-Buelga, Celestino; Mateus, Nuno; de Freitas, Victor

2006-11-17

A combination of column chromatography on Toyopearl gel HW-40 (S) and polyamide resin has been developed for the preparative isolation and further determination of pyranoanthocyanins of oligomeric nature formed after reaction between anthocyanins and different flavanols in a complex wine matrix. Polyamide chromatography was found to be exceptionally useful to separate oligomeric pyanoanthocyanins from other classes of wine flavonoids and polymerized pigments into an advanced state of purity for further identification and quantification by HPLC-diode array detector coupled with electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS). Fractionation on Toyopearl gel chromatography allowed the separation of pyranoanthocyanins bearing the same flavanols (catechin, epicatechin and procyanidin dimers) but with different anthocyanin moieties (either acylated or non-acylated in the glucose residue) in order to allow further isolation of individual oligomeric pigments on C18 chromatography. A quantitative procedure for analyzing the major pyranoanthocyanin-flavanol derivatives in different aged wines is proposed for the first time. Results obtained showed good reproducibility and recovery regarding sample pretreatment and quantitative method for all analyzed oligomeric pyranoanthocyanins. The combination of these two chromatographic separations is likely to be applicable to the preparative isolation of other anthocyanin-derived pigments.

Complex Mixture Analysis of Organic Compounds in Yogurt by NMR Spectroscopy

PubMed Central

Lu, Yi; Hu, Fangyu; Miyakawa, Takuya; Tanokura, Masaru

2016-01-01

NMR measurements do not require separation and chemical modification of samples and therefore rapidly and directly provide non-targeted information on chemical components in complex mixtures. In this study, one-dimensional (1H, 13C, and 31P) and two-dimensional (1H-13C and 1H-31P) NMR spectroscopy were conducted to analyze yogurt without any pretreatment. 1H, 13C, and 31P NMR signals were assigned to 10 types of compounds. The signals of α/β-lactose and α/β-galactose were separately observed in the 1H NMR spectra. In addition, the signals from the acyl chains of milk fats were also successfully identified but overlapped with many other signals. Quantitative difference spectra were obtained by subtracting the diffusion ordered spectroscopy (DOSY) spectra from the quantitative 1H NMR spectra. This method allowed us to eliminate interference on the overlaps; therefore, the correct intensities of signals overlapped with those from the acyl chains of milk fat could be determined directly without separation. Moreover, the 1H-31P HMBC spectra revealed for the first time that N-acetyl-d-glucosamine-1-phosphate is contained in yogurt. PMID:27322339

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Significance of appetite hormone ghrelin and obestatin levels in the assessment of the severity of acute pancreatitis.

PubMed

Kanat, Burhan Hakan; Ayten, Refik; Aydın, Süleyman; Girgin, Mustafa; Cetinkaya, Ziya; Ilhan, Yavuz Selim; Yur, Mesut; Catak, Zekiye

2014-06-01

Due to risk of morbidity and mortality, various tests and scoring systems used in the assessment of the diagnosis and severity of acute pancreatitis disease are gaining more importance every day. Most of the current scoring systems, validated by various parameters, have a sophisticated and complex structure. Research is ongoing to establish a method to diagnose the disease and determine the severity by using different and simple parameters. In this trial, we aimed to investigate the role of the orexigenic "ghrelin" and anorexigenic "obestatin" hormones, if any, on the diagnosis and assessment of the severity of acute pancreatitis. A total of 30 patients hospitalized between September 2009 and September 2010 with a diagnosis of acute pancreatitis (AP) and 25 healthy volunteers were enrolled in the trial with a prospective and randomized design. The patients were classified in two groups, mild (Ranson ≤3 and / or Apache II ≤8) and severe (Ranson >3 and/or Apache II >8) cases, as per the Ranson and Apache-II criteria; the ghrelin and obestatin levels in blood samples obtained from the patients were measured using the ELISA method. Twenty-two of the 30 patients (73%) were regarded as mild pancreatitis cases, while 8 cases (27%) were diagnosed as severe pancreatitis. Comparison of the mild and severe pancreatitis groups did not reveal a statistical difference between the two groups in terms of acylated and de-acylated ghrelin values on presentation and following the initiation of oral feeding. Similarly, no significant difference was found in the comparison of the patient and the control groups in terms of acylated and de-acylated ghrelin values on presentation (p=0.863). On the other hand, acylated and de-acylated ghrelin values after initiation of oral feeding were observed to be higher in the patient group (p=0.001, p=0.000). Comparison of these two groups revealed a significant difference in obestatin values, both on presentation and after initiation of oral feeding (p=0.002 and p=0.000). Consistently high serum ghrelin values during pancreatic inflammation suggest that ghrelin may be used as an adjunctive parameter in the monitoring of the course of the disease. On the other hand, high obestatin values in patients on presentation indicate that this hormone is a more significant parameter in terms of diagnosis. However, no correlation was established between these two peptide hormones and the severity of AP.

Two-Dimensional Nuclear Magnetic Resonance Structure Determination Module for Introductory Biochemistry: Synthesis and Structural Characterization of Lyso-Glycerophospholipids

ERIC Educational Resources Information Center

Garrett, Teresa A.; Rose, Rebecca L.; Bell, Sidney M.

2013-01-01

In this laboratory module, introductory biochemistry students are exposed to two-dimensional [superscript 1]H-nuclear magnetic resonance of glycerophospholipids (GPLs). Working in groups of three, students enzymatically synthesized and purified a variety of 2-acyl lyso GPLs. The structure of the 2-acyl lyso GPL was verified using [superscript…

Enhanced profiling of flavonol glycosides in the fruits of sea buckthorn (Hippophae rhamnoides).

PubMed

Fang, Rui; Veitch, Nigel C; Kite, Geoffrey C; Porter, Elaine A; Simmonds, Monique S J

2013-04-24

Use of enhanced LC-MS/MS methods to identify common glycosyl groups of flavonoid glycosides enabled better characterization of the flavonoids in fruits of sea buckthorn (Hippophae rhamnoides). The saccharide moieties of 48 flavonol O-glycosides detected in a methanol extract were identified by these methods. Several of the flavonol glycosides were acylated, two of which were isolated and found to be new compounds. Their structures were determined using spectroscopic and chemical methods as isorhamnetin 3-O-(6-O-E-sinapoyl-β-D-glucopyranosyl)-(1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside (24) and isorhamnetin 3-O-(6-O-E-feruloyl-β-D-glucopyranosyl)-(1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside (30). Analysis of the acylated glycosyl groups of 24 and 30 by serial mass spectrometry provided evidence to suggest the acylation position of 11 other minor flavonol glycosides acylated with hydroxycinnamic or hydroxybenzoic acids. The nitric oxide scavenging activities of 24 and 30 were compared with those of other flavonoids and with ascorbic acid and the potassium salt of 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO).

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.

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

PubMed Central

Post-Beittenmiller, M A; Schmid, K M; Ohlrogge, J B

1989-01-01

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis. PMID:2535529

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.

"Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis.

PubMed

Sato, Naoki; Awai, Koichiro

2017-11-01

Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called "prokaryotic pathway," which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not "prokaryotic" in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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.

Four-hour infusion of hydrocortisone does not suppress the nocturnal increase of circulating acyl- or desacyl-ghrelin concentrations in healthy young adults.

PubMed

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

2014-09-01

Ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin is found in the circulation in two forms: acyl- and desacyl-ghrelin. Acyl- and desacyl-ghrelin concentrations increase at night, when cortisol concentrations are low. Acute ghrelin administration increases ACTH and cortisol concentrations and a feedback loop between the ghrelin and ACTH-cortisol axis has been postulated. A previous study showed that exogenously induced hypercortisolism for 5 days decreased plasma ghrelin concentrations. The objective of the study was to determine whether a 4-hour infusion of hydrocortisone given at a time of low endogenous cortisol concentrations (11:00 pm to 3:00 am) acutely suppresses acyl- and desacyl-ghrelin. Eight healthy young men aged (mean ± SD) 21.5 ± 2.7 years with a body mass index of 22.4 ± 2.5 kg/m(2) were studied in a single-blind, placebo-controlled study during two separate overnight admissions on the Clinical Research Unit. The volunteers received either a 4-hour (11:00 pm to 3:00 am) infusion of hydrocortisone or a saline infusion. The hydrocortisone infusion rate was 0.3 mg/kg·h for the initial 3 minutes, 0.24 mg/kg·h for 9 minutes, and then 0.135 mg/kg·h until the end of the infusion. Plasma acyl- and desacyl-ghrelin concentrations (in-house two site sandwich assay) and ACTH, cortisol, insulin, GH, and glucose levels were measured every 10 minutes for 16 hours (5:00 pm to 9:00 am). The mean differences (lower 95% limit; upper 95% limit) between the saline infusion and hydrocortisone infusion for acyl- and desacyl-ghrelin concentrations were not significantly different from zero. The infusion period (11:00 pm to 3:00 am) was as follows: acyl-ghrelin, 0.22 (-7.39; 7.83) (P = 1.00); desacyl-ghrelin, -3.36 (-17.66; 10.95) (P = 1.00). The postinfusion period (3:00-7:00 am) was as follows: acyl-ghrelin, 8.68 (1.07; 16.28); (P = .056); desacyl-ghrelin, 8.75 (-5.56; 23.05) (P = .403). A short-term increase in circulating cortisol concentrations by exogenous hydrocortisone infusion does not suppress circulating nocturnal acyl- or desacyl-ghrelin concentrations. Thus, it is likely that the diurnal pattern of ghrelin secretion is under circadian control and not directly regulated by cortisol.

Four-Hour Infusion of Hydrocortisone Does Not Suppress the Nocturnal Increase of Circulating Acyl- or Desacyl-Ghrelin Concentrations in Healthy Young Adults

PubMed Central

Liu, Jianhua; Patrie, James; Pezzoli, Suzan S.; Farhy, Leon S.; Gaylinn, Bruce D.; Thorner, Michael O.

2014-01-01

Background: Ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin is found in the circulation in two forms: acyl- and desacyl-ghrelin. Acyl- and desacyl-ghrelin concentrations increase at night, when cortisol concentrations are low. Acute ghrelin administration increases ACTH and cortisol concentrations and a feedback loop between the ghrelin and ACTH-cortisol axis has been postulated. A previous study showed that exogenously induced hypercortisolism for 5 days decreased plasma ghrelin concentrations. Objective: The objective of the study was to determine whether a 4-hour infusion of hydrocortisone given at a time of low endogenous cortisol concentrations (11:00 pm to 3:00 am) acutely suppresses acyl- and desacyl-ghrelin. Methods: Eight healthy young men aged (mean ± SD) 21.5 ± 2.7 years with a body mass index of 22.4 ± 2.5 kg/m2 were studied in a single-blind, placebo-controlled study during two separate overnight admissions on the Clinical Research Unit. The volunteers received either a 4-hour (11:00 pm to 3:00 am) infusion of hydrocortisone or a saline infusion. The hydrocortisone infusion rate was 0.3 mg/kg·h for the initial 3 minutes, 0.24 mg/kg·h for 9 minutes, and then 0.135 mg/kg·h until the end of the infusion. Plasma acyl- and desacyl-ghrelin concentrations (in-house two site sandwich assay) and ACTH, cortisol, insulin, GH, and glucose levels were measured every 10 minutes for 16 hours (5:00 pm to 9:00 am). Results: The mean differences (lower 95% limit; upper 95% limit) between the saline infusion and hydrocortisone infusion for acyl- and desacyl-ghrelin concentrations were not significantly different from zero. The infusion period (11:00 pm to 3:00 am) was as follows: acyl-ghrelin, 0.22 (−7.39; 7.83) (P = 1.00); desacyl-ghrelin, −3.36 (−17.66; 10.95) (P = 1.00). The postinfusion period (3:00–7:00 am) was as follows: acyl-ghrelin, 8.68 (1.07; 16.28); (P = .056); desacyl-ghrelin, 8.75 (−5.56; 23.05) (P = .403). Conclusions: A short-term increase in circulating cortisol concentrations by exogenous hydrocortisone infusion does not suppress circulating nocturnal acyl- or desacyl-ghrelin concentrations. Thus, it is likely that the diurnal pattern of ghrelin secretion is under circadian control and not directly regulated by cortisol. PMID:24963646

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.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

PubMed

Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

2011-12-01

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

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 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.

Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.

PubMed

Kastaniotis, Alexander J; Autio, Kaija J; Kerätär, Juha M; Monteuuis, Geoffray; Mäkelä, Anne M; Nair, Remya R; Pietikäinen, Laura P; Shvetsova, Antonina; Chen, Zhijun; Hiltunen, J Kalervo

2017-01-01

Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both β-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. Copyright © 2016 Elsevier B.V. All rights reserved.

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 dynamic protein acylation in growth cone- mediated extension of neuronal processes. PMID:8106550

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.

Heteropoly acid encapsulated into zeolite imidazolate framework (ZIF-67) cage as an efficient heterogeneous catalyst for Friedel–Crafts acylation

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

Ammar, Muhammad; Jiang, Sai; Ji, Shengfu, E-mail: jisf@mail.buct.edu.cn

2016-01-15

A new strategy has been developed for the encapsulation of the phosphotungstic heteropoly acid (H{sub 3}PW{sub 12}O{sub 40} denoted as PTA) into zeolite imidazolate framework (ZIF-67) cage and the PTA@ZIF-67(ec) catalysts with different PTA content were prepared. The structure of the catalysts was characterized by XRD, BET, SEM, FT-IR, ICP-AES and TG. The catalytic activity and recovery properties of the catalysts for the Friedel-Crafts acylation of anisole with benzoyl chloride were evaluated. The results showed that 14.6–31.7 wt% PTA were encapsulated in the ZIF-67 cage. The PTA@ZIF-67(ec) catalysts had good catalytic activity for Friedel-Crafts acylation. The conversion of anisole canmore » reach ~100% and the selectivity of the production can reach ~94% over 26.5 wt% PTA@ZIF-67(ec) catalyst under the reaction condition of 120 °C and 6 h. After reaction, the catalyst can be easily separated from the reaction mixture by the centrifugation. The recovered catalyst can be reused five times and the selectivity can be kept over 90%. - Graphical abstract: The PTA@ZIF-67 catalysts with different PTA content were prepared by encapsulating the PTA into ZIF-67 cage and the as-synthesized catalysts exhibited good catalytic activity for the Friedel–Craft acylation of anisole with benzoyl chloride.« less

Crystal structures of the class D beta-lactamase OXA-13 in the native form and in complex with meropenem.

PubMed

Pernot, L; Frénois, F; Rybkine, T; L'Hermite, G; Petrella, S; Delettré, J; Jarlier, V; Collatz, E; Sougakoff, W

2001-07-20

The therapeutic problems posed by class D beta-lactamases, a family of serine enzymes that hydrolyse beta-lactam antibiotics following an acylation-deacylation mechanism, are increased by the very low level of sensitivity of these enzymes to beta-lactamase inhibitors. To gain structural and mechanistic insights to aid the design of new inhibitors, we have determined the crystal structure of OXA-13 from Pseudomonas aeruginosa in the apo form and in complex with the carbapenem meropenem. The native form consisted of a dimer displaying an overall organisation similar to that found in the closely related enzyme OXA-10. In the acyl-enzyme complex, the positioning of the antibiotic appeared to be ensured mainly by (i) the covalent acyl bond and (ii) a strong salt-bridge involving the carboxylate moiety of the drug. Comparison of the structures of OXA-13 in the apo form and in complex with meropenem revealed an unsuspected flexibility in the region of the essential serine 115 residue, with possible consequences for the catalytic properties of the enzyme. In the apo form, the Ser115 side-chain is oriented outside the active site, whereas the general base Lys70 adopts a conformation that seems to be incompatible with the activation of the catalytic water molecule required for the deacylation step. In the OXA-13:meropenem complex, a 3.5 A movement of the backbone of the 114-116 loop towards the side-chain of Lys70 was observed, which seems to be driven by a displacement of the neighbouring 91-104 loop and which results in the repositioning of the side-chain hydroxyl group of Ser115 toward the catalytic centre. Concomitantly, the side-chain of Lys70 is forced to curve in the direction of the deacylating water molecule, which is then strongly bound and activated by this residue. However, a distance of ca 5 A separates the catalytic water molecule from the acyl carbonyl group of meropenem, a structural feature that accounts for the inhibition of OXA-13 by this drug. Finally, the low level of penicillinase activity revealed by the kinetic analysis of OXA-13 could be related to the specific presence in position 73 of a serine residue located close to the general base Lys70, which results in a decrease of the number of hydrogen-bonding interactions stabilising the catalytic water molecule. Copyright 2001 Academic Press.

Environmentally friendly chitosan/PEI-grafted magnetic gelatin for the highly effective removal of heavy metals from drinking water

PubMed Central

Li, Bingbing; Zhou, Feng; Huang, Kai; Wang, Yipei; Mei, Surong; Zhou, Yikai; Jing, Tao

2017-01-01

The development of environmentally friendly sorbents with a high adsorption capacity is an essential problem in the removal of heavy metals from drinking water. In this study, magnetic gelatin was prepared using transglutaminase as a cross-linker, which could only catalyze an acyl-transfer reaction between lysine and glutamine residues of the gelatin and not affect other amino groups. Therefore, it was beneficial for the further modification based on the amino groups, and did not affect the spatial structure of gelatin, which can effectively prevent the embedding of active sites in the polymer matrix. After modification with the chitosan/polyethylenimine copolymers, the numbers of amino groups was greatly increased, and the magnetic composites exhibited a high adsorption capacity, excellent water compatibility and simple magnetic separation. The adsorption capacities of lead and cadmium were 341 mg g−1 and 321 mg g−1, respectively, which could be used for the removal of metal ions in drinking water. PMID:28225082

Environmentally friendly chitosan/PEI-grafted magnetic gelatin for the highly effective removal of heavy metals from drinking water

NASA Astrophysics Data System (ADS)

Li, Bingbing; Zhou, Feng; Huang, Kai; Wang, Yipei; Mei, Surong; Zhou, Yikai; Jing, Tao

2017-02-01

The development of environmentally friendly sorbents with a high adsorption capacity is an essential problem in the removal of heavy metals from drinking water. In this study, magnetic gelatin was prepared using transglutaminase as a cross-linker, which could only catalyze an acyl-transfer reaction between lysine and glutamine residues of the gelatin and not affect other amino groups. Therefore, it was beneficial for the further modification based on the amino groups, and did not affect the spatial structure of gelatin, which can effectively prevent the embedding of active sites in the polymer matrix. After modification with the chitosan/polyethylenimine copolymers, the numbers of amino groups was greatly increased, and the magnetic composites exhibited a high adsorption capacity, excellent water compatibility and simple magnetic separation. The adsorption capacities of lead and cadmium were 341 mg g-1 and 321 mg g-1, respectively, which could be used for the removal of metal ions in drinking water.

Correlation of Intermolecular Acyl Transfer Reactivity with Noncovalent Lattice Interactions in Molecular Crystals: Toward Prediction of Reactivity of Organic Molecules in the Solid State.

PubMed

Krishnaswamy, Shobhana; Shashidhar, Mysore S

2018-04-06

Intermolecular acyl transfer reactivity in several molecular crystals was studied, and the outcome of the reactivity was analyzed in the light of structural information obtained from the crystals of the reactants. Minor changes in the molecular structure resulted in significant variations in the noncovalent interactions and packing of molecules in the crystal lattice, which drastically affected the facility of the intermolecular acyl transfer reactivity in these crystals. Analysis of the reactivity vs crystal structure data revealed dependence of the reactivity on electrophile···nucleophile interactions and C-H···π interactions between the reacting molecules. The presence of these noncovalent interactions augmented the acyl transfer reactivity, while their absence hindered the reactivity of the molecules in the crystal. The validity of these correlations allows the prediction of intermolecular acyl transfer reactivity in crystals and co-crystals of unknown reactivity. This crystal structure-reactivity correlation parallels the molecular structure-reactivity correlation in solution-state reactions, widely accepted as organic functional group transformations, and sets the stage for the development of a similar approach for reactions in the solid state.

A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.

PubMed

Tilvawala, Ronak; Cammarata, Michael; Adediran, S A; Brodbelt, Jennifer S; Pratt, R F

2015-12-22

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

Associations of serum leptin, ghrelin and peptide YY levels with physical activity and cardiorespiratory fitness in adolescent boys with different BMI values

PubMed Central

Tillmann, Vallo; Purge, Priit; Lätt, Evelin; Jürimäe, Jaak

2017-01-01

The aim of this study was to investigate the differences in associations of serum acylated and des-acylated ghrelin, peptide YY (PYY) and leptin levels with physical activity (PA) and cardiorespiratory fitness (CReF) in adolescent boys (mean age of 14.0 years) with overweight (OWB; n=55) and with normal weight (NWB; n=154). Methods: Total PA was measured by 7-day accelerometry (counts/min) and CReF by peak oxygen consumption (VO2peak/kg). Results: No differences were found in serum PYY, acylated ghrelin or des-acyl ghrelin levels, whereas mean leptin (11.6±10.6 vs. 2.0±2.7 ng/ml; p<0.05) and insulin (18.1±8.7 vs. 11.0±6.2 mU/l; p<0.05) levels were significantly higher in OWB compared to NWB. Mean CReF was significantly lower in OWB compared to NWB (39.7±8.7 vs. 50.5±6.8 ml/min/kg; p<0.05). Leptin was negatively correlated with CReF in both groups (r=-0.43; p<0.05), des-acylated ghrelin with CReF only in OWB (r =-0.36; p<0.05). In OWB leptin was negatively correlated with total PA (r=-0.32; p<0.05) and positively with sedentary time of PA (r=0.35; p<0.05). In NWB 28.1% of the variability of CReF was determined by leptin and insulin resistance index (HOMA-IR), whereas in OWB 71.9% was determined by trunk FM and BMI. Conclusions: Leptin concentration was inversely associated with CReF in adolescent boys independently of BMI in both groups, while des-acylated ghrelin was associated with CReF only in OWB. Low PA in OWB was associated with high serum leptin level. PMID:29472737

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.

PubMed

Goblirsch, Brandon R; Jensen, Matthew R; Mohamed, Fatuma A; Wackett, Lawrence P; Wilmot, Carrie M

2016-12-23

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys 143 ) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C 12 and C 14 ) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ 117 ) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Saliva/serum ghrelin, obestatin and homocysteine levels in patients with ischaemic heart disease

PubMed Central

Kilic, Nermin; Dagli, Necati; Aydin, Suleyman; Erman, Fazilet; Bek, Yuksel; Akin, Okhan; Kilic, SS; Erdemli, Haci Kemal; Alacam, Hasan

2017-01-01

Summary Background: We aimed to compare ghrelin, obestatin, homocysteine (Hcy), vitamin B12 and folate levels in the serum and saliva of ischaemic heart disease patients. Methods: Serum and saliva were collected from 33 ischaemic heart disease (IHD) patients and 28 age- and body mass index-matched healthy individuals. Levels of acylated and desacylated ghrelin, obestatin and Hcy were determined using the ELISA method. Results: Acylated ghrelin, desacylated ghrelin and obestatin levels in the saliva were found to be higher than those in the serum of the control group, while acylated and desacylated ghrelin levels in the saliva were significantly lower than those in the serum. Obestatin levels were higher in IHD patients (p = 0.001). Saliva and serum vitamin B12 and folate levels in IHD patients were significantly lower than in the control group (p = 0.001). Conclusions: It was determined that serum ghrelin levels increased in ischaemic heart disease patients, while serum levels of obestatin decreased. PMID:28759087

Disparate effects of oxidation on plasma acyltransferase activities: inhibition of cholesterol esterification but stimulation of transesterification of oxidized phospholipids.

PubMed

Subbaiah, P V; Liu, M

1996-05-31

Oxidation of lipoproteins results in the formation of several polar phospholipids with pro-inflammatory and pro-atherogenic properties. To examine the possible role of lecithin/cholesterol acyltransferase (LCAT) in the metabolism of these oxidized phospholipids, we oxidized whole plasma with either Cu(2+) or a free-radical generator, and determined the various activities of LCAT. Oxidation caused a reduction in plasma phosphatidylcholine (PC), an increase in a short-chain polar PC (SCP-PC), and an inhibition of the transfer of long-chain acyl groups to cholesterol (LCAT activity) or to lyso PC (lysolecithin acyltransferase (LAT) I activity). However, the transfer of short-chain acyl groups from SCP-PC to lyso PCLAT II activity) was stimulated several fold, in direct correlation with the degree of oxidation. LAT II activity was not stimulated by oxidation in LCAT-deficient plasma, showing that it is carried out by LCAT. Oxidized normal plasma exhibited low LCAT activity even in the presence of exogenous proteoliposome substrate, indicating that the depletion of substrate PC was not responsible for the loss of activity. Oxidation of isolated LDL or HDL abolished their ability to support LCAT and LAT I activities of exogenous enzyme, but promoted the LAT II activity. Purified LCAT lost its LCAT and LAT I functions, but not its LAT II function, when oxidized in vitro. These results show that while oxidation of plasma causes a loss of LCAT's ability to transfer long-chain acyl groups, its ability to transfer short-chain acyl groups, from SCP-PC is retained, and even stimulated, suggesting that LCAT may have a physiological role in the metabolism of oxidized PC in plasma.

A photoreactive derivative of radiolabeled GM1 ganglioside: Preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

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

Sonnino, S.; Chigorno, V.; Acquotti, D.

1989-01-10

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with ({sup 3}H)acetic anhydride of very high specific radioactivity. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 for different timesmore » and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2 h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins. When the incubation was prolonged to 24 h, photolabeled GM1 underwent extensive metabolic processing and gave origins to the corresponding ganglioside derivatives of GM2, GM3, and GD1a. Under these conditions many proteins became radioactive, a consequence of GM1 transfer from the surface to the interior or the cell and of the ready availability of interaction of GM1 and its metabolites.« less

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.

Recent advances in lipid separations and structural elucidation using mass spectrometry combined with ion mobility spectrometry, ion-molecule reactions and fragmentation approaches

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

Zheng, Xueyun; Smith, Richard D.; Baker, Erin S.

Lipids are a vital class of molecules that play important and varied roles in biological processes. Fully understanding lipid roles, however, is extremely difficult since the number and diversity of lipid species is immense, with cells expressing hundreds of enzymes that synthesize tens of thousands of different lipids. While recent advances in chromatography and high resolution mass spectrometry have greatly progressed the understanding of lipid species and functions, effectively separating many lipids still remains problematic. Isomeric lipids have made lipid characterization especially difficult and occur due to subclasses having the same chemical composition, or species having multiple acyl chains connectivitiesmore » (sn-1, sn-2, or sn-3), double bond positions and orientations (cis or trans), and functional group stereochemistry (R versus S). Fully understanding the roles of lipids in biological processes therefore requires separating and evaluating how isomers change in biological and environmental samples. To address this challenge, ion mobility spectrometry separations, ion-molecule reactions and fragmentation techniques have increasingly been added to lipid analysis workflows to improve identifications. In this manuscript, we review the current state of these approaches and their capabilities for improving the identification of specific lipid species.« less

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

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.

Chemoenzymatic synthesis of C-4'-spiro-oxetanoribonucleosides.

PubMed

Sharma, Vivek K; Kumar, Manish; Sharma, Deepti; Olsen, Carl E; Prasad, Ashok K

2014-09-19

Novozyme-435-mediated diastereoselective deacylation of one of the two diastereotopic acyloxymethyl groups in 5-O-acyl-4-C-acyloxymethyl-3-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose has been achieved in quantitative yield. The exclusive selectivity of the lipase for the 5-O-acyl over the 4-C-acyloxymethyl group in the substrate was confirmed by chemical transformation of enzymatically monodeacetylated compound to 1,2-O-isopropylidene-C-4-spiro-oxetanoribofuranose. Further, the selective biocatalytic deacylation methodology has been utilized for the efficient synthesis of C-4'-spiro-oxetanoribonucleosides of uracil (U) and thymine (T) in 37 and 45% overall yields, respectively.

Effect of Fatty Acyl Group and Sterol Composition on Sensitivity of Lecithin Liposomes to Imidazole Antimycotics

PubMed Central

Yamaguchi, Hideyo; Iwata, Kazuo

1979-01-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes. PMID:525988

Effect of fatty acyl group and sterol composition on sensitivity of lecithin liposomes to imidazole antimycotics.

PubMed

Yamaguchi, H; Iwata, K

1979-05-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes.

Regulation of lecithin-cholesterol acyltransferase reaction by acyl acceptors and demonstration of its "idling" reaction.

PubMed

Czarnecka, H; Yokoyama, S

1993-09-15

The mechanism for regulation of cholesterol esterification by lecithin-cholesterol acyltransferase (LCAT) was studied using the highly isolated enzyme from pig plasma. In the reaction with phosphatidylcholine small unilamellar vesicles, cholesterol, water, diacylglycerol, and lysophosphatidylcholine were all potent acceptors of an acyl group cleaved from the sn-2 position of egg phosphatidylcholine, generating cholesteryl ester, free fatty acid, triglyceride, and phosphatidylcholine, respectively. All of these reactions required activation by human apolipoprotein A-I, suggesting that this activation leads to the deacylation of phosphatidylcholine. Those acceptors competed against each other in this vesicle reaction system, and cholesterol was the most potent acyl acceptor. Lysophosphatidylcholine that was endogenously generated by deacylation of phosphatidylcholine in the first step of the LCAT reaction was also a good acyl acceptor, showing that the reaction is always partly "idling." Bovine serum albumin partially inhibited this idling reaction in a concentration-dependent manner up to 80% at 0.60 mM. The above results were essentially reproducible with high density lipoprotein, except that cholesterol is less potent than lysophosphatidylcholine in accepting the acyl group under the condition used. Unlike the apolipoprotein A-I-activated reaction, cholesterol was esterified only slightly by the LCAT reaction on low density lipoprotein and, consequently, did not compete against lysophosphatidylcholine for generation of phosphatidylcholine. Thus, apoB may activate LCAT in a very different manner from apoA-I. The rate of esterification of lysophosphatidylcholine on low density lipoprotein was one-tenth of that on the vesicles and on high density lipoprotein. Thus, LCAT is active on low density lipoprotein but mostly idling as deacylating and reacylating glycerophospholipids.

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.

Mucinous (colloid) adenocarcinomas secrete distinct O-acylated forms of sialomucins: a histochemical study of gastric, colorectal and breast adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Poveda, M A; Segura, D I

2001-12-01

Mucinous (colloid) adenocarcinomas represent a distinct group of tumours defined by the presence of large amounts of extracellular mucins. By using histochemical methods, we analysed mucins secreted by mucinous versus non-mucinous adenocarcinomas and looked for differential secretion profiles. Sixty-four adenocarcinomas were studied (23 colorectal, 17 gastric, and 24 breast tumours). Thirty-two tumours were of the colloid type. The following methods were applied to paraffin tissue sections: (i) Alcian blue (pH 2.5) and periodic acid-Schiff (PAS); (ii) high iron diamine and Alcian blue (pH 2.5); (iii) periodic acid borohydride, potassium hydroxide, and PAS; (iv) periodic acid-thionine Schiff, potassium hydroxide, and PAS; and (v) periodic acid-borohydride and PAS. Most adenocarcinomas secreted acidic mucins, with sialomucins predominating over sulfomucins, except for non-mucinous adenocarcinomas of the breast which showed predominant neutral mucins. All mucinous adenocarcinomas contained C9-O-acyl sialic acid as mono, di(C8,C9)-, or tri(C7,C8,C9)-O-acyl forms. Acidic mucins secreted by the majority of non-colloid adenocarcinomas consisted of non-O-acylated sialomucins. C9-O-acylation of sialic acid is a characteristic feature of mucinous adenocarcinomas and can be readily detected by histochemical methods.

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 transfers during acylation are also given.« less

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

Acyl guanidine inhibitors of β-secretase (BACE-1): optimization of a micromolar hit to a nanomolar lead via iterative solid- and solution-phase library synthesis.

PubMed

Gerritz, Samuel W; Zhai, Weixu; Shi, Shuhao; Zhu, Shirong; Toyn, Jeremy H; Meredith, Jere E; Iben, Lawrence G; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Tebben, Andrew J; Muckelbauer, Jodi K; Camac, Daniel M; Metzler, William; Cook, Lynda S; Padmanabha, Ramesh; Lentz, Kimberley A; Sofia, Michael J; Poss, Michael A; Macor, John E; Thompson, Lorin A

2012-11-08

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.

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.

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

PubMed

Horrevoets, A J; Hackeng, T M; Verheij, H M; Dijkman, R; de Haas, G H

1989-02-07

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.

Exploration and Pharmacokinetic Profiling of Phenylalanine Based Carbamates as Novel Substance P 1–7 Analogues

PubMed Central

2014-01-01

The bioactive metabolite of Substance P, the heptapeptide SP1–7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1–7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1–7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1–7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide. PMID:25516784

Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

PubMed

Fransson, Rebecca; Nordvall, Gunnar; Bylund, Johan; Carlsson-Jonsson, Anna; Kratz, Jadel M; Svensson, Richard; Artursson, Per; Hallberg, Mathias; Sandström, Anja

2014-12-11

The bioactive metabolite of Substance P, the heptapeptide SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1-7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1-7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1-7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide.

Chemoselective O-acylation of hydroxyamino acids and amino alcohols under acidic reaction conditions: History, scope and applications

PubMed Central

2015-01-01

Summary Amino acids, whether natural, semisynthetic or synthetic, are among the most important and useful chiral building blocks available for organic chemical synthesis. In principle, they can function as inexpensive, chiral and densely functionalized starting materials. On the other hand, the use of amino acid starting materials routinely necessitates protective group chemistry, and in reality, large-scale preparations of even the simplest side-chain derivatives of many amino acids often become annoyingly strenuous due to the necessity of employing protecting groups, on one or more of the amino acid functionalities, during the synthetic sequence. However, in the case of hydroxyamino acids such as hydroxyproline, serine, threonine, tyrosine and 3,4-dihydroxyphenylalanine (DOPA), many O-acyl side-chain derivatives are directly accessible via a particularly expedient and scalable method not commonly applied until recently. Direct acylation of unprotected hydroxyamino acids with acyl halides or carboxylic anhydrides under appropriately acidic reaction conditions renders possible chemoselective O-acylation, furnishing the corresponding side-chain esters directly, on multigram-scale, in a single step, and without chromatographic purification. Assuming a certain degree of stability under acidic reaction conditions, the method is also applicable for a number of related compounds, such as various amino alcohols and the thiol-functional amino acid cysteine. While the basic methodology underlying this approach has been known for decades, it has evolved through recent developments connected to amino acid-derived chiral organocatalysts to become a more widely recognized procedure for large-scale preparation of many useful side-chain derivatives of hydroxyamino acids and related compounds. Such derivatives are useful in peptide chemistry and drug development, as amino acid amphiphiles for asymmetric catalysis, and as amino acid acrylic precursors for preparation of catalytically active macromolecular networks in the form of soluble polymers, crosslinked polymer beads or nanoparticulate systems. The objective of the present review is to increase awareness of the existence and convenience of this methodology, assess its competitiveness compared to newer and more elaborate procedures for chemoselective O-acylation reactions, spur its further development, and finally to chronicle the informative, but poorly documented history of its development. PMID:25977719

Enantioselective rhodium/ruthenium photoredox catalysis en route to chiral 1,2-aminoalcohols.

PubMed

Ma, Jiajia; Harms, Klaus; Meggers, Eric

2016-08-09

A rhodium-based chiral Lewis acid catalyst combined with [Ru(bpy)3](PF6)2 as a photoredox sensitizer allows for the visible-light-activated redox coupling of α-silylamines with 2-acyl imidazoles to afford, after desilylation, 1,2-amino-alcohols in yields of 69-88% and with high enantioselectivity (54-99% ee). The reaction is proposed to proceed via an electron exchange between the α-silylamine (electron donor) and the rhodium-chelated 2-acyl imidazole (electron acceptor), followed by a stereocontrolled radical-radical reaction. Substrate scope and control experiments reveal that the trimethylsilyl group plays a crucial role in this reductive umpolung of the carbonyl group.

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.

Analysis of acylcarnitines as their N-demethylated ester derivatives by gas chromatography-chemical ionization mass spectrometry.

PubMed

Huang, Z H; Gage, D A; Bieber, L L; Sweeley, C C

1991-11-15

A novel approach to the analysis of acylcarnitines has been developed. It involves a direct esterification using propyl chloroformate in aqueous propanol followed by ion-pair extraction with potassium iodide into chloroform and subsequent on-column N-demethylation of the resulting acylcarnitine propyl ester iodides. The products, acyl N-demethylcarnitine propyl esters, are volatile and are easily analyzed by gas chromatography-chemical ionization mass spectrometry. For medium-chain-length (C4-C12) acylcarnitine standards, detection limits are demonstrated to be well below 1 ng starting material using selected ion monitoring. Well-separated gas chromatographic peaks and structure-specific mass spectra are obtained with samples of synthetic and biological origin. Seven acylcarnitines have been characterized in the urine of a patient suffering from medium-chain acyl-CoA dehydrogenase deficiency.

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

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.

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil1[OPEN

PubMed Central

Stymne, Sten

2017-01-01

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C. sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine. PMID:28235891

Interactions of acylated methylglucoside derivatives with CO2: simulation and calculations.

PubMed

Chang, H H; Cao, R X; Yang, C C; Wei, W L; Pang, X Y; Qiao, Y

2016-01-01

Carbohydrates have drawn considerable interest from researchers recently due to their affinity for CO2. However, most of the research in this field has focused on peracetylated derivatives. Compared with acetylated carbohydrates, which have already been studied in depth, methyl D-glucopyranoside derivatives are more stable and could have additional applications. Thus, in the present work, ab initio calculations were performed to elucidate the characteristics of the interactions of methylglucoside derivatives with CO2, and to investigate how the binding energy (ΔE) is affected by isomerization or the introduction of various acyl groups. Four methyl D-glucopyranosides (each with two anomers) bearing acetyl, propionyl, butyryl, and isobutyryl moieties, respectively, were designed as substrates, and the 1:1 complexes of a CO2 molecule with each of these sugar substrates were modeled. The results indicate that ΔE is mainly influenced by interaction distance and the number of negatively charged donors or interacting pairs in the complex; the structure of the acyl group present in the substrate is a secondary influence. Except in the case of methyl 2-O-acetyl-D-glucopyranose, the ΔE values of the α- and β-anomers of each methylglucoside were found to be almost the same. Therefore, we would expect the CO2 affinities of the four derivatives studied here to be as strong as or even stronger than that of peracetylated D-glucopyranose. Graphical Abstract The binding energy between methyl D-glucopyranoside derivatives with various substituted acyl groups and CO2 are evaluated by ab initio calculations. The strong interaction between these methyl dglucopyranoside derivatives and CO2 showed the potential of their application for CO2 capture.

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

PubMed

Bafor, M; Jonsson, L; Stobart, A K; Stymne, S

1990-11-15

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

PubMed Central

Bafor, M; Jonsson, L; Stobart, A K; Stymne, S

1990-01-01

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids. PMID:2264835

Comparison of the therapeutic effects of sildenafil citrate, heparin and neuropeptides in a rat model of acetic acid-induced gastric ulcer.

PubMed

Kalayci, Mehmet; Kocdor, Mehmet Ali; Kuloglu, Tuncay; Sahin, İbrahim; Sarac, Mehmet; Aksoy, Aziz; Yardim, Meltem; Dalkilic, Semih; Gursu, Onur; Aydin, Suna; Akkoc, Ramazan Fazil; Ugras, Meltem; Artas, Gokhan; Ozercan, İbrahim Hanifi; Ugur, Kader; Aydin, Suleyman

2017-10-01

The purpose of our investigative work has been to determine whether there can be therapeutic roles in the administration of sildenafil citrate, heparin and several neuropeptides on an animal model where gastric ulcers were induced with acetic acid, and to compare their efficacy. The animals were divided into 13 groups, with 4 animals in each. Gastric ulcers was induced in the animals of 12 groups with one untreated group being left as the control (Group I - control; given normal saline (NS)). The other groups were: Group II (ulcer+NS); Group III (5mg/kg sildenafil citrate, low dose); Group IV (10mg/kg sildenafil citrate, high dose); Group V (0.6mg/kg heparin, low dose); Group VI (6mg/kg heparin, high dose); Group VII (20nmol/kg des-acyl ghrelin); Group VIII (40nmol/kg des-acyl ghrelin); Group IX (4nmol/kg acyl ghrelin); Group X (8nmol/kg acly ghrelin); Group XI (20pmol/kg Nesfatin-1); Group XII (15nmol/kg Obestatin) and Group XIII (5nmol/kg Neuropeptide Y). Gastric neuropeptide expression was measured using an immunohistochemical method, and the amount in circulation was detected using ELISA. To compare with no treatment, the controls and other treatment groups, we recorded loss of the surface epithelium of the stomach, erosion, bleeding and inflammatory cell infiltration in the upper halves of the gastric glands. The muscularis and the layers beneath it were, however, apparently normal. The gastric mucosa healed with little or no inflammation when sildenafil citrate, low dose heparin, ghrelin, NUCB2/Nesfatin-1, obestatin, Neuropeptide Y were administered. Overall the data indicate that low dose heparin, and especially sildenafil citrate and neuropeptides, can be used clinically as an alternative approach in the treatment of the gastric ulcer. Copyright © 2017 Elsevier Inc. 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 were produced under mild conditions, in high enantiomeric purity, and in practically quantitative yields.

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 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.

Relationship between adiponectin levels, acylated ghrelin levels, and short-term body mass index changes in children with diabetes mellitus type 1 at diagnosis and after insulin therapy.

PubMed

Martos-Moreno, Gabriel A; Barrios, Vicente; Soriano-Guillén, Leandro; Argente, Jesús

2006-11-01

To determine the effect of the initial metabolic imbalance and its restoration after insulin therapy on adiponectin and acylated ghrelin levels in children with type 1 diabetes mellitus (T1DM). Twenty prepubertal children with newly diagnosed T1DM were prospectively studied at diagnosis and after 1 and 4 months of therapy. Body mass index (BMI) and serum levels of adiponectin, resistin, total and acylated ghrelin, leptin, tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) were determined. The control group comprised 40 healthy prepubertal children. BMI was decreased at diagnosis, normalized at 1 month, and remained so thereafter. Adiponectin levels at diagnosis were similar to controls, increasing significantly after 1 month and normalizing at 4 months. Acylated ghrelin levels were lower at diagnosis, with a significant increase at 1 month and normalizing at 4 months. Resistin levels were normal at all time points. Leptin levels were decreased, while TNF-alpha and IL-6 were increased at diagnosis and normalized at 1 month. These findings suggest that BMI is not the main predictor of acylated ghrelin or adiponectin levels in newly diagnosed T1DM subjects and that these peptides may play an important role in the metabolic adaptation in this disease.

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

Stabilizing function for myristoyl group revealed by the crystal structure of a neuronal calcium sensor, guanylate cyclase-activating protein 1.

PubMed

Stephen, Ricardo; Bereta, Grzegorz; Golczak, Marcin; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2007-11-01

Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins myristoylated at the N terminus that regulate guanylate cyclases in photoreceptor cells and belong to the family of neuronal calcium sensors (NCS). Many NCS proteins display a recoverin-like "calcium-myristoyl switch" whereby the myristoyl group, buried inside the protein in the Ca(2+)-free state, becomes fully exposed upon Ca(2+) binding. Here we present a 2.0 A resolution crystal structure of myristoylated GCAP1 with Ca(2+) bound. The acyl group is buried inside Ca(2+)-bound GCAP1. This is in sharp contrast to Ca(2+)-bound recoverin, where the myristoyl group is solvent exposed. Furthermore, we provide direct evidence that the acyl group in GCAP1 remains buried in the Ca(2+)-free state and does not undergo switching. A pronounced kink in the C-terminal helix and the presence of the myristoyl group allow clustering of sequence elements crucial for GCAP1 activity.

Stabilizing Function for Myristoyl Group Revealed by the Crystal Structure of a Neuronal Calcium Sensor, Guanylate Cyclase-Activating Protein 1

PubMed Central

Stephen, Ricardo; Bereta, Grzegorz; Golczak, Marcin; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2008-01-01

SUMMARY Guanylate cyclase-activating proteins (GCAPs) are Ca2+-binding proteins myristoylated at the N terminus that regulate guanylate cyclases in photoreceptor cells and belong to the family of neuronal calcium sensors (NCS). Many NCS proteins display a recoverin-like “calcium-myristoyl switch” whereby the myristoyl group, buried inside the protein in the Ca2+-free state, becomes fully exposed upon Ca2+ binding. Here we present a 2.0 Å resolution crystal structure of myristoylated GCAP1 with Ca2+ bound. The acyl group is buried inside Ca2+-bound GCAP1. This is in sharp contrast to Ca2+-bound recoverin, where the myristoyl group is solvent exposed. Furthermore, we provide direct evidence that the acyl group in GCAP1 remains buried in the Ca2+-free state and does not undergo switching. A pronounced kink in the C-terminal helix and the presence of the myristoyl group allow clustering of sequence elements crucial for GCAP1 activity. PMID:17997965

The role of negatively charged lipids in lysosomal phospholipase A2 function

PubMed Central

Abe, Akira; Shayman, James A.

2009-01-01

Lysosomal phospholipase A2 (LPLA2) is characterized by increased activity toward zwitterionic phospholipid liposomes containing negatively charged lipids under acidic conditions. The effect of anionic lipids on LPLA2 activity was investigated. Mouse LPLA2 activity was assayed as C2-ceramide transacylation. Sulfatide incorporated into liposomes enhanced LPLA2 activity under acidic conditions and was weakened by NaCl or increased pH. Amiodarone, a cationic amphiphilic drug, reduced LPLA2 activity. LPLA2 exhibited esterase activity when p-nitro-phenylbutyrate (pNPB) was used as a substrate. Unlike the phospholipase A2 activity, the esterase activity was detected over wide pH range and not inhibited by NaCl or amiodarone. Presteady-state kinetics using pNPB were consistent with the formation of an acyl-enzyme intermediate. C2-ceramide was an acceptor for the acyl group of the acyl-enzyme but was not available as the acyl group acceptor when dispersed in liposomes containing amiodarone. Cosedimentation of LPLA2 with liposomes was enhanced in the presence of sulfatide and was reduced by raising NaCl, amiodarone, or pH in the reaction mixture. LPLA2 adsorption to negatively charged lipid membrane surfaces through an electrostatic attraction, therefore, enhances LPLA2 enzyme activity toward insoluble substrates. Thus, anionic lipids present within lipid membranes enhance the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces.—Abe, A., and J. A. Shayman. The role of negatively charged lipids in lysosomal phospholipase A2 function. PMID:19321879

Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry

NASA Astrophysics Data System (ADS)

Barrientos, Rodell C.; Vu, Ngoc; Zhang, Qibin

2017-08-01

Glycosphingolipids are essential biomolecules widely distributed across biological kingdoms yet remain relatively underexplored owing to both compositional and structural complexity. While the glycan head group has been the subject of most studies, there is paucity of reports on the lipid moiety, particularly the location of unsaturation. In this paper, ozone-induced dissociation mass spectrometry (OzID-MS) implemented in a traveling wave-based quadrupole time-of-flight (Q-ToF) mass spectrometer was applied to study unsaturated glycosphingolipids using shotgun approach. Resulting high resolution mass spectra facilitated the unambiguous identification of diagnostic OzID product ions. Using [M+Na]+ adducts of authentic standards, we observed that the long chain base and fatty acyl unsaturation had distinct reactivity with ozone. The reactivity of unsaturation in the fatty acyl chain was about 8-fold higher than that in the long chain base, which enables their straightforward differentiation. Influence of the head group, fatty acyl hydroxylation, and length of fatty acyl chain on the oxidative cleavage of double bonds was also observed. Application of this technique to bovine brain galactocerebrosides revealed co-isolated isobaric and regioisomeric species, which otherwise would be incompletely identified using contemporary collision-induced dissociation (CID) alone. These results highlight the potential of OzID-MS in glycosphingolipids research, which not only provides complementary structural information to existing CID technique but also facilitates de novo structural determination of these complex biomolecules. [Figure not available: see fulltext.

Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

PubMed Central

Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

2013-01-01

Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

PubMed Central

Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

2008-01-01

While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

The chemical nature of the products obtained by the action of cabbage-leaf phospholipase D on lysolecithin: the structure of lysolecithin

PubMed Central

Long, C.; Odavić, R.; Sargent, Elizabeth J.

1967-01-01

1. Lysolecithin, prepared by the action of snake-venom phospholipase A on ovolecithin, when incubated with Savoy-cabbage phospholipase D, in the presence of Ca2+ ions, gave two degradation products (designated A and B) in the form of their calcium salts. 2. These calcium salts were separated quantitatively by solvent fractionation and converted into the corresponding sodium salts. 3. Substance B proved to be a lysophosphatidic acid of conventional structure (1-monoacyl-l-3-glycerophosphoric acid). When the phosphate group was removed by means of prostatic acid phosphomonoesterase, a 1-monoglyceride was formed quantitatively. Alkaline hydrolysis gave the theoretical yield of l-3-glycerophosphate. 4. Substance A, on the other hand, had all the properties expected for a cyclic phosphate of a 1-monoglyceride. It was unaffected by phosphomonoesterase. On alkaline hydrolysis, the acyl group was removed and ring opening of the presumed cyclic phosphate group gave an approximately equimolar mixture of 2- and l-3-glycerophosphates. 5. The structures of substances A and B confirm lysolecithin as 1-monoacyl-l-3-glycerylphosphorylcholine. PMID:4291559

Detailed lipid analysis of yolk platelets of amphibian (Bufo arenarum) oocytes.

PubMed

Buschiazzo, Jorgelina; Bruzzone, Ariana; Alonso, Telma Susana

2003-06-01

Yolk platelets, the principal components of amphibian oocytes, have been generally considered as material reservoirs. Their biochemical composition and function during oogenesis and early development have not been fully elucidated. The aim of this study was to carry out a lipidic characterization of yolk platelets from full-grown Bufo arenarum oocytes. Ovarian oocytes were manually obtained and the subcellular fraction was isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derived by methanolysis, being identified and quantified in a gas-liquid chromatograph. Phospholipid content indicates that phosphatidylcholine and phosphatidylethanolamine are the main phospholipids followed by phosphatidylinositol, sphingomyelin, phosphatidylserine, and phosphatidic acid. Phospholipidic profile is similar to that in whole oocytes except for the absence of diphosphatidylglycerol in yolk platelets. Oleic, palmitic, and linoleic acids are the main fatty acids in phosphatidylcholine, and oleic acid is the principal one in phosphatidylethanolamine. In phosphatidic acid, palmitic, estearic, palmitoleic, and oleic acids represent 68 mol% of the total acyl groups. Phosphatidylinositol, enriched in arachidonic acid, is the most unsaturated phospholipid while sphingomyelin shows the lowest unsaturation index. The acyl group distribution in triacylglycerols is similar when yolk platelets and whole oocytes are compared. Polar and neutral lipids of yolk platelets determine the lipidic profile of the whole oocyte. The presence of unusual fatty acids as 14:0, 15:0, 15:1, 17:0, and 17:1 in phospholipids and triacylglycerols may indicate an oxidation mechanism different from beta-oxidation in yolk platelets and/or a structural and functional relation with mitochondria. Given that yolk platelets in amphibian oocytes may act in a dynamic fashion in development, their role should be reconsidered.

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

DOE PAGES

Bansal, Sunil; Durrett, Timothy P.

2016-11-08

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

PubMed Central

Bansal, Sunil; Durrett, Timothy P.

2016-01-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

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

Bansal, Sunil; Durrett, Timothy P.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

Hyperleptinemia independent of body adiposity in women with fibromyalgia.

PubMed

Homann, Diogo; Carvalho, Humberto Moreira; Stefanello, Joice Mara Facco; Góes, Suelen Meira; Lopes, André Luiz; de Oliveira, Alvaro Reischak; Leite, Neiva

2014-11-01

The prevalence of overweight and obesity in patients with fibromyalgia is high, which makes these patients more likely to trigger metabolic changes. It is also uncertain whether the clinical manifestations of fibromyalgia alter the metabolism in these patients. This study investigates the influence of adiposity indicators and presence of fibromyalgia on leptin and acylated ghrelin levels, which are hormones responsible for controlling energy homeostasis. Seventeen women with fibromyalgia (patients) and fifteen healthy women (controls) were evaluated. Pain intensity, physical activity level characteristics and leptin and acylated ghrelin levels were assessed. General linear models, using a main-effects model, were used to test the effect of fibromyalgia (patients vs. controls) on the relationship of leptin and acylated ghrelin with anthropometric indicators [body mass index, waist circumference (WC) and WC by height]. Patients showed higher leptin levels (controls: 9.1 ± 6.7 vs. patients: 22.4 ± 10.6 ng/mL; p < 0.01) and lower acylated ghrelin levels (controls: 188.7 ± 103.4 vs. patients: 126.7 ± 47.8 pg/mL; p = 0.04). The anthropometric variables, entered into linear models as independent variables, significantly influenced both leptin and acylated ghrelin levels (p < 0.01). The explained variance (R(2)) of the models containing leptin was higher (R(2) = 0.52-0.61) compared to the models containing acylated ghrelin (R(2) = 0.24-0.27). When analyzing the influence of the presence of fibromyalgia (study group: women with fibromyalgia vs. healthy women), only the leptin levels were influenced. High leptin levels independent of adiposity in women with fibromyalgia may be associated with the clinical condition of this syndrome.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis*

PubMed Central

Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

2016-01-01

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe45 in helix II and Phe18 in the α1α2 loop and a hydrogen bonding between Ser15 in helix I and Ile20 in the α1α2 loop, resulting in its high thermal stability. Phe45-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains. PMID:26631734

Separation of actinides from lanthanides

DOEpatents

Smith, B.F.; Jarvinen, G.D.; Ryan, R.R.

1988-03-31

An organic extracting solution and an extraction method useful for separating elements of the actinide series of the periodic table from elements of the lanthanide series, where both are in trivalent form is described. The extracting solution consists of a primary ligand and a secondary ligand, preferably in an organic solvent. The primary ligand is a substituted monothio-1,3-dicarbonyl, which includes a substituted 4-acyl-2-pyrazolin-5-thione, such as 4-benzoyl-2,4- dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT). The secondary ligand is a substituted phosphine oxide, such as trioctylphosphine oxide (TOPO).

Separation of actinides from lanthanides

DOEpatents

Smith, Barbara F.; Jarvinen, Gordon D.; Ryan, Robert R.

1989-01-01

An organic extracting solution and an extraction method useful for separating elements of the actinide series of the periodic table from elements of the lanthanide series, where both are in trivalent form. The extracting solution consists of a primary ligand and a secondary ligand, preferably in an organic solvent. The primary ligand is a substituted monothio-1,3-dicarbonyl, which includes a substituted 4-acyl-2-pyrazolin-5-thione, such as 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT). The secondary ligand is a substituted phosphine oxide, such as trioctylphosphine oxide (TOPO).

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

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

PubMed Central

Steenkamp, D J

1987-01-01

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA. Images Fig. 2. Fig. 3. Fig. 5. PMID:3115254

Peptide affinity labels for thrombin and other trypsin-like proteases

DOEpatents

Shaw, Elliott N.; Kettner, Charles A.

1982-03-09

A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

PubMed Central

Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

1995-01-01

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

Regioselective Synthesis of Cellulose Ester Homopolymers

Treesearch

Daiqiang Xu; Kristen Voiges; Thomas Elder; Petra Mischnick; Kevin J. Edgar

2012-01-01

Regioselective synthesis of cellulose esters is extremely difficult due to the small reactivity differences between cellulose hydroxyl groups, small differences in steric demand between acyl moieties of interest, and the difficulty of attaching and detaching many protecting groups in the presence of cellulose ester moieties without removing the ester groups. Yet the...

Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

PubMed Central

Inaba, Tomohiro; Tokumoto, Yuta; Miyazaki, Yusuke; Inoue, Naoyuki; Maseda, Hideaki; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo

2013-01-01

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species. PMID:24038682

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

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.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

PubMed Central

Wang, Zhen; Benning, Christoph

2011-01-01

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12. Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6. PMID:21445048

Feature construction can improve diagnostic criteria for high-dimensional metabolic data in newborn screening for medium-chain acyl-CoA dehydrogenase deficiency.

PubMed

Ho, Sirikit; Lukacs, Zoltan; Hoffmann, Georg F; Lindner, Martin; Wetter, Thomas

2007-07-01

In newborn screening with tandem mass spectrometry, multiple intermediary metabolites are quantified in a single analytical run for the diagnosis of fatty-acid oxidation disorders, organic acidurias, and aminoacidurias. Published diagnostic criteria for these disorders normally incorporate a primary metabolic marker combined with secondary markers, often analyte ratios, for which the markers have been chosen to reflect metabolic pathway deviations. We applied a procedure to extract new markers and diagnostic criteria for newborn screening to the data of newborns with confirmed medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and a control group from the newborn screening program, Heidelberg, Germany. We validated the results with external data of the screening center in Hamburg, Germany. We extracted new markers by performing a systematic search for analyte combinations (features) with high discriminatory performance for MCADD. To select feature thresholds, we applied automated procedures to separate controls and cases on the basis of the feature values. Finally, we built classifiers from these new markers to serve as diagnostic criteria in screening for MCADD. On the basis of chi(2) scores, we identified approximately 800 of >628,000 new analyte combinations with superior discriminatory performance compared with the best published combinations. Classifiers built with the new features achieved diagnostic sensitivities and specificities approaching 100%. Feature construction methods provide ways to disclose information hidden in the set of measured analytes. Other diagnostic tasks based on high-dimensional metabolic data might also profit from this approach.

Association of 2-acylaminopyridines and benzoic acids. Steric and electronic substituent effect studied by XRD, solution and solid-state NMR and calculations

NASA Astrophysics Data System (ADS)

Ośmiałowski, Borys; Kolehmainen, Erkki; Ejsmont, Krzysztof; Ikonen, Satu; Valkonen, Arto; Rissanen, Kari; Nonappa

2013-12-01

Eight single crystal X-ray structures, solid-state NMR spectroscopic, and theoretical studies utilizing QTAIM methodology were used to characterize the 2-acyl (alkyl in acyl = methyl, ethyl, t-butyl, and 1-adamantyl) amino-6-R-pyridine/4-R‧-benzoic acid (R,R‧ = H or Me) cocrystals. As expected among alkyl groups 1-adamantyl due to its bulkiness has the most significant effect on the relative positions of molecules in cocrystals. In addition, the subtle electronic and steric effects by the methyl substituents were observed. The theoretical calculations with full geometry optimizations are in agreement with the experimental findings (geometry, energy of hydrogen bonds). Based on the crystal structures and calculations it is concluded that p-methyl substituent in benzoic acid increase the hydrogen bond accepting ability of the CO oxygen and decreases the hydrogen bond donating ability of OH proton. The 15N solid-state (CP MAS) NMR chemical shifts prove that molecules in cocrystal are held together by hydrogen bonding. The biggest variation in the 15N chemical shift of acylamino nitrogen can be related with the size of the alkyl group in acyl moiety.

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

PubMed Central

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

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.

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…

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures.

PubMed

Pannkuk, Evan L; Blair, Hannah B; Fischer, Amy E; Gerdes, Cheyenne L; Gilmore, David F; Savary, Brett J; Risch, Thomas S

2014-01-01

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography-mass spectrometry (GC-MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Ghrelin and obestatin levels in rheumatoid arthritis.

PubMed

Koca, Suleyman Serdar; Ozgen, Metin; Aydin, Suleyman; Dag, Sait; Evren, Bahri; Isik, Ahmet

2008-10-01

Ghrelin is a powerful, endogenous orexigenic peptide. In addition, ghrelin has anti-inflammatory effects, and it has been reported that ghrelin down-regulates pro-inflammatory cytokines, including interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Obestatin appears to decrease food intake and appetite, and its potential role in inflammation is not yet clear. The aims of this study were to assess total and acylated (active) ghrelin and obestatin serum levels and their relations with inflammatory status in rheumatoid arthritis (RA) patients. Fasting blood samples were obtained from 37 patients with RA, 29 patients with Behçet's disease (BD) and 28 healthy controls (HC). Total ghrelin and obestatin levels were measured by radioimmunoassay and acylated ghrelin was quantified by enzyme-linked immunosorbent assay. Patients with RA had lower total ghrelin, but higher obestatin levels than patients with BD (p<0.05 for both), but when compared with HC group differences were not significant. There was no difference across groups in terms of acylated ghrelin. Total ghrelin level was not correlated with any study parameters in the all groups. Obestatin level correlated with erythrocyte sedimentation rate and DAS-28 in the RA group, the level of IL-6 in the BD group, and with the level of TNF-alpha in the HC group (r=0.400, p<0.05; r=0.412, p<0.05, r=0.543, p<0.01 and r=0.528, p<0.05, respectively). Our results did not show a significant correlation between circulating ghrelin and clinical or laboratory markers of disease activity in RA. Surprisingly, obestatin correlated with some inflammatory markers. So, obestatin seems to be more valuable than ghrelin in the pathogenesis of RA.

21 CFR 172.665 - Gellan gum.

Code of Federal Regulations, 2014 CFR

2014-04-01

... following prescribed conditions: (a) The additive is a high molecular weight polysaccharide gum produced..., sodium, calcium, and magnesium salt. The polysaccharide may contain acyl (glyceryl and acetyl) groups as...

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.

Direct Acylation of C(sp(3))-H Bonds Enabled by Nickel and Photoredox Catalysis.

PubMed

Joe, Candice L; Doyle, Abigail G

2016-03-14

Using nickel and photoredox catalysis, the direct functionalization of C(sp(3))-H bonds of N-aryl amines by acyl electrophiles is described. The method affords a diverse range of α-amino ketones at room temperature and is amenable to late-stage coupling of complex and biologically relevant groups. C(sp(3))-H activation occurs by photoredox-mediated oxidation to generate α-amino radicals which are intercepted by nickel in catalytic C(sp(3))-C coupling. The merger of these two modes of catalysis leverages nickel's unique properties in alkyl cross-coupling while avoiding limitations commonly associated with transition-metal-mediated C(sp(3))-H activation, including requirements for chelating directing groups and high reaction temperatures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trend‐Analysis of Solid‐State Structures: Low‐Energy Conformational ‘Reactions’ Involving Directed and Coupled Movements in Half‐Sandwich Compounds [CpFe(CO){C(=O)R}PPh3

PubMed Central

2018-01-01

Abstract Trends in solid‐state structures were used to identify preferred intramolecular movements in half‐sandwich compounds [CpFe(CO){C(=O)R}PPh3]. Three weak interactions were analyzed: 1) the CH/π donor–acceptor interaction of phenyl rings in the PPh3 ligand, 2) the PhPPh3 face‐on Cp stabilization, and 3) the hydrogen bond between the oxygen atom of the acyl group and an ortho‐C−H bond of one of the PPh3 phenyl rings. Clockwise and counter‐clockwise rotations established directed and coupled movements of the PPh3 ligand, the acyl group, and the phenyl rings within the PPh3 ligand. PMID:29744282

Optimizing gelling parameters of gellan gum for fibrocartilage tissue engineering.

PubMed

Lee, Haeyeon; Fisher, Stephanie; Kallos, Michael S; Hunter, Christopher J

2011-08-01

Gellan gum is an attractive biomaterial for fibrocartilage tissue engineering applications because it is cell compatible, can be injected into a defect, and gels at body temperature. However, the gelling parameters of gellan gum have not yet been fully optimized. The aim of this study was to investigate the mechanics, degradation, gelling temperature, and viscosity of low acyl and low/high acyl gellan gum blends. Dynamic mechanical analysis showed that increased concentrations of low acyl gellan gum resulted in increased stiffness and the addition of high acyl gellan gum resulted in greatly decreased stiffness. Degradation studies showed that low acyl gellan gum was more stable than low/high acyl gellan gum blends. Gelling temperature studies showed that increased concentrations of low acyl gellan gum and CaCl₂ increased gelling temperature and low acyl gellan gum concentrations below 2% (w/v) would be most suitable for cell encapsulation. Gellan gum blends were generally found to have a higher gelling temperature than low acyl gellan gum. Viscosity studies showed that increased concentrations of low acyl gellan gum increased viscosity. Our results suggest that 2% (w/v) low acyl gellan gum would have the most appropriate mechanics, degradation, and gelling temperature for use in fibrocartilage tissue engineering applications. Copyright © 2011 Wiley Periodicals, Inc.

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta 11- and Delta 9-desaturases with unusual catalytic properties.

PubMed

Liénard, Marjorie A; Lassance, Jean-Marc; Wang, Hong-Lei; Zhao, Cheng-Hua; Piskur, Jure; Johansson, Tomas; Löfstedt, Christer

2010-06-01

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths. (c) 2010 Elsevier Ltd. All rights reserved.

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 data suggest that the amount of TG in a typical meal does not limit the bioaccessibility of carotenoids.

Chemical structures of constituents from the whole plant of Bacopa monniera.

PubMed

Ohta, Tomoe; Nakamura, Seikou; Nakashima, Souichi; Oda, Yoshimi; Matsumoto, Takahiro; Fukaya, Masashi; Yano, Mamiko; Yoshikawa, Masayuki; Matsuda, Hisashi

2016-07-01

Two new dammarane-type triterpene oligoglycosides, bacomosaponins A and B, and three new phenylethanoid glycosides, bacomosides A, B1, and B2, were isolated from the whole plant of Bacopa monniera Wettst. The chemical structures of the new constituents were characterized on the basis of chemical and physicochemical evidence. In the present study, bacomosaponins A and B with acyl groups were obtained from the whole plant of B. monniera. This is the first report of acylated dammarane-type triterpene oligoglycosides isolated from B. monniera. In addition, dammarane-type triterpene saponins significantly inhibited the aggregation of 42-mer amyloid β-protein.

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

The Physiology of Protein S-acylation

PubMed Central

Chamberlain, Luke H.; Shipston, Michael J.

2015-01-01

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

A macrocyclic ellagitannin trimer, oenotherin T(1), from Oenothera species.

PubMed

Taniguchi, Shoko; Imayoshi, Yoko; Yabu-uchi, Ryoko; Ito, Hideyuki; Hatano, Tsutomu; Yoshida, Takashi

2002-01-01

Oenotherin T(1) was isolated from leaves of Oenothera tetraptera as a major ellagitannin. Its structure, that of a macrocyclic trimer with a new acyl group, an isodehydrovaloneoyl group, was established. This compound was also produced by callus tissues induced from O. laciniata leaves.

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.

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies.

PubMed

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20-40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Peptide affinity labels for thrombin and other trypsin-like proteases

DOEpatents

Shaw, E.N.; Kettner, C.A.

1982-03-09

A peptide affinity label is disclosed of the formula (I): as given in the patent wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C[sub 1]--C[sub 4] alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C[sub 1]--C[sub 6] acyl, and Q--(A)--[sub n], wherein Q = hydrogen, aroyl, or C[sub 1]--C[sub 6] acyl, n = 1--10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereofcontaining, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH[sub 2]--, --CH[sub 2]--CH[sub 2]--, --CH[sub 2]--CH[sub 2]--CH[sub 2]--, --CH[double bond]CH-- and --CH(OH)--CH[sub 2]. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent. 2 figs.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

High density DNA loading on the M13 bacteriophage provides access to colorimetric and fluorescent protein microarray biosensors.

PubMed

Domaille, Dylan W; Lee, Ju Hun; Cha, Jennifer N

2013-02-28

We report the synthesis, characterization, and protein sensing capabilities of M13 bacteriophage-DNA bioconjugates. DNA oligonucleotides were conjugated to M13 through acyl hydrazone linkages. In one case, DNAzymes retained their catalytic ability when anchored to the virus coat, and in a separate study, the dynamic nature of the hydrazone allowed for liberation of DNA from the phage under mild conditions.

Phosphatidic Acid Synthesis in Bacteria

PubMed Central

Yao, Jiangwei; Rock, Charles O.

2012-01-01

Membrane phospholipid synthesis is a vital facet of bacterial physiology. Although the spectrum of phospholipid headgroup structures produced by bacteria is large, the key precursor to all of these molecules is phosphatidic acid (PtdOH). Glycerol-3-phosphate derived from the glycolysis via glycerol-phosphate synthase is the universal source for the glycerol backbone of PtdOH. There are two distinct families of enzymes responsible for the acylation of the 1-position of glycerol-3-phosphate. The PlsB acyltransferase was discovered in Escherichia coli, and homologs are present in many eukaryotes. This protein family primarily uses acyl-acyl carrier protein (ACP) endproducts of fatty acid synthesis as acyl donors, but may also use acyl-CoA derived from exogenous fatty acids. The second protein family, PlsY, is more widely distributed in bacteria and utilizes the unique acyl donor, acyl-phosphate, which is produced from acyl-ACP by the enzyme PlsX. The acylation of the 2-position is carried out by members of the PlsC protein family. All PlsCs use acyl-ACP as the acyl donor, although the PlsCs of the γ-proteobacteria also may use acyl-CoA. Phospholipid headgroups are precursors in the biosynthesis of other membrane-associated molecules and the diacylglycerol product of these reactions is converted to PtdOH by one of two distinct families of lipid kinases. The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:22981714

A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

PubMed Central

Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

2012-01-01

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

In Vivo Lipid "Tag and Track" Approach Shows Acyl Editing of Plastid Lipids and Chloroplast Import of Phosphatidylglycerol Precursors in Arabidopsis thaliana.

PubMed

Hurlock, Anna K; Wang, Kun; Takeuchi, Tomomi; Horn, Patrick J; Benning, Christoph

2018-06-19

In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexity in vivo, we developed an in vivo lipid "tag and track" method. Essentially, we probed lipid metabolism in Arabidopsis thaliana by expressing a coding sequence for a fatty acid desaturase from Physcomitrella patens (Δ6D). This enzyme places a double bond after the 6 th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at its sn-2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to "tag and track" the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence of ER-derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this "tag and track" method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

PubMed Central

Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

1994-01-01

Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

Individual Variation in Hunger, Energy Intake, and Ghrelin Responses to Acute Exercise.

PubMed

King, James A; Deighton, Kevin; Broom, David R; Wasse, Lucy K; Douglas, Jessica A; Burns, Stephen F; Cordery, Philip A; Petherick, Emily S; Batterham, Rachel L; Goltz, Fernanda R; Thackray, Alice E; Yates, Thomas; Stensel, David J

2017-06-01

This study aimed to characterize the immediate and extended effect of acute exercise on hunger, energy intake, and circulating acylated ghrelin concentrations using a large data set of homogenous experimental trials and to describe the variation in responses between individuals. Data from 17 of our group's experimental crossover trials were aggregated yielding a total sample of 192 young, healthy males. In these studies, single bouts of moderate to high-intensity aerobic exercise (69% ± 5% V˙O2 peak; mean ± SD) were completed with detailed participant assessments occurring during and for several hours postexercise. Mean hunger ratings were determined during (n = 178) and after (n = 118) exercise from visual analog scales completed at 30-min intervals, whereas ad libitum energy intake was measured within the first hour after exercise (n = 60) and at multiple meals (n = 128) during the remainder of trials. Venous concentrations of acylated ghrelin were determined at strategic time points during (n = 118) and after (n = 89) exercise. At group level, exercise transiently suppressed hunger (P < 0.010, Cohen's d = 0.77) but did not affect energy intake. Acylated ghrelin was suppressed during exercise (P < 0.001, Cohen's d = 0.10) and remained significantly lower than control (no exercise) afterward (P < 0.024, Cohen's d = 0.61). Between participants, there were notable differences in responses; however, a large proportion of this spread lay within the boundaries of normal variation associated with biological and technical assessment error. In young men, acute exercise suppresses hunger and circulating acylated ghrelin concentrations with notable diversity between individuals. Care must be taken to distinguish true interindividual variation from random differences within normal limits.

Triclosan-immobilized polyamide thin film composite membranes with enhanced biofouling resistance

NASA Astrophysics Data System (ADS)

Park, Sang-Hee; Hwang, Seon Oh; Kim, Taek-Seung; Cho, Arah; Kwon, Soon Jin; Kim, Kyoung Taek; Park, Hee-Deung; Lee, Jung-Hyun

2018-06-01

We report on a strategy to improve biofouling resistance of a polyamide (PA) thin-film composite (TFC) reverse osmosis (RO) membrane via chemically immobilizing triclosan (TC), known as a common organic biocide, on its surface. To facilitate covalent attachment of TC on the membrane surface, TC was functionalized with amine moiety to prepare aminopropyl TC. Then, the TC-immobilized TFC (TFC-TC) membranes were fabricated through a one-step amide formation reaction between amine groups of aminopropyl TC and acyl chloride groups present on the PA membrane surface, which was confirmed by high-resolution XPS. Strong stability of the immobilized TC was also confirmed by a hydraulic washing test. Although the TFC-TC membrane showed slightly reduced separation performance compared to the pristine control, it still maintained a satisfactory RO performance level. Importantly, the TFC-TC membrane exhibited excellent antibacterial activity against both gram negative (E. coli and P. aeruginosa) and gram positive (S. aureus) bacteria along with greatly enhanced resistance to biofilm formation. Our immobilization approach offers a robust and relatively benign strategy to control biofouling of functional surfaces, films and membranes.

Utilization of oxygen difluoride for syntheses of fluoropolymers

NASA Technical Reports Server (NTRS)

Toy, M. S. (Inventor)

1976-01-01

The reaction oxygen difluoride, OF2, with ethylenically unsaturated fluorocarbon compounds is examined. Depending upon the fluorocarbon material and reaction conditions, OF2 can chain extend fluoropolyenes, convert functional perfluorovinyl groups to acyl fluoride and/or epoxide groups, and act as a monomer for an addition type copolymerization with diolefins.

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

PubMed Central

Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

2016-01-01

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

PubMed

Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

2015-03-01

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

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

A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

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

McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectralmore » data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.« less

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

Early life disruption to the ghrelin system with over-eating is resolved in adulthood in male rats.

PubMed

Sominsky, Luba; Ziko, Ilvana; Nguyen, Thai-Xinh; Andrews, Zane B; Spencer, Sarah J

2017-02-01

Early life overweight is a significant risk factor for developmental programming of adult obesity due to changes in the availability of metabolic factors crucial for the maturation of brain appetite-regulatory circuitry. The appetite-stimulating hormone, ghrelin, has been recently identified as a major regulator of the establishment of hypothalamic feeding pathways. Ghrelin exists in circulation in two major forms, as acylated and des-acylated ghrelin. While most research has focused on acyl ghrelin, the role of neonatal des-acyl ghrelin in metabolic programming is currently unknown. Here we assessed the influences of early life overfeeding on the ghrelin system, including acyl and des-acyl ghrelin's ability to access the hypothalamus in male rats. Our data show that early life overfeeding influences the ghrelin system short-term, leading to an acute reduction in circulating des-acyl ghrelin and increased expression of the growth hormone secretagogue receptor (GHSR) in the arcuate nucleus of the hypothalamus (ARC). These changes are associated with increased neuronal activation in response to exogenous acyl, but not des-acyl, ghrelin in the ARC and the paraventricular nucleus of the hypothalamus (PVN). Interestingly, while we observed no differences in the accessibility of the ARC to acyl or des-acyl ghrelin, less exogenous acyl ghrelin reaches the PVN in the neonatally overfed. Importantly, the influences of neonatal overfeeding on the ghrelin system were not maintained into adulthood. Therefore, while early life overfeeding results in excess body weight and stimulates acute changes in the brain's sensitivity to metabolic signals, this developmental mal-programming is at least partially alleviated in adulthood. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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 sialomucins in C9. Adenocarcinomas of the colloid type, thought to bear a poor prognosis, are the most heavily O-acylated.

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

Untargeted metabolomics reveals specific withanolides and fatty acyl glycoside as tentative metabolites to differentiate organic and conventional Physalis peruviana fruits.

PubMed

Llano, Sandra M; Muñoz-Jiménez, Ana M; Jiménez-Cartagena, Claudio; Londoño-Londoño, Julián; Medina, Sonia

2018-04-01

The agronomic production systems may affect the levels of food metabolites. Metabolomics approaches have been applied as useful tool for the characterization of fruit metabolome. In this study, metabolomics techniques were used to assess the differences in phytochemical composition between goldenberry samples produced by organic and conventional systems. To verify that the organic samples were free of pesticides, individual pesticides were analyzed. Principal component analysis showed a clear separation of goldenberry samples from two different farming systems. Via targeted metabolomics assays, whereby carotenoids and ascorbic acid were analyzed, not statistical differences between both crops were found. Conversely, untargeted metabolomics allowed us to identify two withanolides and one fatty acyl glycoside as tentative metabolites to differentiate goldenberry fruits, recording organic fruits higher amounts of these compounds than conventional samples. Hence, untargeted metabolomics technology could be suitable to research differences on phytochemicals under different agricultural management practices and to authenticate organic products. Copyright © 2017 Elsevier Ltd. 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

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

Anthocyanin Profiles in Flowers of Grape Hyacinth.

PubMed

Lou, Qian; Wang, Lin; Liu, Hongli; Liu, Yali

2017-04-26

Grape hyacinth ( Muscari spp.) is a popular ornamental bulbous perennial famous for its blue flowers. To understand the chemical basis of the rich blue colors in this plant, anthocyanin profiles of six blue flowering grape hyacinths as well as one pink and one white cultivar were determined using high-performance liquid chromatography and mass spectrometry. Along with two known compounds, eight putative anthocyanins were identified in the tepals of grape hyacinth for the first time. The accumulation and distribution of anthocyanins in the plant showed significant cultivar and flower development specificity. Violet-blue flowers mainly contained simple delphinidin-type anthocyanins bearing one or two methyl-groups but no acyl groups, whereas white and pink flowers synthesised more complex pelargonidin/cyanidin-derivatives with acyl-moieties but no methyl-groups. The results partially reveal why solid blue, orange or red flowers are rare in this plant in nature. In addition, pelargonidin-type anthocyanins were found for the first time in the genus, bringing more opportunities in terms of breeding of flower color in grape hyacinth.

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

PubMed Central

Taylor, Cassandra L.; Bolshan, Yuri

2015-01-01

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups and scaffolds. Traditional methods for the preparation of ynones are associated with drawbacks including harsh conditions, multiple purification steps, and the presence of unwanted byproducts. An alternative method for the straightforward preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts is described herein. The adoption of organotrifluoroborate salts as an alternative to organometallic reagents for the formation of new carbon-carbon bonds has a number of advantages. Potassium organotrifluoroborate salts are shelf stable, have good functional group tolerance, low toxicity, and a wide variety are straightforward to prepare. The title reaction proceeds rapidly at ambient temperature in the presence of a Lewis acid without the exclusion of air and moisture. Fair to excellent yields may be obtained via reaction of various aryl and alkyl acid chlorides with alkynyltrifluoroborate salts in the presence of boron trichloride. PMID:25742169

Using steric hindrance to design new inhibitors of class C beta-lactamases

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

Trehan, Indi; Morandi, F.; Blaszczak, L.C.

{beta}-lactamases confer resistance to {beta}-lactam antibiotics such as penicillins and cephalosporins. However, {beta}-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of {beta}-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R1 side chain of {beta}-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective {beta}-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of themore » penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.« less

Antihypertensive neutral lipid

DOEpatents

Snyder, Fred L.; Blank, Merle L.

1986-01-01

The invention relates to the discovery of a class of neutral acetylated ether-linked glycerolipids having the capacity to lower blood pressure in warm-blooded animals. This physiological effect is structure sensitive requiring a long chain alkyl group at the sn-1 position and a short carbon chain acyl group (acetyl or propionyl) at the sn-2 position, and a hydroxyl group at the sn-3 position.

Antihypertensive neutral lipid

DOEpatents

Snyder, F.L.; Blank, M.L.

1984-10-26

The invention relates to the discovery of a class of neutral acetylated either-linked glycerolipids having the capacity to lower blood presure in warm-blooded animals. This physiological effect is structure sensitive requiring a long chain alkyl group at the sn-1 position and a short carbon chain acyl group (acetyl or propionyl) at the sn-2 position, and a hydroxyl group at the sn-3 position.

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.

Two new phenylpropanoid glycosides with interesterification from Scrophularia dentata Royle ex Benth

NASA Astrophysics Data System (ADS)

Zhang, Liuqiang; Yang, Zhuo; Jia, Qi; Dorje, Gaawe; Zhao, Zhili; Guo, Fujiang; Li, Yiming

2013-10-01

Two new phenylpropanoid glycosides (1-2), along with seven known ones (3-9), were isolated from the whole plant of Scrophularia dentata Royle ex Benth. Their structures were elucidated by spectroscopic methods. Among them, compounds 1 and 2 failed to separated, because they can easily transform into each other by acyl migrant reaction. In this paper, the interesterification mechanism was discussed firstly and the rule can be used in the similar structure elucidation in future.

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.

The utilization of the acyl-CoA and the involvement PDAT and DGAT in the biosynthesis of erucic acid-rich triacylglycerols in Crambe seed oil.

PubMed

Furmanek, Tomasz; Demski, Kamil; Banaś, Walentyna; Haslam, Richard; Napier, Jonathan; Stymne, Sten; Banaś, Antoni

2014-04-01

The triacylglycerol of Crambe abyssinica seeds consist of 95% very long chain (>18 carbon) fatty acids (86% erucic acid; 22:1∆13) in the sn-1 and sn-3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10% of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl-CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0-CoA and 18:1-CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl-CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl-CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl-CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl-CoA by the acyltransferases in the glycerol-3-phosphate pathway.

Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration.

PubMed

Sasaki, Nobuhiro; Nakayama, Toru

2015-01-01

Genetic engineering of roses and other plants of floricultural importance to give them a truly blue petal color is arguably one of the holy grails of plant biotechnology. Toward this goal, bluish carnations and roses were previously engineered by establishing an exclusive accumulation of delphinidin (Dp)-type anthocyanins in their petals via the heterologous expression of a flavonoid 3',5'-hydroxylase gene. Very recently, purple-blue varieties of chrysanthemums were also genetically engineered via a similar biochemical strategy. Although the floral colors of these transgenic plants still lack a true blue color, the basis for the future molecular breeding of truly blue flowers is via the engineering of anthocyanin pathways. Anthocyanins with multiple aromatic acyl groups (often referred to as polyacylated anthocyanins) in the 3'- or 7-position tend to display a more stable blue color than non-acylated anthocyanins. The 7-polyacylation process during the biosynthesis of purple-blue anthocyanins in delphinium (Delphinium grandiflorum) was found to occur in vacuoles using acyl-glucose as both the glucosyl and acyl donor. Glucosyltransferases and acyltransferases involved in anthocyanin 7-polyacylation in delphinium are vacuolar acyl-glucose-dependent enzymes belonging to the glycoside hydrolase family 1 and serine carboxypeptidae-like protein family, respectively. The 7-polyacylation proceeds through the alternate glucosylation and p-hydroxybenzoylation catalyzed by these enzymes. p-Hydroxybenzoyl-glucose serves as the p-hydroxybenzoyl and glucosyl donor to produce anthocyanins modified with a p-hydroxybenzoyl-glucose concatemer at the 7-position. This novel finding has provided a potential breakthrough for the genetic engineering of truly blue flowers, where polyacylated Dp-type anthocyanins are accumulated exclusively in the petals. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Crystal Structures of Covalent Complexes of [beta]-Lactam Antibiotics with Escherichia coli Penicillin-Binding Protein 5: Toward an Understanding of Antibiotic Specificity

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

Nicola, George; Tomberg, Joshua; Pratt, R.F.

Penicillin-binding proteins (PBPs) are the molecular targets for the widely used {beta}-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of Escherichia coli PBP 5 as covalent complexes with imipenem, cloxacillin, and cefoxitin. These antibiotics exhibit very different second-order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the {beta}-lactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with themore » rates of acylation. The same is true for conformational changes, because although a shift of a loop leading to an electrostatic interaction between Arg248 and the {beta}-lactam carboxylate, which occurs completely with cefoxitin and partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 decreased the level of cefoxitin acylation only 2-fold. Together, these data suggest that structures of postcovalent complexes of PBP 5 are unlikely to be useful vehicles for the design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP 5 in complex with a boronic acid peptidomimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the {beta}-lactam. Because the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the {beta}-lactam ring.« less

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.

Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors, pharmacokinetic properties, lead optimization, Lineweaver-Burk plot evaluation and binding analysis.

PubMed

Sajid-Ur-Rehman; Saeed, Aamer; Saddique, Gufran; Ali Channar, Pervaiz; Ali Larik, Fayaz; Abbas, Qamar; Hassan, Mubashir; Raza, Hussain; Fattah, Tanzeela Abdul; Seo, Sung-Yum

2018-06-02

To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a-4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a-4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a-4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC 50 0.251 ± 0.012 µM (standard KH 2 PO 4 4.317 ± 0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a-4j obey Lipinsk's rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues. Copyright © 2018. Published by Elsevier Ltd.

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

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Staphylococcus aureus.

PubMed

Hong, Seung Kon; Kim, Kook Han; Kim, Eunice EunKyeong

2010-01-01

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 A, alpha = gamma = 90, beta = 106.330 degrees . The asymmetric unit contains one SaMCAT molecule.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Synechocystis sp. PCC 6803.

PubMed

Liu, Yinghui; Zhang, Yanming; Cao, Xupeng; Xue, Song

2013-11-01

Malonyl-coenzymeA:acyl-carrier protein transacylase (MCAT), which catalyzes the transfer of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP), is an essential enzyme in type II fatty-acid synthesis. The enzyme MCAT from Synechocystis sp. PCC 6803 (spMCAT), the first MCAT counterpart from a cyanobacterium, was cloned, purified and crystallized in order to determine its three-dimensional crystal structure. A higher-quality crystal with better diffraction was obtained by crystallization optimization. The crystal diffracted to 1.8 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 43.22, b = 149.21, c = 40.59 Å. Matthews coefficient calculations indicated that the crystal contained one spMCAT molecule in the asymmetric unit with a Matthews coefficient of 2.18 Å(3) Da(-1) and a solvent content of 43.65%.

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 chronic inflammatory activity in human obesity.

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:17369415

A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins1

PubMed Central

Rinaldo, Amy R.; Cavallini, Erika; Jia, Yong; Moss, Sarah M.A.; McDavid, Debra A.J.; Hooper, Lauren C.; Robinson, Simon P.; Tornielli, Giovanni B.; Zenoni, Sara; Ford, Christopher M.; Boss, Paul K.; Walker, Amanda R.

2015-01-01

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. PMID:26395841

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

Code of Federal Regulations, 2013 CFR

2013-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

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

Code of Federal Regulations, 2014 CFR

2014-07-01

...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

Ultra(high)-pressure liquid chromatography-electrospray ionization-time-of-flight-ion mobility-high definition mass spectrometry for the rapid identification and structural characterization of flavonoid glycosides from cauliflower waste.

PubMed

Gonzales, Gerard Bryan; Raes, Katleen; Coelus, Sofie; Struijs, Karin; Smagghe, Guy; Van Camp, John

2014-01-03

In this paper, a strategy for the detection and structural elucidation of flavonoid glycosides from a complex matrix in a single chromatographic run using U(H)PLC-ESI-IMS-HDMS/MS(E) is presented. This system operates using alternative low and high energy voltages that is able to perform the task of conventional MS/MS in a data-independent way without re-injection of the sample, which saves analytical time. Also, ion mobility separation (IMS) was employed as an additional separation technique for compounds that are co-eluting after U(H)PLC separation. First, the fragmentation of flavonoid standards were analyzed and criteria was set for structural elucidation of flavonoids in a plant extract. Based on retention times, UV spectra, exact mass, and MS fragment characteristics, such as abundances of daughter ions and the presence of radical ions ([Y0-H](-)), a total 19 flavonoid glycosides, of which 8 non-acylated and 11 acylated, were detected and structurally characterized in a cauliflower waste extract. Kaempferol and quercetin were the main aglycones detected while sinapic and ferulic acid were the main phenolic acids. C-glycosides were also found although their structure could not be elucidated. The proposed method can be used as a rapid screening test for flavonoid identification and for routine analysis of plant extracts, such as these derived from cauliflower waste. The study also confirms that agroindustrial wastes, such as cauliflower leaves, could be seen as a valuable source of different bioactive phenolic compounds. Copyright © 2013 Elsevier B.V. All rights reserved.

Glu-Trp-ONa or its acylated analogue (R-Glu-Trp-ONa) administration enhances the wound healing in the model of chronic skin wounds in rabbits.

PubMed

Shevtsov, Maxim A; Smagina, Larisa V; Kudriavtceva, Tatiana A; Petlenko, Sergey V; Voronkina, Irina V

2015-01-01

The management of chronic skin wounds represents a major therapeutic challenge. The synthesized dipeptide (Glu-Trp-ONa) and its acylated analogue (R-Glu-Trp-ONa) were assessed in the model of nonhealing dermal wounds in rabbits in relation to their healing properties in wound closure. Following wound modeling, the rabbits received a course of intraperitoneal injections of Glu-Trp-ONa or R-Glu-Trp-ONa. Phosphate-buffered saline and Solcoseryl® were applied as negative and positive control agents, respectively. An injection of Glu-Trp-ONa and R-Glu-Trp-ONa decreased the period of wound healing in animals in comparison to the control and Solcoseryl-treated groups. Acylation of Glu-Trp-ONa proved to be beneficial as related to the healing properties of the dipeptide. Subsequent zymography analyses showed that the applied peptides decreased the proteolytic activity of matrix metalloproteinases MMP-9, MMP-8, and MMP-2 in the early inflammatory phase and reversely increased the activity of MMP-9, MMP-8, and MMP-1 in the remodeling phase. Histological analyses of the wound sections (hematoxylin-eosin, Mallory's staining) confirmed the enhanced formation of granulation tissue and re-epithelialization in the experimental groups. By administering the peptides, wound closures increased significantly through the modulation of the MMPs' activity, indicating their role in wound healing.

Genome-wide analysis of the Glycerol-3-Phosphate Acyltransferase (GPAT) gene family reveals the evolution and diversification of plant GPATs

PubMed Central

Waschburger, Edgar; Kulcheski, Franceli Rodrigues; Veto, Nicole Moreira; Margis, Rogerio; Margis-Pinheiro, Marcia; Turchetto-Zolet, Andreia Carina

2018-01-01

Abstract sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is an important enzyme that catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 or sn-2 position of sn-glycerol-3-phosphate (G3P) to generate lysophosphatidic acids (LPAs). The functional studies of GPAT in plants demonstrated its importance in controlling storage and membrane lipid. Identifying genes encoding GPAT in a variety of plant species is crucial to understand their involvement in different metabolic pathways and physiological functions. Here, we performed genome-wide and evolutionary analyses of GPATs in plants. GPAT genes were identified in all algae and plants studied. The phylogenetic analysis showed that these genes group into three main clades. While clades I (GPAT9) and II (soluble GPAT) include GPATs from algae and plants, clade III (GPAT1-8) includes GPATs specific from plants that are involved in the biosynthesis of cutin or suberin. Gene organization and the expression pattern of GPATs in plants corroborate with clade formation in the phylogeny, suggesting that the evolutionary patterns is reflected in their functionality. Overall, our results provide important insights into the evolution of the plant GPATs and allowed us to explore the evolutionary mechanism underlying the functional diversification among these genes. PMID:29583156

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2.

PubMed

Lister, M D; Hancock, A J

1988-10-01

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.

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.

Infrared-thermographic screening of the activity and enantioselectivity of enzymes.

PubMed

Reetz, M T; Hermes, M; Becker, M H

2001-05-01

The infrared radiation caused by the heat of reaction of an enantioselective enzyme-catalyzed transformation can be detected by modern photovoltaic infrared (IR)-thermographic cameras equipped with focal-plane array detectors. Specifically, in the lipase-catalyzed enantioselective acylation of racemic 1-phenylethanol, the (R)- and (S)-substrates are allowed to react separately in the wells of microtiter plates, the (R)-alcohol showing hot spots in the IR-thermographic images. Thus, highly enantioselective enzymes can be identified at kinetic resolution.

Acylation and deacylation mechanism of Helicobacter pylori AmiF formamidase: A computational DFT study

NASA Astrophysics Data System (ADS)

He, Rongxing; Yang, Qinlei; Li, Ming

2014-04-01

The acylation and deacylation mechanisms of Helicobacter pylori AmiF formamidase were investigated using DFT method. In the constructed active site, residues Glu60, Glu141 and His167 were taken into account besides Lys133 and Cys166. Calculations provided insight on the details of mechanism and explained crucial roles played by Glu60, Glu141 and His167. For acetylation, we proposed a new stepwise mechanism in which the thiol group first attacks the carbon atom of formamide and produces tetrahedral intermediate. In deacylation, Glu60 activates a water molecule to perform nucleophilic attack and then forms an intermediate, which is different from the usually suggested mechanism.

Synthesis of peptide .alpha.-thioesters

DOEpatents

Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

2008-08-19

Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

Ghrelin enhancer, rikkunshito, improves postprandial gastric motor dysfunction in an experimental stress model.

PubMed

Harada, Y; Ro, S; Ochiai, M; Hayashi, K; Hosomi, E; Fujitsuka, N; Hattori, T; Yakabi, K

2015-08-01

Functional dyspepsia (FD) is one of the most common disorders of gastrointestinal (GI) diseases. However, no curable treatment is available for FD because the detailed mechanism of GI dysfunction in stressed conditions remains unclear. We aimed to clarify the association between endogenous acylated ghrelin signaling and gastric motor dysfunction and explore the possibility of a drug with ghrelin signal-enhancing action for FD treatment. Solid gastric emptying (GE) and plasma acylated ghrelin levels were evaluated in an urocortin1 (UCN1) -induced stress model. To clarify the role of acylated ghrelin on GI dysfunction in the model, exogenous acylated ghrelin, an endogenous ghrelin enhancer, rikkunshito, or an α2 -adrenergic receptor (AR) antagonist was administered. Postprandial motor function was investigated using a strain gauge force transducer in a free-moving condition. Exogenous acylated ghrelin supplementation restored UCN1-induced delayed GE. Alpha2 -AR antagonist and rikkunshito inhibited the reduction in plasma acylated ghrelin and GE in the stress model. The action of rikkunshito on delayed GE was blocked by co-administration of the ghrelin receptor antagonist. UCN1 decreased the amplitude of contraction in the antrum while increasing it in the duodenum. The motility index of the antrum but not the duodenum was significantly reduced by UCN1 treatment, which was improved by acylated ghrelin or rikkunshito. The UCN1-induced gastric motility dysfunction was mediated by abnormal acylated ghrelin dynamics. Supplementation of exogenous acylated ghrelin or enhancement of endogenous acylated ghrelin secretion by rikkunshito may be effective in treating functional GI disorders. © 2015 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.

Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed omega-3 and omega-6 fatty acids.

PubMed Central

Rustan, A C; Christiansen, E N; Drevon, C A

1992-01-01

Rats were fed, for 3 weeks, high-fat (20% w/w) diets containing sunflower-seed oil, linseed oil or fish oil. Chow-fed rats were used as a low-fat reference. The high-fat diets markedly reduced non-fasting-rat serum triacylglycerol as compared with the low-fat reference, and the highest reduction (85%) was observed with the fish-oil group, which was significantly lower than that of the other high-fat diets. The serum concentration of phospholipids was significantly reduced (30%) only in the fish-oil-fed animals, whereas serum non-esterified fatty acids were reduced 40-50% by both the fish-oil- and linseed-oil-fed groups. The liver content of triacylglycerol showed a 1.7-fold increase with the fish-oil diet and 2-2.5-fold with the other dietary groups when compared with rats fed a low-fat diet, whereas the hepatic content of phospholipids was unchanged. Peroxisomal fatty acid oxidation (acyl-CoA oxidase) was 2-fold increased for the rats fed fish oil; however this was not significantly higher when comparison was made with rats fed the linseed-oil diet. There was no difference in phosphatidate hydrolysis (microsomal and cytosolic fractions) among animals fed the various diets. Acyl-CoA:diacylglycerol acyltransferase activity was increased by all high-fat diets, but the fish-oil-diet-fed group showed a significantly lower enzyme activity than did rats fed the other high-fat diets. A linear correlation between acyl-CoA:diacylglycerol acyltransferase activity and liver triacylglycerol was observed, and the microsomal enzyme activity was decreased 40-50% by incubation in the presence of eicosapentaenoyl-CoA. CoA derivatives of arachidonic, linolenic and linoleic acid had no inhibitory effect when compared with the control. These results indicate that dietary fish oil may have greater triacylglycerol-lowering effect than other polyunsaturated diets, owing to decreased triacylglycerol synthesis caused by inhibition of acyl-CoA:diacylglycerol acyltransferase. In addition, increased peroxisomal fatty acid oxidation and decreased availability of non-esterified fatty acids could also contribute by decreasing the amounts of fatty acids as substrates for triacylglycerol synthesis and secretion. Images Fig. 3. PMID:1349473

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

Kinetic studies of the acylation of pig muscle–d-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism

PubMed Central

Harrigan, P. J.; Trentham, D. R.

1973-01-01

In the presence of NAD+ the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD+ and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H+ indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5–8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results. PMID:4360248

The Ghrelin/GOAT System Regulates Obesity-Induced Inflammation in Male Mice.

PubMed

Harvey, Rebecca E; Howard, Victor G; Lemus, Moyra B; Jois, Tara; Andrews, Zane B; Sleeman, Mark W

2017-07-01

Ghrelin plays a key role in appetite, energy homeostasis, and glucose regulation. Recent evidence suggests ghrelin suppresses inflammation in obesity; however, whether this is modulated by the acylated and/or des-acylated peptide is unclear. We used mice deficient in acylated ghrelin [ghrelin octanoyl-acyltransferase (GOAT) knockout (KO) mice], wild-type (WT) littermates, and C57BL/6 mice to examine the endogenous and exogenous effects of acyl and des-acyl ghrelin on inflammatory profiles under nonobese and obese conditions. We demonstrate that in the spleen, both ghrelin and GOAT are localized primarily in the red pulp. Importantly, in the thymus, ghrelin was predominantly localized to the medulla, whereas GOAT was found in the cortex, implying differing roles in T cell development. Acute exogenous treatment with acyl/des-acyl ghrelin suppressed macrophage numbers in spleen and thymus in obese mice, whereas only acyl ghrelin increased CD3+ T cells in the thymus in mice fed both chow and a high-fat-diet (HFD). Consistent with this result, macrophages were increased in the spleen of KO mice on a HFD. Whereas there was no difference in CD3+ T cells in the plasma, spleen, or thymus of WT vs KO mice, KO chow and HFD-fed mice displayed decreased leukocytes. Our results suggest that the acylation status affects the anti-inflammatory properties of ghrelin under chow and HFD conditions. Copyright © 2017 Endocrine Society.

Identification of N-acyl-fumonisin B1 as new cytotoxic metabolites of fumonisin mycotoxins.

PubMed

Harrer, Henning; Laviad, Elad L; Humpf, Hans Ulrich; Futerman, Anthony H

2013-03-01

Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Code of Federal Regulations, 2014 CFR

2014-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

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.

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.

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.

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.

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.

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

Code of Federal Regulations, 2014 CFR

2014-07-01

...-(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...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

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

Code of Federal Regulations, 2013 CFR

2013-07-01

...-(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...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

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

Code of Federal Regulations, 2012 CFR

2012-07-01

...-(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...-(carboxymethyl)-N,N-dimethyl-, N-soya acyl derivs., inner salts (PMN P-03-46; CAS No. 136504-87-5) is subject to...

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...

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...

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

PubMed

Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

2016-01-15

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides. Copyright © 2015 Elsevier B.V. All rights reserved.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

PubMed

Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

2008-12-01

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

Utilization of sophorolipids as biosurfactants for postemergence herbicides

USDA-ARS?s Scientific Manuscript database

Sophorolipids are carbohydrate-based, amphiphilic biosurfactants produced by several species of the Starmerella yeast clade. Most sophorolipids are partially acetylated sophorose sugars O-ß-glycosidically linked to 17-L-hydroxy-delta9-octadecenoic acid, where typically the acyl carboxyl group forms...

Exogenous lysophospholipids with large head groups perturb clathrin-mediated endocytosis.

PubMed

Ailte, Ieva; Lingelem, Anne Berit D; Kvalvaag, Audun S; Kavaliauskiene, Simona; Brech, Andreas; Koster, Gerbrand; Dommersnes, Paul G; Bergan, Jonas; Skotland, Tore; Sandvig, Kirsten

2017-03-01

In this study, we have investigated how clathrin-dependent endocytosis is affected by exogenously added lysophospholipids (LPLs). Addition of LPLs with large head groups strongly inhibits transferrin (Tf) endocytosis in various cell lines, while LPLs with small head groups do not. Electron and total internal reflection fluorescence microscopy (EM and TIRF) reveal that treatment with lysophosphatidylinositol (LPI) with the fatty acyl group C18:0 leads to reduced numbers of invaginated clathrin-coated pits (CCPs) at the plasma membrane, fewer endocytic events per membrane area and increased lifetime of CCPs. Also, endocytosis of Tf becomes dependent on actin upon LPI treatment. Thus, our results demonstrate that one can regulate the kinetics and properties of clathrin-dependent endocytosis by addition of LPLs in a head group size- and fatty acyl-dependent manner. Furthermore, studies performed with optical tweezers show that less force is required to pull membrane tubules outwards from the plasma membrane when LPI is added to the cells. The results are in agreement with the notion that insertion of LPLs with large head groups creates a positive membrane curvature which might have a negative impact on events that require plasma membrane invagination, while it may facilitate membrane bending toward the cell exterior. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Wheat leaf lipids during heat stress: II. Lipids experiencing coordinated metabolism are detected by analysis of lipid co-occurrence

PubMed Central

Narayanan, Sruthi; Prasad, P.V. Vara; Welti, Ruth

2016-01-01

Identifying lipids that experience coordinated metabolism during heat stress would provide information regarding lipid dynamics under stress conditions and assist in developing heat-tolerant wheat varieties. We hypothesized that co-occurring lipids, which are up-or-down-regulated together through time during heat stress, represent groups that can be explained by coordinated metabolism. Wheat plants (Triticum aestivum L.) were subjected to 12 days of high day and/or night temperature stress, followed by a 4-day recovery period. Leaves were sampled at four time points, and 165 lipids were measured by electrospray ionization-tandem mass spectrometry. Correlation analysis of lipid levels in 160 leaf samples from each of two wheat genotypes revealed 13 groups of lipids. Lipids within each group co-occurred through the high day and night temperature stress treatments. The lipid groups can be broadly classified as groups containing: extraplastidic phospholipids, plastidic glycerolipids, oxidized glycerolipids, triacylglycerols, acylated sterol glycosides, and sterol glycosides. Current knowledge of lipid metabolism suggests that the lipids in each group co-occur because they are regulated by the same enzyme(s). The results suggest that increases in activities of desaturating, oxidizing, glycosylating, and acylating enzymes lead to simultaneous changes in levels of multiple lipid species during high day and night temperature stress in wheat. PMID:26436445

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

Crystal Structures of a Quorum-Quenching Antibody

PubMed Central

Debler, Erik W.; Kaufmann, Gunnar F.; Kirchdoerfer, Robert N.; Mee, Jenny M.; Janda, Kim D.; Wilson, Ian A.

2007-01-01

Summary A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely-related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short and narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow for entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody combining site with a closed binding pocket, suggestive of an induced fit mechanism for ligand binding. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will now aid in further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing. PMID:17400249

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation

PubMed Central

2017-01-01

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response. PMID:28737384

A retrospective review of anesthesia and perioperative care in children with medium-chain acyl-CoA dehydrogenase deficiency.

PubMed

Allen, Claire; Perkins, Russell; Schwahn, Bernd

2017-01-01

Medium-chain acyl-CoA dehydrogenase deficiency is the most common genetically determined disorder of mitochondrial fatty acid oxidation. Decompensation can result in hypoglycemia, seizures, coma, and death but may be prevented by ensuring glycogen stores do not become depleted. Perioperative care is of interest as surgery, fasting, and infection may all trigger decompensation and the safety of anesthetic agents has been questioned. Current guidelines from the British Inherited Metabolic Disease Group advise on administering fluid containing 10% glucose during the perioperative period. To review the management of anesthesia and perioperative care for children with medium-chain acyl-CoA dehydrogenase deficiency and determine the frequency and nature of any complications. A retrospective review of case notes of children with medium-chain acyl-CoA dehydrogenase deficiency undergoing anesthesia between 1997 and 2014. Fourteen patients underwent 21 episodes of anesthesia. In 20 episodes, the patient received a glucose-containing fluid during their perioperative fast, of which eight received fluid containing 10% dextrose throughout the entire perioperative period. No episodes of hypoglycemia or decompensation occurred, but perioperative hyperglycemia occurred in five episodes. A propofol bolus was administered at induction in 16 episodes and volatile agents were administered for maintenance of anesthesia in all episodes without any observed complications. In one episode, delayed offset of atracurium was reported. Perioperative metabolic decompensation and hypoglycemia appear to be uncommon in children who are well and receive glucose supplementation. Hyperglycemia may occur as a consequence of surgery and glucose supplementation. Propofol boluses and volatile anesthetic agents were used without any apparent complications. Prolonged action of atracurium was reported in one case, suggesting that nondepolarizing muscle relaxants may have delayed offset in this patient group. We do not recommend any particular approach to anesthesia but would advise administering glucose supplementation according to current guidelines, frequent monitoring of blood glucose perioperatively, and monitoring of neuromuscular blockade. © 2016 John Wiley & Sons Ltd.

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.

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.

Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables

PubMed Central

Calabriso, Nadia; Berland, Helge; Maiorano, Gabriele; Gerardi, Carmela; Carluccio, Maria Annunziata; Andersen, Øyvind M.

2018-01-01

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure–activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and “Sun Black” tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection. PMID:29316619

Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.

PubMed

Dehesh, K; Edwards, P; Hayes, T; Cranmer, A M; Fillatti, J

1996-01-01

The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.

Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.

PubMed Central

Dehesh, K; Edwards, P; Hayes, T; Cranmer, A M; Fillatti, J

1996-01-01

The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system. PMID:8587983

Comparison of the Deacylase and Deacetylase Activity of Zinc-Dependent HDACs.

PubMed

McClure, Jesse J; Inks, Elizabeth S; Zhang, Cheng; Peterson, Yuri K; Li, Jiaying; Chundru, Kalyan; Lee, Bradley; Buchanan, Ashley; Miao, Shiqin; Chou, C James

2017-06-16

The acetylation status of lysine residues on histone proteins has long been attributed to a balance struck between the catalytic activity of histone acetyl transferases and histone deacetylases (HDAC). HDACs were identified as the sole removers of acetyl post-translational modifications (PTM) of histone lysine residues. Studies into the biological role of HDACs have also elucidated their role as removers of acetyl PTMs from lysine residues of nonhistone proteins. These findings, coupled with high-resolution mass spectrometry studies that revealed the presence of acyl-group PTMs on lysine residues of nonhistone proteins, brought forth the possibility of HDACs acting as removers of both acyl- and acetyl-based PTMs. We posited that HDACs fulfill this dual role and sought to investigate their specificity. Utilizing a fluorescence-based assay and biologically relevant acyl-substrates, the selectivities of zinc-dependent HDACs toward these acyl-based PTMs were identified. These findings were further validated using cellular models and molecular biology techniques. As a proof of principal, an HDAC3 selective inhibitor was designed using HDAC3's substrate preference. This resulting inhibitor demonstrates nanomolar activity and >30 fold selectivity toward HDAC3 compared to the other class I HDACs. This inhibitor is capable of increasing p65 acetylation, attenuating NF-κB activation, and thereby preventing downstream nitric oxide signaling. Additionally, this selective HDAC3 inhibition allows for control of HMGB-1 secretion from activated macrophages without altering the acetylation status of histones or tubulin.

Acyltransferases in Bacteria

PubMed Central

Röttig, Annika

2013-01-01

SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue. PMID:23699259

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles.

PubMed

Borden, Mark A; Pu, Gang; Runner, Gabriel J; Longo, Marjorie L

2004-06-01

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating-cooling followed by compression-expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.

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.

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.

Influence of etanercept on leptin and ghrelin secretion in children with juvenile idiopathic arthritis.

PubMed

Maciejewska-Paszek, Izabela; Grochowska-Niedworok, Elżbieta; Siwiec, Andrzej; Gruenpeter, Anna; Dul, Lechosław; Irzyniec, Tomasz

2017-04-01

Objective To assess possible changes in leptin and ghrelin secretion due to etanercept in juvenile idiopathic arthritis (JIA). Methods 50 patients with JIA and 16 age-matched controls were enrolled into this prospective, cross-sectional study. Serum leptin, total and acyl ghrelin were measured in addition to white blood cell (WBC) and lymphocyte counts. Results 25 patients received etanercept and 25 conventional therapies (including methotrexate) for JIA. There was no difference between treatment and control groups in leptin or ghrelin levels and no evidence of a relationship between leptin and ghrelin in patients with JIA. In all children with JIA there was a correlation between leptin and body mass index (BMI). However, compared with children in the conventional treatment group, children in the etanercept group showed a positive correlation between total ghrelin and BMI and those with a low BMI showed a negative correlation between acyl ghrelin and BMI. Conclusion No differences in leptin and ghrelin concentrations were found when patients with JIA and controls were compared or when patients who received etanercept were compared with those who received conventional treatment for JIA.

Influence of etanercept on leptin and ghrelin secretion in children with juvenile idiopathic arthritis

PubMed Central

Maciejewska-Paszek, Izabela; Grochowska-Niedworok, Elżbieta; Siwiec, Andrzej; Gruenpeter, Anna; Dul, Lechosław

2017-01-01

Objective To assess possible changes in leptin and ghrelin secretion due to etanercept in juvenile idiopathic arthritis (JIA). Methods 50 patients with JIA and 16 age-matched controls were enrolled into this prospective, cross-sectional study. Serum leptin, total and acyl ghrelin were measured in addition to white blood cell (WBC) and lymphocyte counts. Results 25 patients received etanercept and 25 conventional therapies (including methotrexate) for JIA. There was no difference between treatment and control groups in leptin or ghrelin levels and no evidence of a relationship between leptin and ghrelin in patients with JIA. In all children with JIA there was a correlation between leptin and body mass index (BMI). However, compared with children in the conventional treatment group, children in the etanercept group showed a positive correlation between total ghrelin and BMI and those with a low BMI showed a negative correlation between acyl ghrelin and BMI. Conclusion No differences in leptin and ghrelin concentrations were found when patients with JIA and controls were compared or when patients who received etanercept were compared with those who received conventional treatment for JIA. PMID:28415953

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.

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

Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

PubMed

Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

2015-06-01

N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

Structure elucidation and DFT-study on substrate-selective formation of chalcones containing ferrocene and phenothiazine units. Study on ferrocenes, Part 17

NASA Astrophysics Data System (ADS)

Lovász, Tamás; Túrós, György; Găină, Luiza; Csámpai, Antal; Frigyes, Dávid; Fábián, Balázs; Silberg, Ioan A.; Sohár, Pál

2005-09-01

By means of base-catalysed condensation of 1-acyl-/1,1'-diacylferrocenes (acyl dbnd6 formyl or acetyl) with 3-formyl- and 3,7-diacetylphenothiazines a series of novel mono- and bis-chalcones were prepared. The enhanced reactivity of the enolate anions of the mono-chalcone intermediates relative to that of the enolates of the corresponding diacetyl-substituted precursor was interpreted by the electron-releasing effect of the ferrocenyl- or phenothiazinyl group present in the β position of the enone subunit. The structures of the novel products were evidenced by IR, 1H and 13C NMR spectroscopy including 2D-COSY, 2D-HSQC and 2D-HMBC measurements.

A novel biocatalytic approach to acetylation of 1-β-D-arabinofuranosylcytosine by Aspergillus oryzae whole cell in organic solvents.

PubMed

Li, Xiao-Feng; Zhu, Zhen; Zhao, Guang-Lei; Yu, Yi-Gang; Lai, Fu-Rao; Wu, Hui

2012-01-01

Biocatalytic acylation of 1-β-D-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. (13)C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3'-hydroxyl group than 5'-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3'-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3'-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.

Cytotoxic triterpenoid saponins from the fruits of Aesculus pavia L.

PubMed

Zhang, Zhizhen; Li, Shiyou

2007-08-01

Continued chemical investigation on the fruits of North American Aesculus pavia L. resulted in the isolation and identification of 13 polyhydroxyoleanene pentacyclic triterpenoid saponins, named aesculiosides IIe-IIk (1-7), and IIIa-IIIf (8-13), together with 18 known compounds: aesculiosides Ia-Ie (14-18), IIa-IId (19-22), IVa-IVc (23-25), 3-O-[beta-D-galactopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,15 alpha,16 alpha,21 beta,22 alpha,28-hexahydroxyolean-12-ene (26), 3-O-[beta-D-glucopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,16 alpha,21 beta,22 alpha,24 beta,28-hexahydroxyolean-12-ene (27), 3-O-[beta-D-galactopyranosyl(1-->2)]-alpha-L-arabinofuranosyl(1-->3)-beta-D-glucuronopyranosyl-21,22-O-diangeloyl-3beta,16 alpha,21 beta,22 alpha,28-pentahydroxyolean-12-ene (28), R(1)-barrigenol (29), scopolin (30), and 5-methoxyscopolin (31). The structures of these compounds were elucidated by spectroscopic and chemical analyses. Compounds 14-22 and 26-28 were tested in vitro for their activity against 59 cell lines from nine different human cancers including leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast. It was found that compounds with two-acyl groups at C-21 and C-22 had cytotoxic activity for all cell lines tested with GI(50) 0.175-8.71 microM, while compounds without acyl groups at C-21 and C-22 had weak or no cytotoxic activity. These results suggest that the acyl groups at C-21 and C-22 are essential for their activity.

Administration of exogenous acylated ghrelin or rikkunshito, an endogenous ghrelin enhancer, improves the decrease in postprandial gastric motility in an acute restraint stress mouse model

PubMed Central

Nahata, M; Saegusa, Y; Sadakane, C; Yamada, C; Nakagawa, K; Okubo, N; Ohnishi, S; Hattori, T; Sakamoto, N; Takeda, H

2014-01-01

Background Physical or psychological stress causes functional disorders in the upper gastrointestinal tract. This study aims to elucidate the ameliorating effect of exogenous acylated ghrelin or rikkunshito, a Kampo medicine which acts as a ghrelin enhancer, on gastric dysfunction during acute restraint stress in mice. Methods Fasted and postprandial motor function of the gastric antrum was wirelessly measured using a strain gauge force transducer and solid gastric emptying was detected in mice exposed to restraint stress. Plasma corticosterone and ghrelin levels were also measured. To clarify the role of ghrelin on gastrointestinal dysfunction in mice exposed to stress, exogenous acylated ghrelin or rikkunshito was administered, then the mice were subjected to restraint stress. Key Results Mice exposed to restraint stress for 60 min exhibited delayed gastric emptying and increased plasma corticosterone levels. Gastric motility was decreased in mice exposed to restraint stress in both fasting and postprandial states. Restraint stress did not cause any change in plasma acylated ghrelin levels, but it significantly increased the plasma des-acyl ghrelin levels. Administration of acylated ghrelin or rikkunshito improved the restraint stress-induced delayed gastric emptying and decreased antral motility. Ameliorating effects of rikkunshito on stress-induced gastric dysfunction were abolished by simultaneous administration of a ghrelin receptor antagonist. Conclusions & Inferences Plasma acylated/des-acyl ghrelin imbalance was observed in acute restraint stress. Supplementation of exogenous acylated ghrelin or enhancement of endogenous ghrelin signaling may be useful in the treatment of decreased gastric function caused by stress. PMID:24684160

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

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation.

PubMed

Tang, Mingguo; Guschina, Irina A; O'Hara, Paul; Slabas, Antoni R; Quant, Patti A; Fawcett, Tony; Harwood, John L

2012-10-01

Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14)C]acetate, but stimulated that from [U-14C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27±0.06 for Block A and 0.73±0.06 for Block B. The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Synthesis of Highly Polymerized Water-soluble Cellulose Acetate by the Side Reaction in Carboxylate Ionic Liquid 1-ethyl-3-methylimidazolium Acetate

NASA Astrophysics Data System (ADS)

Pang, Jinhui; Liu, Xin; Yang, Jun; Lu, Fachuang; Wang, Bo; Xu, Feng; Ma, Mingguo; Zhang, Xueming

2016-09-01

In the present study, we describe a novel one-step method to prepare water-soluble cellulose acetate (WSCA) with higher degree of polymerization values (DP = 650-680) by in situ activation of carboxyl group in ionic liquid. First of all, cellulose was dissolved in 1-ethyl-3-methylimidazolium acetate (EmimAc) and reacted with dichloroacetyl chloride (Cl2AcCl) in order to make cellulose dichloroacetate. Under various conditions, a series of water soluble products were produced. Elemental analysis and NMR results confirmed that they were cellulose acetate with DS (degree of substitution) values in the range from 0.30 to 0.63. NMR studies demonstrated that Cl2AcCl reacted with acetate anion of EmimAc producing a mixed anhydride that acetylated cellulose. Other acylating reagents such as benzoyl chloride, chloroacetyl chloride can also work similarly. 2D NMR characterization suggested that 6-mono-O-acetyl moiety, 3,6-di-O-acetylcellulose and 2,6-di-O-acetyl cellulose were all synthesized and the reactivity of hydroxyl groups in anhydro-glucose units was in the order C-6>C-3>C-2. This work provides an alternative way to make WSCA, meanwhile, also services as a reminder that the activity of EmimAc toward carbohydrate as acylating reagents could be a problem, because the expected acylated products may not be resulted and recycling of this ionic liquid could also be difficult.

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

PubMed Central

Nile, Aaron H.; Mukund, Susmith; Stanger, Karen; Wang, Weiru; Hannoush, Rami N.

2017-01-01

Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt–FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt–FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function. PMID:28377511

Investigation of some characteristics of polyhydroxy milkweed triglycerides and their acylated derivatives in relation to lubricity.

PubMed

Harry-O'kuru, Rogers E; Biresaw, Girma; Cermak, Steven C; Gordon, Sherald H; Vermillion, Karl

2011-05-11

Most industrial lubricants are derived from nonrenewable petroleum-based sources. As useful as these lubricants are, their unintended consequences are the pollution of the Earth's environment as a result of the slow degradation of the spent materials. Native seed oils, on the other hand, are renewable and are also biodegradable in the environment, but these oils often suffer a drawback in having lower thermal stability and a shorter shelf life because of the intrinsic -C═C- unsaturation in their structures. This drawback can be overcome, yet the inherent biodegradative property retained, by appropriate derivatization of the oil. Pursuant to this, this study investigated derivatized polyhydroxy milkweed oil to assess its suitability as lubricant. The milkweed plant is a member of the Asclepiadaceae, a family with many genera including the common milkweeds, Asclepias syriaca L., Asclepias speciosa L., Asclepias tuberosa L., etc. The seeds of these species contain mainly C-18 triglycerides that are highly unsaturated, 92%. The olefinic character of this oil has been chemically modified by generating polyhydroxy triglycerides (HMWO) that show high viscosity and excellent moisturizing characteristics. In this work, HMWO have been chemically modified by esterifying their hydroxyl groups with acyl groups of various chain lengths (C2-C5). The results of investigation into the effect of the acyl derivatives' chemical structure on kinematic and dynamic viscosity, oxidation stability, cold-flow (pour point, cloud point) properties, coefficient of friction, wear, and elastohydrodynamic film thickness are discussed.

Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

PubMed

Larsen, Jannik B; Kennard, Celeste; Pedersen, Søren L; Jensen, Knud J; Uline, Mark J; Hatzakis, Nikos S; Stamou, Dimitrios

2017-09-19

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Alkyl group effects on CO insertion into coordinatively unsaturated early-transition-metal alkyls. Preparations and the first structural characterizations of tantalum enolate-O and tantalum. eta. sup 2 -acyl complexes

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

Meyer, T.Y.; Garner, L.R.; Baenziger, N.C.

1990-10-03

Low-pressure carbonylation of the mono(peralkylcyclopentadienyl)tantalum(V) alkyls ({eta}-C{sub 5}Me{sub 4}R)TaR{prime}Cl{sub 3} (R = Me, Et; R{prime} = CH{sub 2}C{sub 6}H{sub 4}-p-Me, CH{sub 2}CMe{sub 3}) yields either the O-bound enolate or the {eta}{sup 2}-acyl as shown by ir/NMR spectroscopy and x-ray diffractometry. The p-tolyl enolate ({eta}-C{sub 5}Me{sub 5})Ta(OCH{double bond}CHC{sub 6}H{sub 4}-p-Me)Cl{sub 3}, derived directly from carbonylation of the tantalum 4-methylbenzyl precursor, is shown to possess a cis configuration in solution and in the solid state. Key structural features from a single-crystal x-ray diffraction study of the tetrahydrofuran-ligated enolate complex are reported. The mechanism of formation of the enolate from carbonylation of themore » 4-methylbenzyl complex is discussed. The previously reported acyl ({eta}-C{sub 5}Me{sub 4}R)Ta(C(O)CH{sub 2}CMe{sub 3})Cl{sub 3} has been reexamined and found to possess a symmetric, strongly distorted {eta}{sup 2}-acyl coordination by solution {sup 1}H NMR spectroscopy and solid-state x-ray diffractometry. The molecular structures of ({eta}-C{sub 5}Me{sub 5})Ta(OCH{double bond}CHC{sub 6}H{sub 4}-p-Me)Cl{sub 3} and ({eta}-C{sub 5}Me{sub 5})Ta(C(O)CH{sub 2}CMe{sub 3})Cl{sub 3}, which are reported here, are the first structural determinations of a tantalum enolate and of a tantalum {eta}{sup 2}-acyl. 41 refs., 2 figs., 8 tabs.« less

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter.

PubMed

Rosenberry, Terrone L; Sonoda, Leilani K; Dekat, Sarah E; Cusack, Bernadette; Johnson, Joseph L

2008-12-09

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

Analysis of the reaction of carbachol with acetylcholinesterase with thioflavin T as a coupled fluorescence reporter†

PubMed Central

Rosenberry, Terrone L.; Sonoda, Leilani K.; Dekat, Sarah E.; Cusack, Bernadette; Johnson, Joseph L.

2009-01-01

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC) which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analog of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging, because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site. PMID:19006330

Ion-pair high-speed countercurrent chromatography in fractionation of a high-molecular weight variation of acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra.

PubMed

Wybraniec, Sławomir; Jerz, Gerold; Gebers, Nadine; Winterhalter, Peter

2010-02-15

The natural pigment composition of purple bracts of Bougainvillea glabra (Nyctaginaceae) consists of a highly complex mixture of betacyanins solely differing by the substitution with a variety of acyl-oligoglycoside units. This study was focused on a two-dimensional chromatography approach, a combination of preparative high-speed countercurrent chromatography (HSCCC) and analytical C18-HPLC with ESI-DAD-MS/MS detection which finally enabled a more detailed view into the pigment profile and elucidated the existence of an overwhelming amount of varying betacyanin structures occurring in Bougainvillea bracts. The detected molecular weights of the pigments reached so far unknown high values and ranged up to maximum values of 1653 and 1683 Da for the largest molecules due to oligosaccharide linkage and multiple acyl substitutions. The preparative IP-HSCCC separation yielded 15 complex fractions containing betacyanins of enhanced polarity as well as structures with highly increased lipophilicity. Betacyanin structures extended by large oligosaccharide chains with bigger number of glycoside units and also carrying a reduced number of hydroxycinnamic acid substitutions were characteristic for polar pigments occurring mainly in the early eluting CCC fractions. IP-HSCCC was proven to be extremely effective for fractionating this complex crude betalain pigment extract into more defined 'polarity-windows'. Structural analysis by analytical LC-ESI-MS/MS in the positive ionization mode detected a total sum of 146 different betacyanin pigments in the CCC fractions of reduced complexity. Copyright 2010 Elsevier B.V. All rights reserved.

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

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

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.

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 range of non-acylated flavonoids. Copyright © 2011 Elsevier Ltd. All rights reserved.

Peroxiredoxin 6 in the repair of peroxidized cell membranes and cell signaling.

PubMed

Fisher, Aron B

2017-03-01

Peroxiredoxin 6 represents a widely distributed group of peroxiredoxins that contain a single conserved cysteine in the protein monomer (1-cys Prdx). The cys when oxidized to the sulfenic form is reduced with glutathione (GSH) catalyzed by the π isoform of GSH-S-transferase. Three enzymatic activities of the protein have been described:1) peroxidase with H 2 O 2 , short chain hydroperoxides, and phospholipid hydroperoxides as substrates; 2) phospholipase A 2 (PLA 2 ); and 3) lysophosphatidylcholine acyl transferase (LPCAT). These activities have important physiological roles in antioxidant defense, turnover of cellular phospholipids, and the generation of superoxide anion via initiation of the signaling cascade for activation of NADPH oxidase (type 2). The ability of Prdx6 to reduce peroxidized cell membrane phospholipids (peroxidase activity) and also to replace the oxidized sn-2 fatty acyl group through hydrolysis/reacylation (PLA 2 and LPCAT activities) provides a complete system for the repair of peroxidized cell membranes. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Structure and self-assembly properties of a new chitosan-based amphiphile.

PubMed

Huang, Yuping; Yu, Hailong; Guo, Liang; Huang, Qingrong

2010-06-17

A new chitosan-based amphiphile, octanoyl-chitosan-polyethylene glycol monomethyl ether (acylChitoMPEG), has been prepared using both hydrophobic octanoyl and hydrophilic polyethylene glycol monomethyl ether (MPEG) substitutions. The success of synthesis was confirmed by Fourier transform infrared (FT-IR) and (1)H NMR spectroscopy. The synthesized acylChitoMPEG exhibited good solubility in either aqueous solution or common organic solvents such as ethanol, acetone, and CHCl(3). The self-aggregation behavior of acylChitoMPEG in solutions was studied by a combination of pyrene fluorescence technique, dynamic light scattering, atomic force microscopy, and small-angle X-ray scattering (SAXS). The critical aggregation concentration (CAC) and hydrodynamic diameter were found to be 0.066 mg/mL and 24.4 nm, respectively. SAXS results suggested a coiled structure of the triple helical acylChitoMPEG backbone with the hydrophobic moieties hiding in the center of the backbone, and the hydrophilic MPEG chains surrounding the acylChitoMPEG backbone in a random Gaussian chain conformation. Cytotoxicity results showed that acylChitoMPEG exhibited negligible cytotoxicity even at concentrations as high as 1.0 mg/mL. All results implied that acylChitoMPEG has the potential to be used for biological or medical applications.

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.

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.

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

Extractant composition

DOEpatents

Smith, Barbara F.; Jarvinen, Gordon D.; Ryan, Robert R.

1990-01-01

An organic extracting solution useful for separating elements of the actinide series of the periodic table from elements of the lanthanide series, where both are in trivalent form. The extracting solution consists of a primary ligand and a secondary ligand, preferably in an organic solvent. The primary ligand is a substituted monothio-1,3-dicarbonyl, which includes a substituted 4-acyl-2-pyrazolin-5-thione, such as 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT). The secondary ligand is a substituted phosphine oxide, such as trioctylphosphine oxide (TOPO).

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins1

PubMed Central

Smith, Rebecca A.; Gonzales-Vigil, Eliana; Karlen, Steven D.; Park, Ji-Young; Lu, Fachuang; Wilkerson, Curtis G.; Samuels, Lacey; Ralph, John; Mansfield, Shawn D.

2015-01-01

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production. PMID:26511914

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.

PubMed

Marcella, Aaron M; Jing, Fuyuan; Barb, Adam W

2015-11-01

The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first).

PubMed

Han, Zhen; Wu, Hong; Kim, Sunjoo; Yang, Xiangkun; Li, Qianjin; Huang, He; Cai, Houjian; Bartlett, Michael G; Dong, Aiping; Zeng, Hong; Brown, Peter J; Yang, Xiang-Jiao; Arrowsmith, Cheryl H; Zhao, Yingming; Zheng, Y George

2018-03-02

Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

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.

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

PubMed

Patelski, J; Pioruńska-Stolzmann, M

1985-01-01

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

PubMed Central

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

Equine atypical myopathy caused by hypoglycin A intoxication associated with ingestion of sycamore maple tree seeds.

PubMed

Żuraw, A; Dietert, K; Kühnel, S; Sander, J; Klopfleisch, R

2016-07-01

Evidence suggest there is a link between equine atypical myopathy (EAM) and ingestion of sycamore maple tree seeds. To further evaluate the hypothesis that the ingestion of hypoglycin A (HGA) containing sycamore maple tree seeds causes acquired multiple acyl-CoA dehydrogenase deficiency and might be associated with the clinical and pathological signs of EAM. Case report. Necropsy and histopathology, using hematoxylin and eosin and Sudan III stains, were performed on a 2.5-year-old mare that died following the development of clinical signs of progressive muscle stiffness and recumbency. Prior to death, the animal ingested sycamore maple tree seeds (Acer pseudoplatanus). Detection of metabolites in blood and urine obtained post mortem was performed by rapid ultra-performance liquid chromatography-tandem mass spectrometry. Data from this case were compared with 3 geldings with no clinical history of myopathy. Macroscopic examination revealed fragments of maple tree seeds in the stomach and severe myopathy of several muscle groups including Mm. intercostales, deltoidei and trapezii. Histologically, the affected muscles showed severe, acute rhabdomyolysis with extensive accumulation of finely dispersed fat droplets in the cytoplasm of degenerated skeletal muscle cells not present in controls. Urine and serum concentrations of several acyl carnitines and acyl glycines were increased, and both contained metabolites of HGA, a toxic amino acid present in sycamore maple tree seeds. The study supports the hypothesis that ingestion of HGA-containing maple tree seeds may cause EAM due to acquired multiple acyl-CoA dehydrogenase deficiency. © 2015 EVJ Ltd.

Species-Specific Activation of TLR4 by Hypoacylated Endotoxins Governed by Residues 82 and 122 of MD-2

PubMed Central

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation. PMID:25203747

Species-specific activation of TLR4 by hypoacylated endotoxins governed by residues 82 and 122 of MD-2.

PubMed

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases

DOE PAGES

Stewart, Nichole K.; Smith, Clyde A.; Frase, Hilary; ...

2014-12-08

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to anmore » increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem–GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES β-lactamases. As a result, our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.« less

Elevated Medium-Chain Acylcarnitines Are Associated With Gestational Diabetes Mellitus and Early Progression to Type 2 Diabetes and Induce Pancreatic β-Cell Dysfunction.

PubMed

Batchuluun, Battsetseg; Al Rijjal, Dana; Prentice, Kacey J; Eversley, Judith A; Burdett, Elena; Mohan, Haneesha; Bhattacharjee, Alpana; Gunderson, Erica P; Liu, Ying; Wheeler, Michael B

2018-05-01

Specific circulating metabolites have emerged as important risk factors for the development of diabetes. The acylcarnitines (acylCs) are a family of metabolites known to be elevated in type 2 diabetes (T2D) and linked to peripheral insulin resistance. However, the effect of acylCs on pancreatic β-cell function is not well understood. Here, we profiled circulating acylCs in two diabetes cohorts: 1 ) women with gestational diabetes mellitus (GDM) and 2 ) women with recent GDM who later developed impaired glucose tolerance (IGT), new-onset T2D, or returned to normoglycemia within a 2-year follow-up period. We observed a specific elevation in serum medium-chain (M)-acylCs, particularly hexanoyl- and octanoylcarnitine, among women with GDM and individuals with T2D without alteration in long-chain acylCs. Mice treated with M-acylCs exhibited glucose intolerance, attributed to impaired insulin secretion. Murine and human islets exposed to elevated levels of M-acylCs developed defects in glucose-stimulated insulin secretion and this was directly linked to reduced mitochondrial respiratory capacity and subsequent ability to couple glucose metabolism to insulin secretion. In conclusion, our study reveals that an elevation in circulating M-acylCs is associated with GDM and early stages of T2D onset and that this elevation directly impairs β-cell function. © 2018 by the American Diabetes Association.

Wheat leaf lipids during heat stress: II. Lipids experiencing coordinated metabolism are detected by analysis of lipid co-occurrence.

PubMed

Narayanan, Sruthi; Prasad, P V Vara; Welti, Ruth

2016-03-01

Identifying lipids that experience coordinated metabolism during heat stress would provide information regarding lipid dynamics under stress conditions and assist in developing heat-tolerant wheat varieties. We hypothesized that co-occurring lipids, which are up-regulated or down-regulated together through time during heat stress, represent groups that can be explained by coordinated metabolism. Wheat plants (Triticum aestivum L.) were subjected to 12 days of high day and/or night temperature stress, followed by a 4-day recovery period. Leaves were sampled at four time points, and 165 lipids were measured by electrospray ionization-tandem mass spectrometry. Correlation analysis of lipid levels in 160 leaf samples from each of two wheat genotypes revealed 13 groups of lipids. Lipids within each group co-occurred through the high day and night temperature stress treatments. The lipid groups can be broadly classified as groups containing extraplastidic phospholipids, plastidic glycerolipids, oxidized glycerolipids, triacylglycerols, acylated sterol glycosides and sterol glycosides. Current knowledge of lipid metabolism suggests that the lipids in each group co-occur because they are regulated by the same enzyme(s). The results suggest that increases in activities of desaturating, oxidizing, glycosylating and acylating enzymes lead to simultaneous changes in levels of multiple lipid species during high day and night temperature stress in wheat. © 2015 John Wiley & Sons Ltd.

Angiotensin-converting enzyme: I. New strategies for assay

PubMed Central

Ryan, James W.; Chung, Alfred; Ryan, Una S.

1980-01-01

The disposition of converting enzyme (kininase II) on the luminal surface of pulmonary endothelial cells is well established. Further, it is known that there is a net conversion of angiotensin I into angiotensin II as blood passes through the lungs. However, little is known about modulations of converting enzyme activity that may arise through, e.g., changes in the quality of inhalants, blood flow, or blood oxygenation. There are few data on the effects of lung disease. A major barrier to studies to examine for pathophysiologic modulations of converting enzyme is that of assay. The enzyme can be measured in terms of the rate of formation of angiotensin II from a known quantity of angiotensin I. However, both peptides are biologically active, and lungs contain other enzymes capable of degrading them. We have developed a series of radiolabeled, acylated tripeptides to improve our ability to examine for changes in the net converting enzyme of intact lungs. The enzyme, a dipeptidyl carboxypeptidase, is capable of removing C-terminal dipeptides from a variety of oligopeptides. We have prepared benzoyl-Gly-Gly-Gly (I), benzoyl-Pro-Phe-Arg (II), benzoyl-Gly-His-Leu (III), benzoyl-Phe-Ala-Pro (IV), and benzoyl-Phe-His-Leu (V), each containing a 3H-atom in the para position of the benzoyl moiety. Substrates I and III have been used previously in photometric assays of low sensitivity. II is the acylated C-terminal tripeptide of bradykinin, IV is an acylated tripeptide analog of BPP5a (

Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO).

PubMed

Baba, Takashi; Campbell, J Larry; Le Blanc, J C Yves; Baker, Paul R S

2016-11-01

Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (∼10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Beyond the analysis of lipid standards, this technique was applied to edible oils and natural lipid extracts to demonstrate the power of this method to provide in-depth structural elucidation of TAG molecular species. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

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...

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.

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

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.

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

Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.

PubMed

Grillo, M P; Benet, L Z

2001-08-01

Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl-cysteine products rather than the S-acyl-NAC conjugate.

Sex Differences in Psychiatric Comorbidity and Plasma Biomarkers for Cocaine Addiction in Abstinent Cocaine-Addicted Subjects in Outpatient Settings

PubMed Central

Pedraz, María; Araos, Pedro; García-Marchena, Nuria; Serrano, Antonia; Romero-Sanchiz, Pablo; Suárez, Juan; Castilla-Ortega, Estela; Mayoral-Cleries, Fermín; Ruiz, Juan Jesús; Pastor, Antoni; Barrios, Vicente; Chowen, Julie A.; Argente, Jesús; Torrens, Marta; de la Torre, Rafael; Rodríguez De Fonseca, Fernando; Pavón, Francisco Javier

2015-01-01

There are sex differences in the progression of drug addiction, relapse, and response to therapies. Because biological factors participate in these differences, they should be considered when using biomarkers for addiction. In the current study, we evaluated the sex differences in psychiatric comorbidity and the concentrations of plasma mediators that have been reported to be affected by cocaine. Fifty-five abstinent cocaine-addicted subjects diagnosed with lifetime cocaine use disorders (40 men and 15 women) and 73 healthy controls (48 men and 25 women) were clinically assessed with the diagnostic interview “Psychiatric Research Interview for Substance and Mental Disorders.” Plasma concentrations of chemokines, cytokines, N-acyl-ethanolamines, and 2-acyl-glycerols were analyzed according to history of cocaine addiction and sex, controlling for covariates age and body mass index (BMI). Relationships between these concentrations and variables related to cocaine addiction were also analyzed in addicted subjects. The results showed that the concentrations of chemokine (C-C motif) ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) and chemokine (C-X-C motif) ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) were only affected by history of cocaine addiction. The plasma concentrations of interleukin 1-beta (IL-1β), IL-6, IL-10, and tumor necrosis factor-alpha (TNFα) were affected by history of cocaine addiction and sex. In fact, whereas cytokine concentrations were higher in control women relative to men, these concentrations were reduced in cocaine-addicted women without changes in addicted men. Regarding fatty acid derivatives, history of cocaine addiction had a main effect on the concentration of each acyl derivative, whereas N-acyl-ethanolamines were increased overall in the cocaine group, 2-acyl-glycerols were decreased. Interestingly, N-palmitoleoyl-ethanolamine (POEA) was only increased in cocaine-addicted women. The covariate BMI had a significant effect on POEA and N-arachidonoyl-ethanolamine concentrations. Regarding psychiatric comorbidity in the cocaine group, women had lower incidence rates of comorbid substance use disorders than did men. For example, alcohol use disorders were found in 80% of men and 40% of women. In contrast, the addicted women had increased prevalences of comorbid psychiatric disorders (i.e., mood, anxiety, and psychosis disorders). Additionally, cocaine-addicted subjects showed a relationship between the concentrations of N-stearoyl-ethanolamine and 2-linoleoyl-glycerol and diagnosis of psychiatric comorbidity. These results demonstrate the existence of a sex influence on plasma biomarkers for cocaine addiction and on the presence of comorbid psychopathologies for clinical purposes. PMID:25762940

Organometallic macromolecules with piano stool coordination repeating units: chain configuration and stimulated solution behaviour.

PubMed

Cao, Kai; Ward, Jonathan; Amos, Ryan C; Jeong, Moon Gon; Kim, Kyoung Taek; Gauthier, Mario; Foucher, Daniel; Wang, Xiaosong

2014-09-11

Theoretical calculations illustrate that organometallic macromolecules with piano stool coordination repeating units (Fe-acyl complex) adopt linear chain configuration with a P-Fe-C backbone surrounded by aromatic groups. The macromolecules show molecular weight-dependent and temperature stimulated solution behaviour in DMSO.

The backbone N-(4-azidobutyl) linker for the preparation of peptide chimera.

PubMed

Fernández-Llamazares, Ana I; García, Jesús; Adan, Jaume; Meunier, David; Mitjans, Francesc; Spengler, Jan; Albericio, Fernando

2013-09-06

A robust synthetic strategy for the introduction of the N-(4-azidobutyl) linker into peptides using standard SPPS techniques is described. Based on the example of Cilengitide it is shown that the N-(4-azidobutyl) group exerts similar conformational restraints as a backbone N-Me group and allows conjugation of a desired molecule either via click chemistry or-after azide reduction-via acylation or reductive alkylation.

Salen- Zr(IV) complex grafted into amine-tagged MIL-101(Cr) as a robust multifunctional catalyst for biodiesel production and organic transformation reactions

NASA Astrophysics Data System (ADS)

Hassan, Hassan M. A.; Betiha, Mohamed A.; Mohamed, Shaimaa K.; El-Sharkawy, E. A.; Ahmed, Emad A.

2017-08-01

The synthesis of metal-organic frameworks (MOFs), porous coordination polymers with functional groups has received immense interest due to the functional groups can offer desirable properties and allow post-synthetic modification. Herein, for the first time, Zr(IV)-Sal Schiff base complex incorporated into amino-functionalized MIL-101(Cr) framework by salicylaldehyde condensing to amino group, and coordinating Zr(IV) ion have been successfully synthesized. The worthiness of the synthesized material as a catalyst has been examined for the esterification of oleic acid (free fatty acid) with methanol producing biodiesel (methyl oleate), Knoveonagel condensation reaction of aldehydes and Friedel-Crafts acylation of anisole. Our findings demonstrated that Salen-Zr(IV) grafted to framework of NH2-MIL-101(Cr) as a solid acid catalyst exhibited distinct catalytic performance for the production of biodiesel by esterification of oleic acid with methanol, Knoveonagel condensation and Friedel-Crafts acylation. These could be attributed to high surface area which allow high distribution of Zr(IV) species lead to a sufficient contact with the reactants species. Furthermore, the catalyst showed excellent recycling efficiency due to the strong interaction between the Zr(IV) ions and chelating groups in the NH2-MIL-101(Cr)-Sal.

Analysis of amino acids and acyl carnitine profiles in low birth weight, preterm, and small for gestational age neonates.

PubMed

Liu, Qian; Wu, Jing; Shen, Wen; Wei, Ran; Jiang, Jianhui; Liang, Jinqun; Chen, Min; Zhong, Mei; Yin, Aihua

2017-11-01

To analyze the amino acids (AA) and acyl carnitine (AC) profiles in dry blood spot (DBS) specimens of low birth weight (LBW), preterm birth (PTB), and small for gestational age (SGA), and to compare the concentration difference of AA and AC with those without above. This is a retrospectively study. Eight thousand nine hundred and seventy-nine uncomplicated pregnant newborns were enrolled into the study. DBS were collected on the third day of life, and concentrations of 11 types of AA, free carnitine and 30 types of AC were detected by using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). Shapiro-Wilk test and Kruskal-Wallis rank test were applied in statistical analysis. Concentrations of most AA and AC in infants born in SGA were significantly higher than those in non-SGA group, while lower in LBW and PTB groups than those in non-LBW and non-PTB groups (p < 0.05). The difference of concentration of AA and AC in the subgroups suggested there may be a dysutilization of AA and AC in SGA, but an inborn insufficient of AA and AC in LBW and PTB neonates.

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

RAPID 2D NMR METHOD FOR DETERMINING P-COUMARATE AND FERULATE LEVELS IN CORN (AND OTHER GRASS) CELL WALLS

USDA-ARS?s Scientific Manuscript database

Grass cell wall components are acylated by the hydroxycinnamates p-coumarate and ferulate. p-Coumarates largely acylate lignin sidechains, exclusively at the gamma-position, whereas ferulates primarily acylate the arabinosyl C5-position of arabinoxylans. Such components can be quantified as the corr...

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.

Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals

PubMed Central

Xu, Peng; Qiao, Kangjian; Ahn, Woo Suk; Stephanopoulos, Gregory

2016-01-01

Harnessing lipogenic pathways and rewiring acyl-CoA and acyl-ACP (acyl carrier protein) metabolism in Yarrowia lipolytica hold great potential for cost-efficient production of diesel, gasoline-like fuels, and oleochemicals. Here we assessed various pathway engineering strategies in Y. lipolytica toward developing a yeast biorefinery platform for sustainable production of fuel-like molecules and oleochemicals. Specifically, acyl-CoA/acyl-ACP processing enzymes were targeted to the cytoplasm, peroxisome, or endoplasmic reticulum to generate fatty acid ethyl esters and fatty alkanes with tailored chain length. Activation of endogenous free fatty acids and the subsequent reduction of fatty acyl-CoAs enabled the efficient synthesis of fatty alcohols. Engineering a hybrid fatty acid synthase shifted the free fatty acids to a medium chain-length scale. Manipulation of alternative cytosolic acetyl-CoA pathways partially decoupled lipogenesis from nitrogen starvation and unleashed the lipogenic potential of Y. lipolytica. Taken together, the strategies reported here represent promising steps to develop a yeast biorefinery platform that potentially upgrades low-value carbons to high-value fuels and oleochemicals in a sustainable and environmentally friendly manner. PMID:27621436

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

PubMed

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Saturated Heterocyclic Aminosulfonyl Fluorides: New Scaffolds for Protecting-Group-Free Synthesis of Sulfonamides.

PubMed

Zhersh, Sergey A; Blahun, Oleksandr P; Sadkova, Iryna V; Tolmachev, Andrey A; Moroz, Yurii S; Mykhailiuk, Pavel K

2018-06-12

Cyclic saturated aminosulfonyl fluorides were synthesized as their HCl salts. The compounds were found to be stable upon storage and could be used for the protecting-group-free synthesis of sulfonamides. In the presence of the -SO 2 F group, the nitrogen atom could be modified by means of acylation, arylation, or reductive amination to give products that have high potential for the synthesis of bioactive compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acyl chain conformational ordering of individual components in liquid-crystalline bilayers of mixtures of phosphatidylcholines and phosphatidic acids. A comparative FTIR and 2H NMR study

NASA Astrophysics Data System (ADS)

Ziegler, Wolfgang; Blume, Alfred

1995-09-01

The conformational ordering of the acyl chains of all possible binary 1:1 mixtures containing the phospholipids DMPC, DMPA, DPPC, and DPPA was investigated using FTIR and 2H NMR spectroscopy. One of the components was always labelled with perdeuterated chains to be able to observe the individual behaviour of the two components. From the temperature dependence of the frequencies of the symmetric and antisymmetric CH 2- and CD 2-stretching vibrations the transition temperatures were determined. The integral intensities of the conformation sensitive CH 2-wagging bands at ca. 1368 cm -1(gtg' and gtg sequences), 1356 cm -1 (double gauche), and 1342 cm -1 (end gauche) can be converted to numbers of gauche conformers per acyl chain using calibration factors published by Senak et al. J. Phys. Chem. 95 (1991) 2565. The 2H NMR quadrupolar splittings of the CD 2-segments of the perdeuterated lipid components are affected not only by trans-gauche isomerizations but also by long axis rotations and restricted wobbling motions of the acyl chains. The values of the average gauche probability overlinep3 from FTIR spectroscopy and the average order parameters overlineSCD, the order parameter of the terminal methyl groups SCDCD 3 and the average order parameter for the plateau region overlineSCDPlat of components in the mixtures are compared to those of the pure lipids evaluated in a previous publication Tuchtenhagen et al. Eur. Biophys. J. 23 (1994) 323. The conformational behaviour of lipids in mixtures is mainly influenced by head groups interactions, PAs always being more ordered than the corresponding PCs. Depending on absolute chain length and on chain length differences between the two components different conformational behaviour is observed for the two components in the mixtures, indicating non-ideal mixing and formation of micro-domains in the liquid-crystalline phase. Increases in order at the chain ends with a concomitant decrease in probabilities for end gauche conformations give hints to chain interdigitation in the liquid-crystalline phase.

The gut hormone ghrelin partially reverses energy substrate metabolic alterations in the failing heart.

PubMed

Mitacchione, Gianfranco; Powers, Jeffrey C; Grifoni, Gino; Woitek, Felix; Lam, Amy; Ly, Lien; Settanni, Fabio; Makarewich, Catherine A; McCormick, Ryan; Trovato, Letizia; Houser, Steven R; Granata, Riccarda; Recchia, Fabio A

2014-07-01

The gut-derived hormone ghrelin, especially its acylated form, plays a major role in the regulation of systemic metabolism and exerts also relevant cardioprotective effects; hence, it has been proposed for the treatment of heart failure (HF). We tested the hypothesis that ghrelin can directly modulate cardiac energy substrate metabolism. We used chronically instrumented dogs, 8 with pacing-induced HF and 6 normal controls. Human des-acyl ghrelin [1.2 nmol/kg per hour] was infused intravenously for 15 minutes, followed by washout (rebaseline) and infusion of acyl ghrelin at the same dose. (3)H-oleate and (14)C-glucose were coinfused and arterial and coronary sinus blood sampled to measure cardiac free fatty acid and glucose oxidation and lactate uptake. As expected, cardiac substrate metabolism was profoundly altered in HF because baseline oxidation levels of free fatty acids and glucose were, respectively, >70% lower and >160% higher compared with control. Neither des-acyl ghrelin nor acyl ghrelin significantly affected function and metabolism in normal hearts. However, in HF, des-acyl and acyl ghrelin enhanced myocardial oxygen consumption by 10.2±3.5% and 9.9±3.7%, respectively (P<0.05), and cardiac mechanical efficiency was not significantly altered. This was associated, respectively, with a 41.3±6.7% and 32.5±10.9% increase in free fatty acid oxidation and a 31.3±9.2% and 41.4±8.9% decrease in glucose oxidation (all P<0.05). Acute increases in des-acyl or acyl ghrelin do not interfere with cardiac metabolism in normal dogs, whereas they enhance free fatty acid oxidation and reduce glucose oxidation in HF dogs, thus partially correcting metabolic alterations in HF. This novel mechanism might contribute to the cardioprotective effects of ghrelin in HF. © 2014 American Heart Association, Inc.

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 Identification and Quantification of Suberin Monomers of Root and Tuber Periderm from Potato (Solanum tuberosum) as Fatty Acyl tert-Butyldimethylsilyl Derivatives.

PubMed

Company-Arumí, Dolors; Figueras, Mercè; Salvadó, Victoria; Molinas, Marisa; Serra, Olga; Anticó, Enriqueta

2016-11-01

Protective plant lipophilic barriers such as suberin and cutin, with their associated waxes, are complex fatty acyl derived polyesters. Their precise chemical composition is valuable to understand the specific role of each compound to the physiological function of the barrier. To develop a method for the compositional analysis of suberin and associated waxes by gas chromatography (GC) coupled to ion trap-mass spectrometry (IT-MS) using N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) as sylilating reagent, and apply it to compare the suberin of the root and tuber periderm of potato (Solanum tuberosum). Waxes and suberin monomers from root and periderm were extracted subsequently using organic solvents and by methanolysis, and subjected to MTBSTFA derivatisation. GC analyses of periderm extracts were used to optimise the chromatographic method and the compound identification. Quantitative data was obtained using external calibration curves. The method was fully validated and applied for suberin composition analyses of roots and periderm. Wax and suberin compounds were successfully separated and compound identification was based on the specific (M-57) and non-specific ions in mass spectra. The use of calibration curves built with different external standards provided quantitative accurate data and showed that suberin from root contains shorter chained fatty acyl derivatives and a relative predominance of α,ω-alkanedioic acids compared to that of the periderm. We present a method for the analysis of suberin and their associated waxes based on MTBSTFA derivatisation. Moreover, the characteristic root suberin composition may be the adaptive response to its specific regulation of permeability to water and gases. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Interconversion of ephedrine and pseudoephedrine during chemical derivatization.

PubMed

Wong, Colton H F; Ho, Emmie N M; Kwok, W H; Leung, David K K; Leung, Gary N W; Tang, Francis P W; Wong, April S Y; Wong, Jenny K Y; Yu, Nola H; Wan, Terence S M

2012-12-01

Gas chromatography-mass spectrometry (GC-MS) analysis after heptafluorobutyric anhydride (HFBA) derivatization was one of the published methods used for the quantification of ephedrine (EP) and pseudoephedrine (PE) in urine. This method allows the clear separation of the derivatized diastereoisomers on a methyl-silicone-based column. Recently the authors came across a human urine sample with apparently high levels (µg/ml) of EP and PE upon initial screening. However, duplicate analyses of this sample using the HFBA-GC-MS method revealed an unusual discrepancy in the estimated levels of EP and PE, with the area response ratios of EP/PE at around 29% on one occasion and around 57% on another. The same sample was re-analyzed for EP and PE using other techniques, including GC-MS after trimethylsilylation and ultra-high-performance liquid chromatography-tandem mass spectrometry. Surprisingly, the concentration of EP in the sample was determined to be at least two orders of magnitude less than what was observed with the HFBA-GC-MS method. A thorough investigation was then conducted, and the results showed that both substances could interconvert during HFBA derivatization. Similar diastereoisomeric conversion was also observed using other fluorinated acylating agents (e.g. pentafluoropropionic anhydride and trifluoroacetic anhydride). The extent of interconversion was correlated with the degree of fluorination of the acylating agents, with HFBA giving the highest conversion. This conversion has never been reported before. A mechanism for the interconversion was proposed. These findings indicated that fluorinated acylating agents should not be used for the unequivocal identification or quantification of EP and PE as the results obtained can be erroneous. Copyright © 2012 John Wiley & Sons, Ltd.

Utilization of acidic α-amino acids as acyl donors: an effective stereo-controllable synthesis of aryl-keto α-amino acids and their derivatives.

PubMed

Wang, Lei; Murai, Yuta; Yoshida, Takuma; Okamoto, Masashi; Tachrim, Zetryana Puteri; Hashidoko, Yasuyuki; Hashimoto, Makoto

2014-05-16

Aryl-keto-containing α-amino acids are of great importance in organic chemistry and biochemistry. They are valuable intermediates for the construction of hydroxyl α-amino acids, nonproteinogenic α-amino acids, as well as other biofunctional components. Friedel-Crafts acylation is an effective method to prepare aryl-keto derivatives. In this review, we summarize the preparation of aryl-keto containing α-amino acids by Friedel-Crafts acylation using acidic α-amino acids as acyl-donors and Lewis acids or Brönsted acids as catalysts.

Lipid and glycolipid isomer analyses using ultra-high resolution ion mobility spectrometry separations

DOE PAGES

Wojcik, Roza; Webb, Ian K.; Deng, Liulin; ...

2017-01-18

Understanding the biological mechanisms related to lipids and glycolipids is challenging due to the vast number of possible isomers. Mass spectrometry (MS) measurements are currently the dominant approach for studying and providing detailed information on lipid and glycolipid structures. However, difficulties in distinguishing many structural isomers (e.g. distinct acyl chain positions, double bond locations, as well as glycan isomers) inhibit the understanding of their biological roles. Here we utilized ultra-high resolution ion mobility spectrometry (IMS) separations based upon the use of traveling waves in a serpentine long path length multi-pass Structures for Lossless Manipulations (SLIM) to enhance isomer resolution. Themore » multi-pass arrangement allowed separations ranging from ~16 m (1 pass) to ~470 m (32 passes) to be investigated for the distinction of lipids and glycolipids with extremely small structural differences. Lastly, these ultra-high resolution SLIM IMS-MS analyses provide a foundation for exploring and better understanding isomer specific biological and disease processes.« less

Enantioselective separation of defined endocrine-disrupting nonylphenol isomers.

PubMed

Acir, Ismail-Hakki; Wüst, Matthias; Guenther, Klaus

2016-08-01

Nonylphenol is in the focus of worldwide endocrine-disrupter research and accounted for as a priority hazardous substance of the Water Framework Directive of the European Union. Technical nonylphenol consists of a very complex mixture of isomers and enantiomers. As estrogenic effect and degradation behavior in environmental processes of single nonylphenols are heavily dependent on the structure of the nonyl side chain, it is absolutely necessary to consider the nonylphenol problem from an isomer and enantiomer-specific viewpoint. In this study, an enantiomer-specific separation of eight defined synthesized nonylphenol isomers by five different special chiral cyclodextrin columns was performed underivatized and after methylation, silylation, and acylation. This work demonstrates that three columns out of the investigated five show an excellent separation behavior for the studied different nonylphenol isomers and can be used for the enantiomer-specific determination of nonylphenols in food, other biological matrices, and environmental samples in the future. Graphical abstract Enantiomeric pair of 4-NP170 (4-[1-ethyl-1,3,3-trimethylbutyl]phenol).

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.

Environmentally friendly surface modification of silk fiber: Chitosan grafting and dyeing

NASA Astrophysics Data System (ADS)

Davarpanah, Saideh; Mahmoodi, Niyaz Mohammad; Arami, Mokhtar; Bahrami, Hajir; Mazaheri, Firoozmehr

2009-01-01

In this paper, the surface modification of silk fiber using anhydrides to graft the polysaccharide chitosan and dyeing ability of the grafted silk were studied. Silk fiber was degummed and acylated with two anhydrides, succinic anhydride (SA) and phthalic anhydride (PA), in different solvents (dimethyl sulfoxide (DMSO) and N, N-dimethyl formamide (DMF)). The effects of anhydrides, solvents, anhydride concentration, liquor ratio (L:R) and reaction time on acylation of silk were studied. The polysaccharide chitosan was grafted to the acylated silk fiber and dyed by acid dye (Acid Black NB.B). The effects of pH, chitosan concentration, and reaction time on chitosan grafting of acylated silk were investigated. The physical properties show sensible changes regardless of weight gain. Scanning electron microscopy (SEM) analysis showed the presence of foreign materials firmly attached to the surface of silk. FTIR spectroscopy provided evidence that chitosan was grafted onto the acylated silk through the formation of new covalent bonds. The dyeing of the chitosan grafted-acylated silk fiber indicated the higher dye ability in comparison to the acylated and degummed silk samples. The mechanism of chitosan grafting over degummed silk through anhydride linkage was proposed. The findings of this research support the potential production of new environmentally friendly textile fibers. It is worthwhile to mention that the grafted samples have antibacterial potential due to the antibacterial property of chitosan molecules.

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.

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.

Application of selected ion monitoring to the analysis of triacylglycerols in olive oil by high temperature-gas chromatography/mass spectrometry.

PubMed

Ruiz-Samblás, C; González-Casado, A; Cuadros-Rodríguez, L; García, F P Rodríguez

2010-06-30

The analysis of the triacylglycerol (TAG) composition of oils is a very challenging task, since the TAGs have very similar physico-chemical properties. In this work, a high temperature-gas chromatographic method coupled to electron ionization-mass spectrometry (HT-GC/EI-MS), in the Selected Ion Monitoring (SIM) mode, method was developed for the analysis of TAGs in the olive oil; this is a method suitable for routine analysis. This method was developed using commercially available standard TAGs. The TAGs studied were separated according to their equivalent carbon number and degree of unsaturation. The peak assignment was carried out by locating the characteristic fragment ions having the same retention time on the SIM profile such as [RCO+74](+) and [RCO+128](+) ions, due to the fatty acyl residues on sn-1, sn-2 and sn-3 positions of the TAG molecule and the [M-OCOR](+) ions corresponding to the acyl ions. The developed method was very useful to eliminate the interferences that appeared in the mass spectrum since electron ionization can prevent satisfactory interpretation of spectra. Copyright 2010 Elsevier B.V. All rights reserved.

Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium.

PubMed

Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Sa, Tongmin

2007-02-01

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).

Identification of predictive biomarkers of disease state in transition dairy cows.

PubMed

Hailemariam, D; Mandal, R; Saleem, F; Dunn, S M; Wishart, D S; Ametaj, B N

2014-05-01

In dairy cows, periparturient disease states, such as metritis, mastitis, and laminitis, are leading to increasingly significant economic losses for the dairy industry. Treatments for these pathologies are often expensive, ineffective, or not cost-efficient, leading to production losses, high veterinary bills, or early culling of the cows. Early diagnosis or detection of these conditions before they manifest themselves could lower their incidence, level of morbidity, and the associated economic losses. In an effort to identify predictive biomarkers for postpartum or periparturient disease states in dairy cows, we undertook a cross-sectional and longitudinal metabolomics study to look at plasma metabolite levels of dairy cows during the transition period, before and after becoming ill with postpartum diseases. Specifically we employed a targeted quantitative metabolomics approach that uses direct flow injection mass spectrometry to track the metabolite changes in 120 different plasma metabolites. Blood plasma samples were collected from 12 dairy cows at 4 time points during the transition period (-4 and -1 wk before and 1 and 4 wk after parturition). Out of the 12 cows studied, 6 developed multiple periparturient disorders in the postcalving period, whereas the other 6 remained healthy during the entire experimental period. Multivariate data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation between healthy controls and diseased cows at all 4 time points. This analysis allowed us to identify several metabolites most responsible for separating the 2 groups, especially before parturition and the start of any postpartum disease. Three metabolites, carnitine, propionyl carnitine, and lysophosphatidylcholine acyl C14:0, were significantly elevated in diseased cows as compared with healthy controls as early as 4 wk before parturition, whereas 2 metabolites, phosphatidylcholine acyl-alkyl C42:4 and phosphatidylcholine diacyl C42:6, could be used to discriminate healthy controls from diseased cows 1 wk before parturition. A 3-metabolite plasma biomarker profile was developed that could predict which cows would develop periparturient diseases, up to 4 wk before clinical symptoms appearing, with a sensitivity of 87% and a specificity of 85%. This is the first report showing that periparturient diseases can be predicted in dairy cattle before their development using a multimetabolite biomarker model. Further research is warranted to validate these potential predictive biomarkers. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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 CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

USDA-ARS?s Scientific Manuscript database

Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

Molecular design of flotation collectors: A recent progress.

PubMed

Liu, Guangyi; Yang, Xianglin; Zhong, Hong

2017-08-01

The nature of froth flotation is to selectively hydrophobize valuable minerals by collector adsorption so that the hydrophobized mineral particles can attach air bubbles. In recent years, the increasing commercial production of refractory complex ores has been urgent to develop special collectors for enhancing flotation separation efficiency of valuable minerals from these ores. Molecular design methods offer an effective way for understanding the structure-property relationship of flotation collectors and developing new ones. The conditional stability constant (CSC), molecular mechanics (MM), quantitative structure-activity relationship (QSAR), and first-principle theory, especially density functional theory (DFT), have been adopted to build the criteria for designing flotation collectors. Azole-thiones, guanidines, acyl thioureas and thionocarbamates, amide-hydroxamates, and double minerophilic-group surfactants such as Gemini, dithiourea and dithionocarbamate molecules have been recently developed as high-performance collectors. To design hydrophobic groups, the hydrophilic-hydrophobic balance parameters have been extensively used as criteria. The replacement of aryl group with aliphatic group or CC single bond(s) with CC double bond(s), reduction of carbon numbers, introduction of oxygen atom(s) and addition of trisiloxane to the tail terminal have been proved to be useful approaches for adjusting the surface activity of collectors. The role of molecular design of collectors in practical flotation applications was also summarized. Based on the critical review, some comments and prospects for further research on molecular design of flotation collectors were also presented in the paper. Copyright © 2017 Elsevier B.V. All rights reserved.

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

PubMed

Levine, L; Gjika, H B; Van Vunakis, H

1989-11-30

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Synthesis and biological activities of turkesterone 11α-acyl derivatives

PubMed Central

Dinan, Laurence; Bourne, Pauline; Whiting, Pensri; Tsitsekli, Ada; Saatov, Ziyadilla; Dhadialla, Tarlochan S.; Hormann, Robert E.; Lafont, René; Coll, Josep

2003-01-01

Turkesterone is a phytoecdysteroid possessing an 11α-hydroxyl group. It is an analogue of the insect steroid hormone 20-hydroxyecdysone. Previous ecdysteroid QSAR and molecular modelling studies predicted that the cavity of the ligand binding domain of the ecdysteroid receptor would possess space in the vicinity of C-11/C-12 of the ecdysteroid. We report the regioselective synthesis of a series of turkesterone 11α-acyl derivatives in order to explore this possibility. The structures of the analogues have been unambiguously determined by spectroscopic means (NMR and low-resolution mass spectrometry). Purity was verified by HPLC. Biological activities have been determined in Drosophila melanogaster BII cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially-expressed D. melanogaster EcR/USP receptor proteins. The 11α-acyl derivatives do retain a significant amount of biological activity relative to the parent ecdysteroid. Further, although activity initially drops with the extension of the acyl chain length (C2 to C4), it then increases (C6 to C10), before decreasing again (C14 and C20). The implications of these findings for the interaction of ecdysteroids with the ecdysteroid receptor and potential applications in the generation of affinity-labelled and fluorescently-tagged ecdysteroids are discussed. Abbreviation: CoMFA comparative molecular field analysis DCM dichloromethane DMF dimethylformamide DMP 2,2-dimethoxypropane 4D-QSAR 4-dimensional quantitative structure-activity relationship EcR ecdysteroid receptor EcRE ecdysteroid response element HPLC high-performance liquid chromatography LBD ligand-binding domain NMR nuclear magnetic resonance ponA ponasterone A QSAR quantitative structure-activity relationship RXR retinoid X receptor SAR structure-activity relationship SPE solid-phase extraction THF tetrahydrofuran TLC thin-layer chromatography p-TsOH para-toluenesulphonic acid USP ultraspiracle UV-VIS ultraviolet-visible PMID:15841223

Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa.

PubMed

González, C Ruth; Vázquez, María J; López, Miguel; Diéguez, Carlos

2008-12-01

The most unique feature of ghrelin is the acyl-modification of a hydroxyl group of the Ser3 in the N-terminus. The Ser3 is commonly modified by n-octanoic acid in vertebrates being needed for its biological effects, at least in terms of feeding. Therefore, a critical question regarding the role of ghrelin was to characterize the mechanism involved in its acylation. The acyltransferase that catalyzes ghrelin octanoylation has been recently identified and named ghrelin O-acyltransferase (GOAT). The aim of this study was to clarify the physiological implications of GOAT in the regulation of energy balance, by assessing the effect of undernutrition, as well as fasting in adult male rats. We have determined GOAT mRNA expression levels by real time-PCR in the stomach mucosa. Our results show that chronic food restriction led to an increase in GOAT mRNA, particularly following long-term chronic malnutrition (21 days). Furthermore, following 48 h complete fasting, a situation with high-circulating ghrelin levels, we found similar mRNA expression of GOAT in fed and fasted rats; exogenous leptin administration markedly increase GOAT mRNA levels in the stomach mucosa of fasted rats. These findings suggest that increased GOAT mRNA levels may have a role in mediating the physiological responses to chronic undernutrition and could represent an adaptive response to prevent long-lasting alterations in energy balance and body weight homeostasis. Furthermore, our data also offer mechanistic insights into the reason why during fasting acylated ghrelin levels are not increased at a time when a marked increase in an orexigenic signal as important as acylated ghrelin will be expected.

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.

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.

Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae).

PubMed

Vink, Cor J; Paterson, Adrian M

2003-09-01

Datasets from the mitochondrial gene regions NADH dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (COI) of the 20 species in the New Zealand wolf spider (Lycosidae) genus Anoteropsis were generated. Sequence data were phylogenetically analysed using parsimony and maximum likelihood analyses. The phylogenies generated from the ND1 and COI sequence data and a previously generated morphological dataset were significantly congruent (p<0.001). Sequence data were combined with morphological data and phylogenetically analysed using parsimony. The ND1 region sequenced included part of tRNA(Leu(CUN)), which appears to have an unstable amino-acyl arm and no TpsiC arm in lycosids. Analyses supported the existence of five species groups within Anoteropsis and the monophyly of species represented by multiple samples. A radiation of Anoteropsis species within the last five million years is inferred from the ND1 and COI likelihood phylograms, habitat and geological data, which also indicates that Anoteropsis arrived in New Zealand some time after it separated from Gondwana.

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.

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.

Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380

USDA-ARS?s Scientific Manuscript database

Liamocins are polyol-lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a D-mannitol head-group ester linked to 3,5-dihydroxydecanoate acyl chains,...

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

NASA Astrophysics Data System (ADS)

Xie, Bo; Luo, Xiaoyang; Zhao, Cheng; Priest, Christina Marie; Chan, Shiu-Yung; O'Connor, Peter B.; Kirschner, Daniel A.; Costello, Catherine E.

2007-12-01

Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS compact myelin.

Mitochondrial lipids in Bufo arenarum full-grown oocytes.

PubMed

Gili, Valeria; Alonso, Telma S

2004-05-01

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.

Neutrally Charged Gas/Liquid Interface by a Catanionic Langmuir Monolayer

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

Vaknin, David; Bu, Wei

Surface-sensitive synchrotron X-ray scattering and spectroscopic experiments were performed to explore the characteristics of Langmuir monolayers of oppositely charged mixed amphiphiles. A premixed (molar 1:1 stearic acid/stearylamine) solution was spread as a monolayer at the gas/liquid interface on pure water and on mono- and divalent salt solutions, revealing that the negatively charged carboxyl groups and positively charged amine groups are miscible into one another and tend to bond together to form a nearly neutral surface. Similar control experiments on pure stearic acid (SA) and stearylamine (ST) were also conducted for comparison. Due to the strong bonding, hexagonal structures in smallmore » domains with acyl-chains normal to the liquid surface are formed at zero surface pressures, that is, at molecular areas much larger than those of the densely packed acyl chains. In-plane X-ray diffraction indicates that the catanionic surface is highly ordered and modifies the structure of the water surface and thus can serve as a model system for interactions of an amino acid template with solutes.« less

Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module.

PubMed

Skiba, Meredith A; Sikkema, Andrew P; Moss, Nathan A; Lowell, Andrew N; Su, Min; Sturgis, Rebecca M; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2018-05-08

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

Tile-based self-assembly of a triple-helical polysaccharide into cell wall-like mesoporous nanocapsules.

PubMed

Wu, Chaoxi; Wang, Xiaoying; Wang, Jianjing; Zhang, Zhen; Wang, Zhiping; Wang, Yifei; Tang, Shunqing

2017-07-20

Tile-based self-assembly is a robust system in the construction of three-dimensional DNA nanostructures but it has been rarely applied to other helical biopolymers. β-Glucan is an immunoactive natural polymer which exists in a triple helical conformation. Herein, we report that β-glucan, after modification using two types of short chain acyl groups, can self-assemble into tiles with inactivated sticky ends at the interface of two solvents. These tiles consist of a single layer of helices laterally aligned, and the sticky ends can be activated when a few acyl groups at the ends are removed; these tiles can further pack into mesoporous nanocapsules, in a similar process as the sticky DNA tiles pack into complex polyhedral nano-objects. These nanocapsules were found to have targeted effects to antigen presenting cells in a RAW264.7 cell model. Our study suggests that tile-based self-assembly can be a general strategy for helical biopolymers, and on fully exploiting this strategy, various new functional nanostructures will become accessible in the future.

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...

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…

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...

Fat Metabolism in Higher Plants

PubMed Central

Jacobson, Bruce S.; Jaworski, J. G.; Stumpf, P. K.

1974-01-01

Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds. PMID:16658913

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.

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.

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.

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

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

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 has the capacity to inhibit the macrophage-dependent induction of aromatase, and provides a novel mechanism for potential effects of des-acyl ghrelin to break the linkage between obesity and breast cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel acyloxy derivatives of branched mono- and polyol esters of sal fat: multiviscosity grade lubricant base stocks.

PubMed

Kamalakar, Kotte; Sai Manoj, Gorantla N V T; Prasad, Rachapudi B N; Karuna, Mallampalli S L

2014-12-10

Sal fat, a nontraditional seed oil, was chemically modified to obtain base stocks with a wide range of specifications that can replace mineral oil base stocks. Sal fatty acids were enriched to 72.6% unsaturation using urea adduct method and reacted with branched mono alcohol, 2-ethylhexanol (2-EtH), and polyols namely neopentyl glycol (NPG) and trimethylolpropane (TMP) to obtain corresponding esters. The esters were hydroxylated and then acylated using propionic, butyric, and hexanoic anhydrides to obtain corresponding acylated derivatives. The acylated TMP esters exhibited very high viscosities (427.35-471.93 cSt at 40 °C) similar to those of BS 150 mineral oil base stock range, ISO VG 460, while the acylated NPG esters (268.81-318.84 cSt at 40 °C) and 2-EtH esters viscosities (20.94-24.44 cSt at 40 °C) exhibited viscosities in the range of ISO VG 320 and 22 respectively with good viscosity indices. Acylated NPG esters were found suitable for high temperature and acylated 2-ethylhexyl esters for low viscosity grade industrial applications. It was observed that the thermo-oxidative stabilities of all acylated products were found better compared to other vegetable oil based base stocks. Overall, all the sal fat based lubricant base stocks are promising candidates with a wide range of properties, which can replace most of the mineral oil base stocks with appropriate formulations.

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

PubMed Central

Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

2014-01-01

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

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.

Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract.

PubMed

Saker, Safwan; Almousa Almaksour, Ziade; Chorin, Anne-Claire; Lebrihi, Ahmed; Mathieu, Florence

2014-01-01

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.

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

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Photocurable acrylic composition, and U.V. curing with development of U.V. absorber

DOEpatents

McKoy, Vincent B.; Gupta, Amitava

1992-01-01

In-situ development of an ultraviolet absorber is provided by a compound such as a hydroxy-phenyl-triazole containing a group which protects the absorber during actinically activated polymerization by light at first frequency. After polymerization the protective group is removed by actinic reaction at a second frequency lower than the first frequency. The protective group is formed by replacing the hydrogen of the hydroxyl group with an acyl group containing 1 to 3 carbon atoms or an acryloxy group of the formula: ##STR1## where R.sup.1 is either an alkyl containing 1 to 6 carbon atoms or --CH.dbd.CH.sub.2.

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.

Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli.

PubMed

Bouhss, A; Dementin, S; van Heijenoort, J; Parquet, C; Blanot, D

1999-06-18

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.

The Effects of Space Flight on Some Liver Enzymes Concerned with Carbohydrate and Lipid Metabolism in Rats

NASA Technical Reports Server (NTRS)

Abraham, S.; Lin, C. Y.; Klein, H. P.; Volkmann, C.

1978-01-01

The activities of about 30 enzymes concerned with carbohydrate and lipid metabolism and the levels of glycogen and of individual fatty acids were measured in livers of rats ex- posed to prolonged space flight (18.5 days) aboard COSMOS 986 Biosatellite. When flight stationary, (FS) and flight centrifuged (FC) rats were compared at recovery (R(sub 0)), decrceases in the activities of glycogen phosphorylase, alpha glycerphosphate, acyl transferase, diglyceride acyl transferase, acconitase and Epsilon-phosphogluconate dehydrogenase were noted in the weightless group (FS). The significance of these findings was strengthened since all activities, showing alterations at R(sub 0), returned to normal 25 days post-flight. Differences were also seen in levels of two liver constituents. When glycogen and total fatty acids of the two groups of flight animals were determined, differences that could be attributed to reduced gravity were observed, the FS group at R(sub 0) contained, on the average, more than twice the amount of glycogen than did controls ad a remarkable shift in the ratio of palmitate to palmitoleate were noted. These metabolic alterations appear to be unique to the weightless condition. Our data justify the conclusion that centrifugation during space flight is equivalent to terrestrial gravity.

Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

NASA Astrophysics Data System (ADS)

Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2016-02-01

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

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.

Changes in acyl and total ghrelin concentrations and their association with dry matter intake, average daily gain, and feed efficiency of finishing beef steers and heifers

USDA-ARS?s Scientific Manuscript database

Ghrelin is a peptide hormone produced in the gut that is implicated in signaling appetite and regulating DMI. The objective of this experiment was to determine the change in acyl ghrelin, total ghrelin, and the ghrelin ratio (acyl ghrelin/total ghrelin) over an 84-d DMI and ADG measurement period a...

Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

PubMed Central

Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

2016-01-01

Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

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

Behavioral and neurosensory responses of the boll weevil,Anthonomus grandis Boh. (Coleoptera: Curculionidae), to fluorinated analogs of aldehyde components of its pheromone.

PubMed

Dickens, J C; Prestwich, G D; Sun, W C

1991-06-01

Competitive field tests with α-fluorinated analogs of compounds III and IV (III-α-F and IV-α-F, respectively) of the boll weevil,Anthonomus grandis Boh., aggregation pheromone showed these compounds, when combined with the other pheromone components [(±)-I and II], to be as attractive as grandlure [(+)-I, II, and III+IV]. Dose-response curves constructed from electroantennograms of male boll weevils to serial stimulus loads of III, IV, III-α-F, IV-α-F, and the corresponding acyl fluorinated analogs (III-acyl-F and IV-acyl-F) showed the α-fiuorinated analogs to be as active as the pheromone components (threshold=0.1 μg), while the acyl fluorinated analogs had a 10-100 x higher threshold (=1-10 μg). Single-neuron recordings showed that IV neurons and II neurons (Dickens, 1990) responded to IV-α-F and III-α-F, respectively, while IV-acyl-F and III-acyl-F were inactive. Since a previous study showed compounds I, II, and IV to be essential for behavioral responses in the field, it seems likely that the activity of the α-fluorinated analogs observed here is due to the stimulation of IV neurons by IV-α-F as indicated in single neuron recordings.

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

WRINKLED Transcription Factors Orchestrate Tissue-Specific Regulation of Fatty Acid Biosynthesis in Arabidopsis[W

PubMed Central

To, Alexandra; Joubès, Jérôme; Barthole, Guillaume; Lécureuil, Alain; Scagnelli, Aurélie; Jasinski, Sophie; Lepiniec, Loïc; Baud, Sébastien

2012-01-01

Acyl lipids are essential constituents of all cells, but acyl chain requirements vary greatly and depend on the cell type considered. This implies a tight regulation of fatty acid production so that supply fits demand. Isolation of the Arabidopsis thaliana WRINKLED1 (WRI1) transcription factor established the importance of transcriptional regulation for modulating the rate of acyl chain production. Here, we report the isolation of two additional regulators of the fatty acid biosynthetic pathway, WRI3 and WRI4, which are closely related to WRI1 and belong to the APETALA2–ethylene-responsive element binding protein family of transcription factors. These three WRIs define a family of regulators capable of triggering sustained rates of acyl chain synthesis. However, expression patterns of the three WRIs differ markedly. Whereas only WRI1 activates fatty acid biosynthesis in seeds for triacylglycerol production, the three WRIs are required in floral tissues to provide acyl chains for cutin biosynthesis and prevent adherence of these developing organs and subsequent semisterility. The targets of these WRIs encode enzymes providing precursors (acyl chain and glycerol backbones) for various lipid biosynthetic pathways, but not the subsequent lipid-assembling enzymes. These results provide insights into the developmental regulation of fatty acid production in plants. PMID:23243127

Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

PubMed Central

Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

2012-01-01

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

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

Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry.

PubMed

Schmidt, Susanne; Zietz, Michaela; Schreiner, Monika; Rohn, Sascha; Kroh, Lothar W; Krumbein, Angelika

2010-07-30

Kale is a member of the Brassicaceae family and has a complex profile of flavonoid glycosides. Therefore, kale is a suitable matrix to discuss in a comprehensive study the different fragmentation patterns of flavonoid glycosides. The wide variety of glycosylation and acylation patterns determines the health-promoting effects of these glycosides. The aim of this study is to investigate the naturally occurring flavonoids in kale. A total of 71 flavonoid glycosides of quercetin, kaempferol and isorhamnetin were identified using a high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry (HPLC-DAD/ESI-MS(n)) method. Of these 71 flavonol glycosides, 27 were non-acylated, 30 were monoacylated and 14 were diacylated. Non-acylated flavonol glycosides were present as mono-, di-, tri- and tetraglycosides. This is the first time that the occurrence of four different fragmentation patterns of non-acylated flavonol triglycosides has been reported in one matrix simultaneously. In addition, 44 flavonol glycosides were acylated with p-coumaric, caffeic, ferulic, hydroxyferulic or sinapic acid. While monoacylated glycosides existed as di-, tri- and tetraglycosides, diacylated glycosides occurred as tetra- and pentaglycosides. To the best of our knowledge, 28 compounds in kale are reported here for the first time. These include three acylated isorhamnetin glycosides (isorhamnetin-3-O-sinapoyl-sophoroside-7-O-D-glucoside, isorhamnetin-3-O-feruloyl-sophoroside-7-O-diglucoside and isorhamnetin-3-O-disinapoyl-triglucoside-7-O-diglucoside) and seven non-acylated isorhamnetin glycosides. Copyright 2010 John Wiley & Sons, Ltd.

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

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.

Correlation between colonic secretion and colonic motility in rats: Role of ghrelin

PubMed Central

Huang, Hsien-Hao; Ting, Ching-Heng; Syu, Yu-Fong; Chang, Shi-Chuan; Chen, Chih-Yen

2016-01-01

AIM To explore the relationship between colonic secretory function and colonic motility. METHODS Using a rat model chronically implanted with intracerebroventricular (ICV) and cecal catheters, we validated the correlation between colonic secretion and colonic motor functions, as well as the role of ICV injection volume. RESULTS Compared to saline controls (5 μL/rat), ICV acyl ghrelin at 1 nmol/5 μL enhanced the total fecal weight, accelerated the colonic transit time, and increased the fecal pellet output during the first hour post-injection, while ICV des-acyl ghrelin at 1 nmol/5 μL only accelerated the colonic transit time. These stimulatory effects on colonic motility and/or secretion from acyl ghrelin and des-acyl ghrelin disappeared when the ICV injection volume increased to 10 μL compared with saline controls (10 μL/rat). Additionally, the ICV injection of 10 μL of saline significantly shortened the colonic transit time compared with the ICV injection of 5 μL of saline. The total fecal weight during the first hour post-injection correlated with the colonic transit time and fecal pellet output after the ICV injection of acyl ghrelin (1 nmol/5 μL), whereas the total fecal weight during the first hour post-injection correlated with the fecal pellet output but not the colonic transit time after the ICV injection of des-acyl ghrelin (1 nmol/5 μL). CONCLUSION Colonic secretion does not always correlate with colonic motility in response to different colonic stimulations. Acyl ghrelin stimulates colonic secretion. PMID:28028362

Indirect selection of industrial tomato genotypes that are resistant to spider mites (Tetranychus urticae).

PubMed

Baier, J E; Resende, J T V; Faria, M V; Schwarz, K; Meert, L

2015-01-16

Acyl sugars present in the tomato Solanum lycopersicum 'LA-716' accession confer good levels of resistance to arthropod pests. The objective of the present study was to select F₂ plants from the interspecific cross Solanum pennellii 'LA-716' x Solanum lycopersicum 'Redenção' to assess resistance to spider mites (Tetranychus urticae) based on the leaf acyl sugar content and repellence tests. Four genotypes were selected with high leaflet acyl sugar content (RVTA-2010 pl#31, RVTA-2010 pl#75, RVTA-2010 pl#83, and RVTA-2010 pl#94), and an additional three genotypes with low acyl sugar content were also selected (RVTA-2010 pl#33, RVTA-2010 pl#39, and RVTA-2010 pl#73). The results from the in vivo tests used to confirm the selection of plants resistant to mites indicated that the genotypes with high acyl sugars content did not differ from the resistant parent LA-716. The negative correlation between acyl sugar content and the distance run by the mite along the leaflet surface confirmed the association between high and low allelochemical content and resistance. The medium degree of dominance (MDD) was estimated (MDD = -0.83), indicating that the high acyl sugar content was due to incomplete dominance of a recessive allele. A value of 81.85% was found for the broad sense heritability estimate, which suggests that most among-plant variation in the F2 generation is genetically based. Furthermore, 0.69 genes were estimated, which presumably confirms monogenic inheritance. Thus, indirect selection was an efficient method used to obtain industrial tomato plants that are resistant to spider mites.

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

PubMed Central

Zhang, Xinxing; Jones, Rachel A.; Bruner, Steven D.; Butcher, Rebecca A.

2016-01-01

Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state. PMID:27551084

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices.

PubMed

Mokhir, A A; Connors, W H; Richert, C

2001-09-01

A total of 16 oligodeoxyribonucleotides of general sequence 5'-TCTTCTZTCTTTCT-3', where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5'-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3', where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an 'interrupting' T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4 degrees C higher than that of the triplex with 5'-TCTTCTGTCTTTCT-3' as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.

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

Two-dimensional nuclear magnetic resonance structure determination module for introductory biochemistry: synthesis and structural characterization of lyso-glycerophospholipids.

PubMed

Garrett, Teresa A; Rose, Rebecca L; Bell, Sidney M

2013-01-01

In this laboratory module, introductory biochemistry students are exposed to two-dimensional (1) H-nuclear magnetic resonance of glycerophospholipids (GPLs). Working in groups of three, students enzymatically synthesized and purified a variety of 2-acyl lyso GPLs. The structure of the 2-acyl lyso GPL was verified using (1) H-correlation spectroscopy. Students scored significantly higher on an assessment of NMR knowledge after having participated in this lab module and in comparison to a similar cohort who did not participate. Inaddition, student confidence in their NMR knowledge and abilities increased 62% following the module and correlated with their ability to apply their NMR knowledge. Based on these results, the laboratory module was very effective at providing students with a more extensive understanding of the underlying concepts of NMR as a tool for structural determination. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Acyl-homoserine lactone quorum sensing: from evolution to application.

PubMed

Schuster, Martin; Sexton, D Joseph; Diggle, Stephen P; Greenberg, E Peter

2013-01-01

Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.

PubMed

Fan, Pengxiang; Miller, Abigail M; Liu, Xiaoxiao; Jones, A Daniel; Last, Robert L

2017-12-12

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

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.

The synthesis of amphipathic prodrugs of 1,2-diol drugs with saccharide conjugates by high regioselective enzymatic protocol.

PubMed

Quan, Jing; Chen, Zhichun; Han, Chengyou; Lin, Xianfu

2007-02-15

A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different carbon chain length was performed under the catalysis of Candida antarctica lipase acrylic resin and Lipozyme in anhydrous acetone at 50 degrees C, respectively. A series of lipophilic derivatives with vinyl groups of mephenesin and chlorphenesin were prepared. The influences of different organic solvents, enzyme sources, reaction time, and the acylation reagents on the synthesis of vinyl esters were investigated. And then, protease-catalyzed high regioselective acylation of D-glucose and D-mannose with vinyl esters of mephenesin and chlorphenesin gave drug-saccharide derivatives in good yields. The studies of lipophilicity and hydrolysis in vitro of prodrugs verified that drug-saccharide derivatives had amphipathic properties, and both lipophilic and amphipathic drug derivatives had obvious controlled release characteristics.

Specificity of lecithin:cholesterol acyltransferase and atherogenic risk: comparative studies on the plasma composition and in vitro synthesis of cholesteryl esters in 14 vertebrate species.

PubMed

Liu, M; Bagdade, J D; Subbaiah, P V

1995-08-01

To determine whether the specificity of lecithin: cholesterol acyltransferase (LCAT) influences the susceptibility to atherosclerosis, we compared the composition and in vitro synthesis of cholesteryl ester (CE) in the plasmas of 14 vertebrate species with varying predisposition to atherosclerosis. The susceptible species (Group I) had significantly higher ratios of 16:0 CE/20:4 CE in their plasma than the resistant species (Group II). The in vitro formation of labeled CE species in native plasma from labeled cholesterol correlated highly with the mass composition, showing that the LCAT reaction is the predominant source of plasma CE in all the animal species examined. Isolated LCATs from Group I species also synthesized CE with higher ratios of 16:0/20:4 than LCATs from Group II when egg phosphatidylcholine (PC) was used as the acyl donor. In addition, the Group I LCATs exhibited lower specificity towards sn-2-20:4 and sn-2-22:6 PCs, and higher specificity towards sn-2-18:2 PC species than Group II LCATs. With 16:0-20:4 PC as the substrate, all Group I LCATs synthesized more 16:0 CE than 20:4 CE, whereas all Group II LCATs, with the exception of dog enzyme, synthesized predominantly 20:4 CE, showing that the two types of LCAT have different positional specificities towards this PC. These results suggest that there are two classes of LCAT in nature that differ from each other in their substrate and positional specificities, possibly because of differences in their active-site architectures. We propose that the presence of one type of LCAT, which cannot efficiently transfer certain long chain polyunsaturated acyl groups and which consequently synthesizes more saturated CE, may increase the risk of atherosclerosis.

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.

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.

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

Wojcik, Roza; Webb, Ian K.; Deng, Liulin

Understanding the biological mechanisms related to lipids and glycolipids is challenging due to the vast number of possible isomers. Mass spectrometry (MS) measurements are currently the dominant approach for studying and providing detailed information on lipid and glycolipid structures. However, difficulties in distinguishing many structural isomers (e.g. distinct acyl chain positions, double bond locations, as well as glycan isomers) inhibit the understanding of their biological roles. Here we utilized ultra-high resolution ion mobility spectrometry (IMS) separations based upon the use of traveling waves in a serpentine long path length multi-pass Structures for Lossless Manipulations (SLIM) to enhance isomer resolution. Themore » multi-pass arrangement allowed separations ranging from ~16 m (1 pass) to ~470 m (32 passes) to be investigated for the distinction of lipids and glycolipids with extremely small structural differences. Lastly, these ultra-high resolution SLIM IMS-MS analyses provide a foundation for exploring and better understanding isomer specific biological and disease processes.« less

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

PubMed Central

Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S.

2017-01-01

ABSTRACT The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. PMID:28794042

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion.

PubMed

Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Sodt, Alexander; Schwartz, Cindi; Nair, Vinod; Blank, Paul S; Zimmerberg, Joshua

2017-11-01

The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion. Copyright © 2017 American Society for Microbiology.

Altered Appetite-Mediating Hormone Concentrations Precede Compensatory Overeating After Severe, Short-Term Energy Deprivation in Healthy Adults.

PubMed

O'Connor, Kristie L; Scisco, Jenna L; Smith, Tracey J; Young, Andrew J; Montain, Scott J; Price, Lori Lyn; Lieberman, Harris R; Karl, J Philip

2016-02-01

Adaptive responses of appetite-mediating hormones to negative energy balance are thought to contribute to a counterregulatory response that drives weight regain, but they have not been studied while controlling for reduced diet volume. In this secondary analysis, we aimed to determine the effects of short-term, severe energy deprivation (ED) on appetite and appetite-mediating hormone concentrations. Twenty-one adults with a mean ± SD age of 21 ± 3 y and body mass index of 25 ± 3 kg/m(2) consumed isovolumetric diets provided over separate 48-h periods while increasing habitual energy expenditure by 1683 ± 329 kcal/d through light- and moderate-intensity exercise. Energy intake was matched to energy expenditure to maintain energy balance (EB) (-44 ± 92 kcal/d) or was <10% of energy expenditure to generate a -3696 ± 742-kcal/d energy deficit. Postprandial appetite, glucose, insulin, acyl ghrelin, peptide YY, pancreatic polypeptide (PP), and glucagon-like peptide-1 (GLP-1) responses and ad libitum energy intake were measured as secondary outcomes after both experimental periods. Fasting insulin (-56% ± 42%) and acyl ghrelin (-60% ± 17%) concentrations decreased during ED but not during EB (condition-by-time interaction; P-interaction ≤ 0.01), whereas fasting leptin concentrations decreased more during ED compared with during EB (-47% ± 27% compared with -20% ± 27%; P-interaction = 0.05). Postprandial insulin (57% ± 63%; P < 0.001), GLP-1 (14% ± 28%; P = 0.04), and PP (54% ± 52%; P < 0.001) areas under the curve (AUCs) were higher, whereas the acyl ghrelin AUC was lower (-56% ± 13%; P < 0.001) after ED compared with after EB. After ED, self-rated appetite was greater, and ad libitum energy intake was 811 kcal/36 h (95% CI: 184, 1439 kcal/36 h) higher relative to after EB (P = 0.01). Short-term, severe ED suppressed acyl ghrelin concentrations and increased postprandial anorexigenic hormone concentrations. These effects preceded compensatory overeating, suggesting that in adults without obesity, altered sensitivity to appetite-mediating hormones may contribute to an adaptive counterregulatory response during the initial stages of negative EB. This trial was registered at clinicaltrials.gov as NCT01603550. © 2016 American Society for Nutrition.

Exploiting the acylating nature of the imide-Ugi intermediate: a straightforward synthesis of tetrahydro-1,4-benzodiazepin-2-ones.

PubMed

Mossetti, Riccardo; Saggiorato, Dèsirèe; Tron, Gian Cesare

2011-12-16

We describe a simple and novel protocol for the synthesis of tetrahydro-1,4-benzodiazepin-2-ones with three points of diversity, exploiting the acylating properties of the recently rediscovered Ugi-imide. The final compounds can be easily prepared in three synthetic steps using a multicomponent reaction, a Staudinger reduction, and an acylative protocol, with good to excellent yields for each synthetic step.

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.

Recent developments in the long-term preservation of red blood cells.

PubMed

Greenwalt, T J

1997-11-01

The first study to suggest the successful prolongation of the useful shelf life of red blood cells (RBCs) used a hypotonic additive solution containing glycerol. It was necessary to use twice the volume of this solution than the commercial additive solution per unit of packed RBCs. The final concentration of glycerol in the units was approximately 0.69% (75 mmol/L). A subsequent study demonstrated that the membranes of the exocytic microvesicles shed during storage had less of bands 3 and 4.1 than those in Adsol (Fenwal Laboratories, Deerfield, IL). Band 4.1 is important for strengthening the bonds between spectrin and actin in the cytoskeleton. In another study, glutamine or glutamine plus phosphate was used in a hypotonic additive solution, otherwise similar to the glycerol-containing additive. In the latter medium, phosphatidylethanolamine was less accessible to phospholipase action than in Adsol or when glutamine alone was added. Another group reported encouraging data regarding the action of L-carnitine when added to AS-3. Acylation of phosphatidylethanolamine mediated by the action of carnitine fatty acid transferase with acyl coenzyme A (acyl-CoA) occurred. Adenosine-5'-triphosphate levels and red cell recovery were better in the test units. In the last paper reviewed, the authors demonstrated that oxidant damage of erythrocytes was less if the donors were given a mixture of antioxidants for 10 days prior to donating.

Synthesis and evaluation of cationic nanomicelles for in vitro and in vivo gene delivery

NASA Astrophysics Data System (ADS)

Mandke, Rhishikesh Subhash

The goal of proposed study was to contribute towards the development of a nano size, high efficiency and low toxicity non-viral polymeric vector for gene delivery in vitro and in vivo. A series of fatty acid grafted low-molecular-weight chitosan (N-acyl LMWCs) were synthesized, purified and characterized for their physicochemical properties using various analytical techniques such as infrared spectroscopy, elemental analysis and dynamic light scattering. The formulation parameters including pH, sonication duration, and filtration altered the physicochemical characteristics of N-acyl LMWC nanomicelles. The acyl chain length and degree of unsaturation in fatty acids also had an impact on the physicochemical properties and the transfection efficiency of nanomicelles. N-acyl LMWC nanomicelles showed efficient in vitro transfection as visualized and quantified using a reporter plasmid (encoding green fluorescent protein), and therapeutic plasmids (encoding for interleukin-4 and interleukin-10), respectively. The in vitro transfection efficiencies of N-acyl LMWCs with 18:1 and 18:2 grafts (oleic and linoleic acids) were comparable with FuGENERTM HD (marketed non-viral vector) but were ˜8-fold and 35-fold higher as compared to LMWC and naked DNA, respectively. The in vivo transfection efficiency of N-acyl LMWC to deliver plasmids individually encoding IL-4 and IL-10 as well as a bicistronic plasmid encoding both IL-4 and IL-10 was studied in a multiple, low-dose streptozotocin induced diabetic mouse model. The transfection efficiency of pDNA/N-acyl LMWC polyplexes injected via intramuscular route showed significant improvement (p<0.05) over passive (naked DNA) or positive (FuGENE HD) controls. Additionally, a sustained and efficient expression of IL-4 and IL-10 was observed, accompanied by a reduction in interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF-alpha) levels. The pancreas of pDNA/N-acyl LMWC polyplex treated animals exhibited protection from streptozotocin-induced insulitis and the delivery systems were biocompatible. Histological studies revealed that there were no signs of chronic inflammation at the injection site. The bicistronic plasmid exhibited significantly (p<0.05) greater expression of IL-4 and IL-10, and demonstrated the feasibility of bicistronic IL-4/IL-10 plasmid/N-acyl LMWC nanomicelles-based polyplexes as an efficient and biocompatible system for the prevention of autoimmune diabetes.

Xanthomonas campestris RpfB is a Fatty Acyl-CoA Ligase Required to Counteract the Thioesterase Activity of the RpfF Diffusible Signal Factor (DSF) Synthase

PubMed Central

Bi, Hongkai; Yu, Yonghong; Dong, Huijuan; Wang, Haihong; Cronan, John E.

2014-01-01

SUMMARY In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signaling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli β-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalyzing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids. PMID:24866092

Rapid Analysis of Microalgal Triacylglycerols with Direct-Infusion Mass Spectrometry

DOE PAGES

Christensen, Earl; Sudasinghe, Nilusha; Dandamudi, Kodanda Phani Raj; ...

2015-09-01

Cultivation of microalgae has the potential to provide lipid-derived feedstocks for conversion to liquid transportation fuels. Lipid extracts from microalgae are significantly more complex than those of traditional seed oils, and their composition changes significantly throughout the microalgal growth period. With three acyl side chains per molecule, triglycerides (TAGs) are an important fuel precursor, and the distribution of acyl chain composition for TAGs has a significant impact on fuel properties and processing. Therefore, determination of the distribution of microalgal TAG production is needed to assess the value of algal extracts designed for fuel production and to optimize strain, cultivation, andmore » harvesting practices. Methods utilized for TAG speciation commonly involve complicated and time-consuming chromatographic techniques. Here we present a method for TAG speciation and quantification based on direct-infusion mass spectrometry, which provides rapid characterization of TAG profiles without chromatographic separation. Specifically, we utilize Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to provide a reference library of TAGs for the microalgae Nannochloropsis sp. that provides the basis for high-throughput TAG quantitation by time-of-flight mass spectrometry (TOF MS). In conclusion, we demonstrate the application of this novel approach for lipid characterization with respect to TAG compound distribution, which informs both immediate and future strain and process optimization strategies.« less

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

Christensen, Earl; Sudasinghe, Nilusha; Dandamudi, Kodanda Phani Raj

Cultivation of microalgae has the potential to provide lipid-derived feedstocks for conversion to liquid transportation fuels. Lipid extracts from microalgae are significantly more complex than those of traditional seed oils, and their composition changes significantly throughout the microalgal growth period. With three acyl side chains per molecule, triglycerides (TAGs) are an important fuel precursor, and the distribution of acyl chain composition for TAGs has a significant impact on fuel properties and processing. Therefore, determination of the distribution of microalgal TAG production is needed to assess the value of algal extracts designed for fuel production and to optimize strain, cultivation, andmore » harvesting practices. Methods utilized for TAG speciation commonly involve complicated and time-consuming chromatographic techniques. Here we present a method for TAG speciation and quantification based on direct-infusion mass spectrometry, which provides rapid characterization of TAG profiles without chromatographic separation. Specifically, we utilize Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to provide a reference library of TAGs for the microalgae Nannochloropsis sp. that provides the basis for high-throughput TAG quantitation by time-of-flight mass spectrometry (TOF MS). In conclusion, we demonstrate the application of this novel approach for lipid characterization with respect to TAG compound distribution, which informs both immediate and future strain and process optimization strategies.« less

Ghrelin, leptin and insulin in cirrhotic children and adolescents: relationship with cirrhosis severity and nutritional status.

PubMed

Dornelles, Cristina T L; Goldani, Helena A S; Wilasco, Maria Inês A; Maurer, Rafael L; Kieling, Carlos O; Porowski, Marilene; Ferreira, Cristina T; Santos, Jorge L; Vieira, Sandra M G; Silveira, Themis R

2013-01-10

Ghrelin, leptin, and insulin concentrations are involved in the control of food intake and they seem to be associated with anorexia-cachexia in cirrhotic patients. The present study aimed to investigate the relationship between the nutritional status and fasting ghrelin, leptin and insulin concentrations in pediatric cirrhotic patients. Thirty-nine patients with cirrhosis and 39 healthy controls aged 0-15 years matched by sex and age were enrolled. Severity of liver disease was assessed by Child-Pugh classification, and Pediatric for End Stage Liver Disease (PELD) or Model for End-stage Liver Disease (MELD) scores. Blood samples were collected from patients and controls to assay total ghrelin, acyl ghrelin, leptin and insulin by using a commercial ELISA kit. Anthropometry parameters used were standard deviation score of height-for-age and triceps skinfold thickness-for-age ratio. A multiple linear regression analysis was used to determine the correlation between dependent and independent variables. Acyl ghrelin was significantly lower in cirrhotic patients than in controls [142 (93-278) pg/mL vs 275 (208-481) pg/mL, P=0.001]. After multiple linear regression analysis, total ghrelin and acyl ghrelin showed an inverse correlation with age; acyl ghrelin was associated with the severity of cirrhosis and des-acyl ghrelin with PELD or MELD scores ≥15. Leptin was positively correlated with gender and anthropometric parameters. Insulin was not associated with any variable. Low acyl ghrelin and high des-acyl ghrelin concentrations were associated with cirrhosis severity, whereas low leptin concentration was associated with undernourishment in children and adolescents with cirrhosis. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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.

Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors.

PubMed

Caldo, Kristian Mark P; Acedo, Jeella Z; Panigrahi, Rashmi; Vederas, John C; Weselake, Randall J; Lemieux, M Joanne

2017-10-01

Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT1 1-113 ) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

PubMed

Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

2013-07-01

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.

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.

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

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.

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

Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH wasmore » assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.« less

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.

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 HAMSB

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169*

PubMed Central

Allen, James W.; DiRusso, Concetta C.; Black, Paul N.

2017-01-01

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. PMID:27903654

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

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.

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169.

PubMed

Allen, James W; DiRusso, Concetta C; Black, Paul N

2017-01-06

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO 2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U- 13 C]glucose, 13 CO 2 , or D 2 O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Rhodium-catalyzed enantioselective cyclizations of γ-alkynylaldehydes with acyl phosphonates: ligand- and substituent-controlled C-P or C-H bond cleavage.

PubMed

Masuda, Kengo; Sakiyama, Norifumi; Tanaka, Rie; Noguchi, Keiichi; Tanaka, Ken

2011-05-11

It has been established that a cationic rhodium(I)/(R)-H(8)-BINAP or (R)-Segphos complex catalyzes two modes of enantioselective cyclizations of γ-alkynylaldehydes with acyl phosphonates via C-P or C-H bond cleavage. The ligands of the Rh(I) complexes and the substitutents of both γ-alkynylaldehydes and acyl phosphonates control these two different pathways. © 2011 American Chemical Society

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

Pfleger, Brian F.; Youngquist, Tyler J.

Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

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.

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

Structure of a bacterial toxin-activating acyltransferase.

PubMed

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

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.

Biocatalytic Synthesis of Flavonoid Esters by Lipases and Their Biological Benefits.

PubMed

de Araújo, Maria Elisa M B; Franco, Yollanda E M; Messias, Marcia C F; Longato, Giovanna B; Pamphile, João A; Carvalho, Patricia de O

2017-01-01

Several studies have described important biological activities of flavonoids such as coronary heart disease prevention, hepatoprotective, anti-inflammatory and anticancer activities, enzyme inhibition activity, and antibacterial, antifungal, and antiviral activities. Flavonoids show promising activity as natural plant-based antioxidants due to their antioxidant and free radical scavenging properties. However, their primary applications as antioxidants in the pharmaceutical, cosmetic, and food industries are limited because of their moderately hydrophilic nature. Enzymatic acylation of natural polyphenols with fatty acids or other acyl donors has been suggested for improving the lipophilic nature of the glycosylated flavonoids. This approach increases flavonoid solubility and stability in lipophilic systems. Acylation of flavonoids with different acyl donors may also introduce beneficial properties to the molecule, such as penetration through the cell membrane and improved antioxidant, antimicrobial, anti-inflammatory, antiproliferative, cytogenetic, and enzyme inhibition activities. Chemical methods for the synthesis of flavonoid esters lead to the formation of side products and the simultaneous decomposition of the flavonoids due to harsh reaction conditions. In contrast, biocatalytic acylation of flavonoids by lipases offers advantages associated to the wide availability of these enzymes, their low cost, chemo-, regio-, and enantioselectivity, mild condition processing and non-requirement of cofactors. This article is focused on the recent development of lipase-catalyzed synthesis of flavonoid esters and the impact of the acylation reaction on their biological activities. Georg Thieme Verlag KG Stuttgart · New York.

Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

PubMed Central

Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

2013-01-01

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations.

PubMed

Narayanan, Sruthi; Tamura, Pamela J; Roth, Mary R; Prasad, P V Vara; Welti, Ruth

2016-04-01

Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate-resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization-tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat-tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3-containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd-numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0-acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0-ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT. © 2015 John Wiley & Sons Ltd.

Synthesis and Characterization of 9-Hydroxyphenalenone Using 2D NMR Techniques

ERIC Educational Resources Information Center

Caes, Benjamin; Jensen, Dell, Jr.

2008-01-01

9-Hydroxyphenalenone is a planar multicyclic [beta]-keto-enol, which is synthesized via a Friedel-Crafts acylation followed by acid-catalyzed intramolecular Michael addition with the loss of a phenyl group in a one-pot reaction during a four-hour lab period. Tautomerization of the [beta]-keto-enol results in C[subscript 2v] symmetry on the NMR…

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.

Effect of chloroquine on intestinal lipid metabolism

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

Mansbach, C.M. 2d.; Arnold, A.; Garrett, M.

1987-11-01

Most studies that have quantitated recovery of infused lipid in the intestinal mucosa and mesenteric lymph have only been able to recapture 50-75%. One possibility is that the missing lipid enters a triacylglycerol (TG) storage pool in the enterocyte and is hydrolyzed by lysosomal lipase, and the free fatty acid released is transported by the portal vein. This postulate was tested by comparing glyceryl trioleate (TO)-infused rats pretreated with the lysosomotropic drug, chloroquine (6.3 mg.kg-1.h-1) with saline controls. Chloroquine increased mucosal TG from 94 +/- 6 to 128 +/- 8 mumol. Additionally, the specific activity of the mucosal TG relativemore » to the infused (/sup 3/H)TO was reduced in the treated rats. The mucosal TG increase was not due to impaired TG output, which remained the same as controls. We conclude that the TG in the acid lipase-sensitive pool derives most of its glyceride-glycerol from endogenous sources. Furthermore, the increment in mucosal TG caused by chloroquine is not enough to explain the majority of the acyl groups unaccounted for in the mucosa and lymph after a TG infusion. For these a direct passage of acyl groups through the enterocyte is postulated.« less

Chemical and pharmacological properties of diflunisal.

PubMed

Shen, T Y

1983-01-01

Diflunisal, 5-(2',4'-difluorophenyl) salicylic acid, was discovered as a potent antiinflammatory analgesic agent after an extensive investigation of more than 500 salicylic acid analogs. The addition of a difluorophenyl substituent at the C5 position of salicylic acid yielded a new molecule with much improved lipophilicity, a longer duration of action, and a molecular configuration that is optimal for greater antiinflammatory and analgesic activities. The difluorophenyl group is metabolically stable; the acyl and phenolic glucuronides of the intact diflunisal are major urinary metabolites. The absence of an O-acetyl group in this novel salicylate analog circumvents the well-known in vivo acylation potential of aspirin and renders diflunisal a reversible cyclooxygenase inhibitor with a secondary oxygen radical scavenging effect. In a variety of analgesic, acute, and chronic antiinflammatory models, diflunisal is active at 10 mg/kg, approximately 5 to 10 times more potent than aspirin. It has a relatively low potential to cause gastrointestinal irritation as indicated by the lack of acute effect on the integrity of gastric mucosa barrier, no change of intragastric potential, and no disturbance of prostaglandin production by the gastric tissue. Diflunisal holds promise as a new clinically useful analgesic and antiinflammatory drug with good tolerance and a long duration of action.

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

PubMed

Chu, Weihua; Vattem, Dhiraj A; Maitin, Vatsala; Barnes, Mary B; McLean, Robert J C

2011-01-01

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

PubMed

Vandavasi, Venu Gopal; Langan, Patricia S; Weiss, Kevin L; Parks, Jerry M; Cooper, Jonathan B; Ginell, Stephan L; Coates, Leighton

2017-01-01

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. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not 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 class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found 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, as previously proposed. Copyright © 2016 Vandavasi et al.

Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

PubMed Central

Mullaney, Brendan; Ashrafi, Kaveh

2010-01-01

Summary Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3 derived long chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans. PMID:20889131