New Complexity-Building Reactions of Alpha-Keto Esters

NASA Astrophysics Data System (ADS)

Bartlett, Samuel L.

I. Introduction: Importance of Asymmetric Catalysis and the Reactivity Patterns of alpha-Keto Esters. II. Synthesis of Complex Tertiary Glycolates by Enantioconvergent Arylation of Stereochemically Labile alpha-Keto Esters. Enantioconvergent arylation reactions of boronic acids and racemic ?-stereogenic alpha-keto esters have been developed. The reactions are catalyzed by a chiral (diene)Rh(I) complex and provide a wide array of beta-stereogenic tertiary aryl glycolate derivatives with high levels of diastereo- and enantioselectivity. Racemization studies employing a series of sterically differentiated tertiary amines suggest that the steric nature of the amine base additive exerts a significant influence on the rate of substrate racemization. III. Palladium-Catalyzed beta-Arylation of alpha-Keto Esters . A catalyst system derived from commercially available Pd2(dba) 3 and PtBu3 has been applied to the coupling of alpha-keto ester enolates and aryl bromides. The reaction provides access to an array of beta-stereogenic alpha-keto ester derivatives. When the air stable ligand precursor PtBu 3˙HBF4 is employed, the reaction can be carried out without use of a glovebox. The derived products are of broad interest given the prevalence of the alpha-keto acid substructure in biologically important molecules. IV. Catalytic Enantioselective [3+2] Cycloaddition of alpha-Keto Ester Enolates and Nitrile Oxides. An enantioselective [3+2] cycloaddition reaction between nitrile oxides and transiently generated enolates of alpha-keto esters has been developed. The catalyst system was found to be compatible with in situ nitrile oxide generation conditions. A versatile array of nitrile oxides and alpha-keto esters could participate in the cycloaddition, providing novel 5-hydroxy-2-isoxazolines in high chemical yield with high levels of diastereo- and enantioselectivity. Notably, the optimal reaction conditions circumvented concurrent reaction via O-imidoylation and hetero-[3+2] pathways.

Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages.

PubMed Central

Lolait, S J; Clements, J A; Markwick, A J; Cheng, C; McNally, M; Smith, A I; Funder, J W

1986-01-01

We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31. Images PMID:2423557

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…

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

PubMed

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-04-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

Mechanism of antiinflammatory actions of curcumine and boswellic acids.

PubMed

Ammon, H P; Safayhi, H; Mack, T; Sabieraj, J

1993-03-01

Curcumine from Curcuma longa and the gum resin of Boswellia serrata, which were demonstrated to act as anti-inflammatories in in vivo animal models, were studied in a set of in vitro experiments in order to elucidate the mechanism of their beneficial effects. Curcumine inhibited the 5-lipoxygenase activity in rat peritoneal neutrophils as well as the 12-lipoxygenase and the cyclooxygenase activities in human platelets. In a cell free peroxidation system curcumine exerted strong antioxidative activity. Thus, its effects on the dioxygenases are probably due to its reducing capacity. Boswellic acids were isolated from the gum resin of Boswellia serrata and identified as the active principles. Boswellic acids inhibited the leukotriene synthesis via 5-lipoxygenase, but did not affect the 12-lipoxygenase and the cyclooxygenase activities. Additionally, boswellic acids did not impair the peroxidation of arachidonic acid by iron and ascorbate. The data suggest that boswellic acids are specific, non-redox inhibitors of leukotriene synthesis either interacting directly with 5-lipoxygenase or blocking its translocation.

Sesquiterpenoids and phenolics from roots of Taraxacum udum.

PubMed

Michalska, Klaudia; Marciniuk, Jolanta; Kisiel, Wanda

2010-07-01

From roots of Taraxacum udum, two new and four known sesquiterpene lactones were isolated, together with five known phenolic compounds. The new compounds were characterized as 11beta, 13-dihydrotaraxinic acid and taraxinic acid 6-O-acetyl-beta-glucopyranosyl ester by spectroscopic methods, especially 1D and 2D NMR, and by comparison with structurally related compounds. The plant material was shown to be a good source of taraxinic acid derivatives. Copyright 2009 Elsevier B.V. All rights reserved.

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

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

Rakus,J.; Fedorov, A.; Fedorov, E.

2007-01-01

The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain andmore » a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only conserved residues in the enolase superfamily, establishing the primary functional importance of the Mg2+-assisted strategy for stabilizing the enolate anion intermediate.« less

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

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

Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductasemore » 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. - Highlights: • We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum. • 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress. • KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression. • KetoC promoted the expression of antioxidative enzymes in mice organs. • The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.« less

Protective effect of boswellic acids versus pioglitazone in a rat model of diet-induced non-alcoholic fatty liver disease: influence on insulin resistance and energy expenditure.

PubMed

Zaitone, Sawsan A; Barakat, Bassant M; Bilasy, Shymaa E; Fawzy, Manal S; Abdelaziz, Eman Z; Farag, Noha E

2015-06-01

Non-alcoholic fatty liver disease (NAFLD) is closely linked to insulin resistance, oxidative stress, and cytokine imbalance. Boswellic acids, a series of pentacyclic triterpene molecules that are produced by plants in the genus Boswellia, has been traditionally used for the treatment of a variety of diseases. This study aimed at evaluating the protective effect of boswellic acids in a model of diet-induced NAFLD in rats in comparison to the standard insulin sensitizer, pioglitazone. Rats were fed with a high-fat diet (HFD) for 12 weeks to induce NAFLD. Starting from week 5, rats received boswellic acids (125 or 250 mg/kg) or pioglitazone parallel to the HFD. Feeding with HFD induced hepatic steatosis and inflammation in rats. In addition, liver index, insulin resistance index, activities of liver enzymes, and serum lipids deviated from normal. Further, serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and cyclooxygenase 2 were elevated; this was associated with an increase in hepatic expression of inducible nitric oxide synthase (iNOS) and formation of 4-hydroxy-2-nonenal (HNE). Rats treated with boswellic acids (125 or 250 mg/kg) or pioglitazone showed improved insulin sensitivity and a reduction in liver index, activities of liver enzymes, serum TNF-α and IL-6 as well as hepatic iNOS expression and HNE formation compared to HFD group. Furthermore, at the cellular level, boswellic acids (250 mg/kg) ameliorated the expression of thermogenesis-related mitochondrial uncoupling protein-1 and carnitine palmitoyl transferase-1 in white adipose tissues. Data from this study indicated that boswellic acids might be a promising therapy in the clinical management of NAFLD if appropriate safety and efficacy data are available.

[HPLC investigation of antioxidant components in Solidago herba].

PubMed

Apáti, Pál; Houghton, Peter J; Kéry, Agnes

2004-01-01

Representatives of Solidago species have been used in European phytotheraphy for centuries as a component of urological and antiphlogistical remedies. Solidago canadensis L. (Asteraceae) contains a wide range of active ingredients, such as flavonoids, saponins, hydroxycinnamates and mineral elements, which are responsible for its characteristic anti-inflammatory, spasmolytic and diuretic properties. Quality control of collected Solidaginis herba were performed according to the instructions of the X. German Pharmacopoea, while different LC-MS technologies were applied to evaluate the exact phenoloid composition. Three flavonol aglycons (quercetin, kaempferol and isorhamnetin) connected to several sugar components (glucose, rhamnose, galactose and rutinose), caffeoylquinic acid and a caffeoyl-shikimic acid glycoside were identified in the samples. Quercetin-3-O-beta-glucoside (isoquercitrin), quercetin-3-O-beta-galactoside (hyperoside), quercetin-3-O-beta-rhamnoside (quercitrin), quercetin-3-O-beta-rutinoside (rutin), kaempferol-3-O-beta-rhamnoside (afzelin), kaempferol-3-O-beta-rutinoside (nicotiflorin), caffeoil-quinic acid (chlorogenic acid) were identified in sample "A", while the presence of quercetin, quercetin-3-O-beta-glucoside (isoquercitrin), quercetin-3-/6"-O-acetyl-/-beta-glucopiranoside, quercetin-3-O-beta-rutinoside (rutin), kaempferol, kaempferol-3-O-beta-glucoside (astragalin), kaempferol-3-/6"-O-acetyl-/-beta-glucopiranoside, isorhamnetin, isorhamnetin-3-/6"-O-acetyl-/-beta-glucopiranoside, isorhamnetin-3-O-beta-rutinoside (narcissin), caffeoil-quinic acid (chlorogenic acid), caffeoil-shikimic acid-glucoside (dattelic acid-glucoside) were confirmed in sample "B". According to the occurrence of acetyl-glycosides and the diversity of sugar component of flavonoid glycosides Solidaginis herba samples chemotaxonomically were classified into different varieties. Incidence of acetyl-glycosidic flavonoids and absence of flavonoid galactosides and rhamnosides in the sample "B" together give support for the taxonomic recognition of varietases Solidago canadensis L. var. canadensis and var. scabra. Sample "A" was identified as Solidago canadensis L. var. canadensis, while sample "B" has proved to be belong to variety Solidago canadensis L. var. scabra. Due to the same flavonoid aglycons and the large amounts of flavonol glycosides occurring in each drug, phytochemical characteristics of investigated samples proved to be very similar.

Unexpected Diversity of Escherichia coli Sialate O-Acetyl Esterase NanS

PubMed Central

Rangel, Ariel; Steenbergen, Susan M.

2016-01-01

ABSTRACT The sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, where N-acetyl- or N-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations to N-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria with O-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity of Escherichia coli NanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show that E. coli strain O157:H7 Stx prophage or prophage remnants invariably include paralogs of nanS often located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialate O-acetyl esterases, as shown by complementation of an E. coli strain K-12 nanS mutant and the unimpaired growth of an E. coli O157 nanS mutant on O-acetylated sialic acid. We further demonstrate that nanS homologs in Streptococcus spp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialate O-acetyl esterase. IMPORTANCE The sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show that nanS homologs exist in bacteria other than Escherichia coli, as well as part of toxigenic E. coli prophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies of nanS to include mucosal pathogens, prophage, and prophage remnants. This expansion of the nanS superfamily suggests important, although as-yet-unknown, functions in host-microbe interactions. PMID:27481927

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

Aldo-keto Reductase Family 1 B10 as a Novel Target for Breast Cancer Treatment

DTIC Science & Technology

2010-08-01

overexpressed in tested human breast cancer tissues and mediates acetyl-CoA carboxylase-α ( ACCA ) stability, affecting fatty acid de novo synthesis and...9703; Fax. 217-545-3227; E-mail: dcao@siumed.edu Running title: AKR1B10 as a new risk factor for breast cancer Abbreviations used: ACCA , acetyl...The effect of AKR1B10 expression in cancer tissue on patient survival was evaluated with Kaplan - Meier plots, and results showed that AKR1B10

Anomalous regioselective four-member multicomponent Biginelli reaction II: one-pot parallel synthesis of spiro heterobicyclic aliphatic rings.

PubMed

Byk, Gerardo; Kabha, Eihab

2004-01-01

In a previous preliminary study, we found that a cyclic five-member ring beta-keto ester (lactone) reacts with one molecule of urea and two of aldehyde to give a new family of spiro heterobicyclic aliphatic rings in good yields with no traces of the expected dihydropyrimidine (Biginelli) products. The reaction is driven by a regiospecific condensation of two molecules of aldehyde with urea and beta-keto-gamma-lactone to afford only products harboring substitutions exclusively in a syn configuration (Byk, G.; Gottlieb, H. E.; Herscovici, J.; Mirkin, F. J. Comb. Chem. 2000, 2, 732-735). In the present work ((a) Presented in part at ISCT Combitech, October 15, 2002, Israel, and Eurocombi-2, Copenhagen 2003 (oral and poster presentation). (b) Also in American Peptide Society Symposium, Boston, 2003 (poster presentation). (c) Abstract in Biopolymers 2003, 71 (3), 354-355), we report a large and exciting extension of this new reaction utilizing parallel organic synthesis arrays, as demonstrated by the use of chiral beta-keto-gamma-lactams, derived from natural amino acids, instead of tetronic acid (beta-keto-gamma-lactone) and the potential of the spirobicyclic products for generating "libraries from libraries". Interestingly, we note an unusual and important anisotropy effect induced by perpendicular interactions between rigid pi systems and different groups placed at the alpha position of the obtained spirobicyclic system. Stereo/regioselectivity of the aldehyde condensation is driven by the nature of the substitutions on the starting beta-keto-gamma-lactam. Aromatic aldehydes can be used as starting reagents with good yields; however, when aliphatic aldehydes are used, the desired products are obtained in poor yields, as observed in the classical Biginelli reaction. The possible reasons for these poor yields are addressed and clarify, to some extent, the complexity of the Biginelli multicomponent reaction mechanism and, in particular, the mechanism of the present reaction. Finally, we have investigated and proposed a mechanism for this new reaction by intercepting several intermediates.

Disease-modifying effect of anthraquinone prodrug with boswellic acid on collagenase-induced osteoarthritis in Wistar rats.

PubMed

Dhaneshwar, Suneela; Dipmala, Patil; Abhay, Harsulkar; Prashant, Bhondave

2013-08-01

Diacerein and its active metabolite rhein are promising disease modifying agents for osteoarthritis (OA). Boswellic acid is an active ingredient of Gugglu; a herbal medicine commonly administered in osteoarthritis. Both of them possess excellent anti-inflammatory and anti-arthritic activities. It was thought interesting to conjugate rhein and boswellic acid into a mutual prodrug (DSRB) and evaluate its efficacy on collagenase-induced osteoarthritis in rats wherein the conjugate, rhein, boswellic acid and their physical mixture, were tested based on various parameters. Oral administration of 3.85 mg of rhein, 12.36 mg of boswellic acid and 15.73 mg of DSRB which would release equimolar amounts of rhein and boswellic acid, exhibited significant restoration in rat body weight as compared to the untreated arthritic control group. Increase in knee diameter (mm), due to edema was observed in group injected with collagenase, which reduced significantly with the treatment of conjugate. The hematological parameters (Hb, RBC, WBC and ESR) and biochemical parameters (CRP, SALP, SGOT and SGPT) in the osteoarthritic rats were significantly brought back to normal values on treatment with conjugate. It also showed better anti-ulcer activity than rhein. Further the histopathological studies revealed significant anti-arthritic activity of conjugate when compared with the arthritic control group. In conclusion, the conjugate at the specified dose level of 15.73 mg/kg, p. o. (BID) showed reduction in knee diameter and it could significantly normalize the hematological and biochemical abnormalities in collagenase-induced osteoarthritis in rats. Further the histopathological studies confirmed the additive anti-arthritic effect of DSRB as compared to plain rhein.

Chiral discrimination in cyclodextrin complexes of amino acid derivatives: beta-cyclodextrin/N-acetyl-L-phenylalanine and N-acetyl-D-phenylalanine complexes.

PubMed

Alexander, Jennifer M; Clark, Joanna L; Brett, Tom J; Stezowski, John J

2002-04-16

In a systematic study of molecular recognition of amino acid derivatives in solid-state beta-cyclodextrin (beta-CD) complexes, we have determined crystal structures for complexes of beta-cyclodextrin/N-acetyl-L-phenylalanine at 298 and 20 K and for N-acetyl-D-phenylalanine at 298 K. The crystal structures for the N-acetyl-L-phenylalanine complex present disordered inclusion complexes for which the distribution of guest molecules at room temperature is not resolvable; however, they can be located with considerable confidence at low temperature. In contrast, the complex with N-acetyl-D-phenylalanine is well ordered at room temperature. The latter complex presents an example of a complex in this series in which a water molecule is included deeply in the hydrophobic torus of the extended dimer host. In an effort to understand the mechanisms of molecular recognition giving rise to the dramatic differences in crystallographic order in these crystal structures, we have examined the intermolecular interactions in detail and have examined insertion of the enantiomer of the D-complex into the chiral beta-CD complex crystal lattice.

Green acetylation of solketal and glycerol formal by heterogeneous acid catalysts to form a biodiesel fuel additive.

PubMed

Dodson, Jennifer R; Leite, Thays d C M; Pontes, Nathália S; Peres Pinto, Bianca; Mota, Claudio J A

2014-09-01

A glut of glycerol has formed from the increased production of biodiesel, with the potential to integrate the supply chain by using glycerol additives to improve biodiesel properties. Acetylated acetals show interesting cold flow and viscosity effects. Herein, a solventless heterogeneously catalyzed process for the acetylation of both solketal and glycerol formal to new products is demonstrated. The process is optimized by studying the effect of acetylating reagent (acetic acid and acetic anhydride), reagent molar ratios, and a variety of commercial solid acid catalysts (Amberlyst-15, zeolite Beta, K-10 Montmorillonite, and niobium phosphate) on the conversion and selectivities. High conversions (72-95%) and selectivities (86-99%) to the desired products results from using acetic anhydride as the acetylation reagent and a 1:1 molar ratio with all catalysts. Overall, there is a complex interplay between the solid catalyst, reagent ratio, and acetylating agent on the conversion, selectivities, and byproducts formed. The variations are discussed and explained in terms of reactivity, thermodynamics, and reaction mechanisms. An alternative and efficient approach to the formation of 100% triacetin involves the ring-opening, acid-catalyzed acetylation from solketal or glycerol formal with excesses of acetic anhydride. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

78 FR 21818 - Schedules of Controlled Substances: Placement of Methylone Into Schedule I

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-12

...- methylenedioxymethamphetamine (MDMA), cathinone and other related substances. The addition of a beta-keto ([beta]-ketone) substituent to the phenethylamine core structure produces a group of substances that have [beta]-keto-phenethylamine as the core structure. Methylone has a [beta]-keto-phenethylamine core structure. Methylone has...

Synthesis of 18,19-dihydroxycorticosterone.

PubMed

Harnik, M; Carmely, S; Cojocaru, M; Kashman, Y

1986-01-01

A four-step synthesis of 18,19-dihydroxycorticosterone 5c, starting with 19,21-dihydroxy-3,20-dioxopregn-5-ene-18,11 beta-lactone-di-(ethylene ketal) 2, is presented. Reduction of 2 with sodium aluminum bis-(methoxyethoxy)hydride gave 11 beta,18,19,21-tetrahydroxy-pregn-5-ene-3,20-dione-di-(ethylene ketal) 3a. Acetylation furnished the corresponding 18,19,21-triacetate 3b, which on treatment with a mixture of perchloric and acetic acids gave 18,19-dihydroxycorticosterone 18,19,21-triacetate 4b. Mild saponification yielded the title compound which, on the basis of ir and nmr spectra, exists as one C-20 isomer of the hemiacetal structure 5c. Periodate oxidation of 5c gave the expected 11 beta, 19-dihydroxy-3-oxoandrost-4-ene-17 beta, 18-carbolactone 6b.

Efficacy and safety of curcumin and its combination with boswellic acid in osteoarthritis: a comparative, randomized, double-blind, placebo-controlled study.

PubMed

Haroyan, Armine; Mukuchyan, Vahan; Mkrtchyan, Nana; Minasyan, Naira; Gasparyan, Srbuhi; Sargsyan, Aida; Narimanyan, Mikael; Hovhannisyan, Areg

2018-01-09

The aim of this clinical trial was to assess the efficacy and safety of curcuminoid complex extract from turmeric rhizome with turmeric volatile oil (CuraMed®) and its combination with boswellic acid extract from Indian frankincense root (Curamin®) vs placebo for the treatment of 40- to 70-year-old patients with osteoarthritis (OA). The effects of CuraMed® 500-mg capsules (333 mg curcuminoids) and Curamin® 500-mg capsules (350 mg curcuminoids and 150 mg boswellic acid) taken orally three times a day for 12 weeks in 201 patients was investigated in a three-arm, parallel-group, randomized, double-blinded, placebo-controlled trial. Primary outcome efficacy measures included OA physical function performance-based tests, the WOMAC recommended index of joint pain, morning stiffness, limitations of physical function, and the patients' global assessment of disease severity. Favorable effects of both preparations compared to placebo were observed after only 3 months of continuous treatment. A significant effect of Curamin® compared to placebo was observed both in physical performance tests and the WOMAC joint pain index, while superior efficacy of CuraMed vs placebo was observed only in physical performance tests. The effect size compared to placebo was comparable for both treatment groups but was superior in the Curamin® group. The treatments were well tolerated. Twelve-week use of curcumin complex or its combination with boswellic acid reduces pain-related symptoms in patients with OA. Curcumin in combination with boswellic acid is more effective. Combining Curcuma longa and Boswellia serrata extracts in Curamin® increases the efficacy of OA treatment presumably due to synergistic effects of curcumin and boswellic acid. This trial is registered at the database www.clinicaltrials.gov . https://clinicaltrials.gov/ct2/show/NCT02390349?term=EuroPharma&rank=1 . Study registration number: NCT02390349 .

Effects of carnitine and its congeners on eicosanoid discharge from rat cells: implications for release of TNFα

PubMed Central

Elliott, Graham R.; Pruimboom, Wanda M.; Zijlstra, Freek J.; Bonta, Iván L.

1993-01-01

THE acyl carrier coenzyme A (CoA) is involved in fatty acid metabolism. The carnitine/CoA ratio is of particular importance in regulating the transport of long-chain fatty acids into mitochondria for oxidation. Also CoA has a role in the formation and breakdown of products from both the cyclooxygenase and lipoxygenase pathways of the precursor arachidonic acid. In the present study the effect of 4 days feeding of 300 mg/kg/day of L-carnitine, acetyl Lcarnitine and propionyl L-carnitine on the basal and calcium ionophore (A23187) stimulated release of arachidonic acid metabolites from rat carrageenin elicited peritoneal cells was investigated. There were two series of experiments carried out. In the first, the harvested peritoneal cell population consisted of less than 90% macrophages and additional polymorphonuclear (PMN) leucocytes. The basal release of prostaglandin E2 (PGE2), 6-ketoprostaglandin F1α (6-keto-PGF1α) and leukotriene B4 (LTB4) was stimulated by all treatments. The A23187 stimulated release of 6-keto-PGF1α and LTB4 was increased by all three compounds. The 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 ratios were increased by carnitine treatment. These results suggested that carnitine could modify the macrophage component of an inflammatory site in vivo. In the second series of experiments the harvested cell population was highly purified (>95% macrophages) and none of the compounds fed to the rats caused a change of either eicosanoid or TNFα formation. Moreover the 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 ratios were not enhanced by any of the compounds tested. It is conceivable that in the first series the increased ratios 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 reflected the effect of carnitine or its congeners on PMN leucocytes rather than on macrophages. PMID:18475573

Selenium-mediated synthesis of biaryls through rearrangement.

PubMed

Shahzad, Sohail A; Vivant, Clotilde; Wirth, Thomas

2010-03-19

A new cyclization of beta-keto ester substituted stilbene derivatives using selenium electrophiles in the presence of Lewis acids is described. Substituted naphthols are obtained through cyclization and subsequent 1,2-rearrangement of aryl groups under very mild reaction conditions.

A precursor to the beta-pyranosides of 3-amino-3,6-dideoxy-D-mannose (mycosamine).

PubMed

Alais, J; David, S

1992-06-04

SN2-type reaction of 3-O-(1-imidazyl)sulfonyl-1,2:5,6-di-O-isopropylidene-alpha-D-gluco furanose with benzoate gave the 3-O-benzoyl-alpha-D-allo derivative 2, which was hydrolysed to give the 5,6-diol 3. Compound 3 was converted into the 6-deoxy-6-iodo derivative 4 which was reduced with tributylstannane, and then position 5 was protected by benzyloxymethylation, to give 3-O-benzoyl-5-O-benzyloxymethyl-6-deoxy-1,2-O-isopropylidene-alpha -D- allofuranose (6). Debenzoylation of 6 gave 7, (1-imidazyl)sulfonylation gave 8, and azide displacement gave 3-azido-5-O-benzyloxymethyl-3,6-dideoxy- 1,2-O-isopropylidene-alpha-D-glucofuranose (9, 85%). Acetolysis of 9 gave 1,2,4-tri-O-acetyl-3-azido-3,6-dideoxy-alpha,beta-D-glucopyranose (10 and 11). Selective hydrolysis of AcO-1 in the mixture of 10 and 11 with hydrazine acetate (----12), followed by conversion into the pyranosyl chloride 13, treatment with N,N-dimethylformamide dimethyl acetal in the presence of tetrabutylammonium bromide, and benzylation gave 3-azido-4-O-benzyl-3,6-dideoxy-1,2-O-(1-methoxyethylidene)-alpha-D -glucopyranose (15). Treatment of 15 with dry acetic acid gave 1,2-di-O-acetyl-3-azido-4-O-benzyl-3,6-dideoxy-beta-D-glucopyranose (16, 86% yield) that was an excellent glycosyl donor in the presence of trimethylsilyl triflate, allowing the synthesis of cyclohexyl 2-O-acetyl-3-azido-4-O-benzyl-3,6-dideoxy-beta-D-glucopyranoside (17, 90%).(ABSTRACT TRUNCATED AT 250 WORDS)

Decarboxylative aldol reactions of allyl beta-keto esters via heterobimetallic catalysis.

PubMed

Lou, Sha; Westbrook, John A; Schaus, Scott E

2004-09-22

Mild and selective heterobimetallic-catalyzed decarboxylative aldol reactions involving allyl beta-keto esters have been developed. The reaction is promoted by Pd(0)- and Yb(III)-DIOP complexes at room temperature and involves the in situ formation of a ketone enolate from allyl beta-keto esters followed by addition of the enolate to aldehydes. The reaction is a new example of heterobimetallic catalysis in which the optimized reaction conditions require the addition of both metals.

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1.

PubMed

Pace-Asciak, C R; Domazet, Z

1984-11-14

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.

5-(Tetradecyloxy)-2-furancarboxylic acid and related hypolipidemic fatty acid-like alkyloxyarylcarboxylic acids.

PubMed

Parker, R A; Kariya, T; Grisar, J M; Petrow, V

1977-06-01

5-(Tetradecyloxy)-2-furancarboxylic acid (91, RMI 14514) was found to lower blood lipids and to inhibit fatty acid synthesis with minimal effects on liver weight and liver fat content. This fatty acid-like compound represents a new class of hypolipidemic agent; it is effective in rats and monkeys. The compound resulted from discovery of hypolipidemic activity in certain beta-keto esters, postulation and confirmation of the corresponding benzoic acids as active metabolites, and systematic exploration of the structure--activity relationships.

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

PubMed Central

Siemoneit, U; Koeberle, A; Rossi, A; Dehm, F; Verhoff, M; Reckel, S; Maier, TJ; Jauch, J; Northoff, H; Bernhard, F; Doetsch, V; Sautebin, L; Werz, O

2011-01-01

BACKGROUND AND PURPOSE Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood. EXPERIMENTAL APPROACH A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E2 synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo. KEY RESULTS Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H2 to PGE2 mediated by mPGES1 (IC50 = 3–10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE2 generation and, in human whole blood, β-BA reduced lipopolysaccharide-induced PGE2 biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF1α and thromboxane B2. Intraperitoneal or oral administration of β-BA (1 mg·kg−1) suppressed rat pleurisy, accompanied by impaired levels of PGE2 and β-BA (1 mg·kg−1, given i.p.) also reduced mouse paw oedema, both induced by carrageenan. CONCLUSIONS AND IMPLICATIONS Suppression of PGE2 formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties. PMID:20840544

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense.

PubMed

Siemoneit, U; Koeberle, A; Rossi, A; Dehm, F; Verhoff, M; Reckel, S; Maier, T J; Jauch, J; Northoff, H; Bernhard, F; Doetsch, V; Sautebin, L; Werz, O

2011-01-01

Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood. A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E(2) synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo. Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H(2) to PGE(2) mediated by mPGES1 (IC(50) = 3-10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE(2) generation and, in human whole blood, β-BA reduced lipopolysaccharide-induced PGE(2) biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF(1α) and thromboxane B(2) . Intraperitoneal or oral administration of β-BA (1 mg·kg(-1) ) suppressed rat pleurisy, accompanied by impaired levels of PGE(2) and β-BA (1 mg·kg(-1) , given i.p.) also reduced mouse paw oedema, both induced by carrageenan. Suppression of PGE(2) formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties. © 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.

Aldo-keto reductase family 1 B10 affects fatty acid synthesis by regulating the stability of acetyl-CoA carboxylase-alpha in breast cancer cells.

PubMed

Ma, Jun; Yan, Ruilan; Zu, Xuyu; Cheng, Ji-Ming; Rao, Krishna; Liao, Duan-Fang; Cao, Deliang

2008-02-08

Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (approximately 40 kDa) bound to DEAE-Sepharose column and protein complexes (approximately 300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.

Acyl derivatives of boswellic acids as inhibitors of NF-κB and STATs.

PubMed

Kumar, Ajay; Shah, Bhahwal A; Singh, Samar; Hamid, Abid; Singh, Shashank K; Sethi, Vijay K; Saxena, Ajit K; Singh, Jaswant; Taneja, Subhash C

2012-01-01

Boswellic acid acylates including their epimers were synthesized and screened against a panel of human cancer cell lines. They exhibited a range of cytotoxicity against various human cancer cell lines thereby leading to the development of a possible SAR. One of the identified lead compounds was found to be an inhibitor of the NF-κB and STAT proteins, warranting further investigations to be developed into a potential anticancer lead. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

PubMed Central

Kim, Dockyu; Chae, Jong-Chan; Zylstra, Gerben J.; Kim, Young-Soo; Kim, Seong-Ki; Nam, Myung Hee; Kim, Young Min; Kim, Eungbin

2004-01-01

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively. PMID:15574904

Cyclization of arylacetoacetates to indene and dihydronaphthalene derivatives in strong acids. Evidence for involvement of further protonation of O,O-diprotonated beta-ketoester, leading to enhancement of cyclization.

PubMed

Kurouchi, Hiroaki; Sugimoto, Hiromichi; Otani, Yuko; Ohwada, Tomohiko

2010-01-20

The chemical features, such as substrate stability, product distribution, and substrate generality, and the reaction mechanism of Brønsted superacid-catalyzed cyclization reactions of aromatic ring-containing acetoacetates (beta-ketoesters) were examined in detail. While two types of carbonyl cyclization are possible, i.e., keto cyclization and ester cyclization, the former was found to take place exclusively. The reaction constitutes an efficient method to synthesize indene and 3,4-dihydronapthalene derivatives. Acid-base titration monitored with (13)C NMR spectroscopy showed that the acetoacetates are fully O(1),O(3)-diprotonated at H(0) = -11. While the five-membered ring cyclization of the arylacetoacetates proceeded slowly at H(0) = -11, a linear increase in the rate of the cyclization was found with increasing acidity in the high acidity region of H(0) = -11.8 to -13.3. Therefore, the O(1),O(3)-diprotonated acetoacetates exhibited some cyclizing reactivity, but they are not the reactive intermediates responsible for the acceleration of the cyclization in the high acidity region. The reactive cationic species might be formed by further protonation (or protosolvation) of the O(1),O(3)-diprotonated acetoacetates; i.e., they may be tricationic species. Thermochemical data on the acid-catalyzed cyclization of the arylacetoacetates showed that the activation energy is decreased significantly as compared with that of the related acid-catalyzed cyclization reaction of a compound bearing a single functional group, such as a ketone. These findings indicate that intervention of the trication contributes to the activation of the cyclization of arylacetoacetates in strong acid, and the electron-withdrawing nature of the O-protonated ester functionality significantly increases the electrophilicity of the ketone moiety.

Pharmacologic activation of peroxisome proliferator-activating receptor-α accelerates hepatic fatty acid oxidation in neonatal pigs

PubMed Central

Shim, Kwanseob; Jacobi, Sheila; Odle, Jack; Lin, Xi

2018-01-01

Up-regulation of peroxisome proliferator-activating receptor-α (PPARα) and increasing fatty acid oxidation are important for reducing pre-weaning mortality of pigs. We examined the time-dependent regulatory effects of PPARα activation via oral postnatal clofibrate administration (75 mg/(kg-BW·d) for up to 7 days) on mitochondrial and peroxisomal fatty acid oxidation in pigs, a species with limited hepatic fatty acid oxidative capacity due to low ketogenesis. Hepatic oxidation was increased by 44-147% (depending on fatty acid chain-length) and was attained after only 4 days of clofibrate treatment. Acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) activities accelerated in parallel. The increase in CPTI activity was accompanied by a rapid reduction in the sensitivity of CPTI to malonyl-CoA inhibition. The mRNA abundance of CPTI and ACO, as well as peroxisomal keto-acyl-CoA thiolase (KetoACoA) and mitochondrial malonyl-CoA decarboxylase (MCD), also were augmented greatly. However, the increase in ACO activity and MCD expression were different from CPTI, and significant interactions were observed between postnatal age and clofibrate administration. Furthermore, the expression of acetyl-CoA carboxylase β (ACCβ) decreased with postnatal age and clofibrate had no effect on its expression. Collectively these results demonstrate that the expression of PPARα target genes and the increase in fatty acid oxidation induced by clofibrate are time- and age-dependent in the liver of neonatal pigs. Although the induction patterns of CPTI, MCD, ACO, KetoACoA, and ACCβ are different during the early postnatal period, 4 days of exposure to clofibrate were sufficient to robustly accelerate fatty acid oxidation.

Isolation of a new anti-inflammatory 20, 21, 22, 23, 24, 25, 26, 27-octanorcucurbitacin-type triterpene from Ibervillea sonorae.

PubMed

Jardón-Delgado, Angel; Magos-Guerrero, Gil Alfonso; Martínez-Vázquez, Mariano

2014-01-01

A new cucurbitane-type triterpene, 20, 21, 22, 23, 24, 25, 26, 27-octanorcucurbita-5-ene-3, 11, 16-trione (1), named kinoin D, was isolated from the roots of the medicinal plant Ibervillea sonorae, (wereque). The structure of 1 was established on the basis of extensive NMR and MS studies. In addition, the known kinoins B (3) and C (5) were isolated, as were 16alpha-20,25-trihydroxy-3alpha-(2-O-alpha-L-rhamnopyranosiyl-D-glucopyranosyloxy)-(10alpha)-cucurbit-5-en-11,22-dione (6), (22S)-16alpha,22-diacetoxy-20,25-dihydroxy-3alpha-[3,4,6-tri-O-acetyl-2-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-beta-glucopyranosyl]-(10alpha)-cucurbita-5,23t-dien-11-one (7) and 16alpha-acetoxy-20,25-dihydroxy-3alpha-[3,4,6-tri-O-acetyl-2-O-(2,3,4,-tri-O-acetyl-alpha-L-rhamnopyranosyl)-beta-D-glucopyranosyl]-(10alpha)-cucurbita-5-ene-11,22-dione (8). Compound 1 exhibited anti-inflammatory activity in TPA-induced edema in mice.

Triterpene constituents from the seedling of Aronia melanocarpa.

PubMed

Yu, M; Li, X; Zhao, C-C; Xu, J; Zhang, P

2007-01-01

Two new triterpene saponins, 16-O-acetyl-21-O-angeloyltheasapogenol A 3-O-[beta-D-galactopyranosyl(1 --> 2)][beta-D-xylopyranosyl(1 --> 2)-alpha-L-arabinopyranosyl (1 --> 3)]-beta-D-glucopyranosiduronic acid (1) and 16,28-O-diacetyl-21-O-tigloyltheasapogenol A 3-O-[beta-D-galactopyranosyl(1 --> 2)][beta-D-xylopyranosyl(1 --> 2)-alpha-L-arabinopyranosyl (1 --> 3)]-beta-D-glucopyranosiduronic acid (2), together with four known triterpenenes, have been isolated from the dried seedling of Aronia melanocarpa, and their structures established by spectroscopic and chemical evidence.

Glycoconjugate sugar residues in the chick embryo developing lung: a lectin histochemical study.

PubMed

Gheri, G; Sgambati, E; Bryk, S G

2000-03-01

A lectin histochemical study was performed to investigate the distribution and changes of the oligosaccharidic component of the glycoconjugates in the lung of chick embryos, of 1-day-old chick, and of the adult animal. For this purpose, a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, Con A, LTA, and UEA I) were employed. During the first phase of parabronchi and atria formation, D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine, alpha-D-mannose, and sialic acid, present at the level of the surface and of cytoplasmic granules of the lining epithelial cells, seem to play a role in regulating morphogenetic phenomena. In the subsequent phases, the parabronchial lumen and the atrial cavities were characterized by the presence of lectin-reactive material rich in terminal D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine and alpha-D-mannose. From day 18 onwards and immediately after hatching, the free border of the cells lining the air capillaries was characterized by the presence of beta-N-acetyl-D-galactosamine and alpha-D-mannose. The appearance of these sugar residues was concomitant with the beginning of respiratory activity. Copyright 2000 Wiley-Liss, Inc.

Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

PubMed

Pace-Asciak, C R; Domazet, Z; Carrara, M

1977-05-25

Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.

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

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

Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel, E-mail: mfessop@sun.ac.za

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

The use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review.

PubMed

Harvey, Cliff J D C; Schofield, Grant M; Williden, Micalla

2018-01-01

Adaptation to a ketogenic diet (keto-induction) can cause unpleasant symptoms, and this can reduce tolerability of the diet. Several methods have been suggested as useful for encouraging entry into nutritional ketosis (NK) and reducing symptoms of keto-induction. This paper reviews the scientific literature on the effects of these methods on time-to-NK and on symptoms during the keto-induction phase. PubMed, Science Direct, CINAHL, MEDLINE, Alt Health Watch, Food Science Source and EBSCO Psychology and Behavioural Sciences Collection electronic databases were searched online. Various purported ketogenic supplements were searched along with the terms "ketogenic diet", "ketogenic", "ketosis" and ketonaemia (/ ketonemia). Additionally, author names and reference lists were used for further search of the selected papers for related references. Evidence, from one mouse study, suggests that leucine doesn't significantly increase beta-hydroxybutyrate (BOHB) but the addition of leucine to a ketogenic diet in humans, while increasing the protein-to-fat ratio of the diet, doesn't reduce ketosis. Animal studies indicate that the short chain fatty acids acetic acid and butyric acid, increase ketone body concentrations. However, only one study has been performed in humans. This demonstrated that butyric acid is more ketogenic than either leucine or an 8-chain monoglyceride. Medium-chain triglycerides (MCTs) increase BOHB in a linear, dose-dependent manner, and promote both ketonaemia and ketogenesis. Exogenous ketones promote ketonaemia but may inhibit ketogenesis. There is a clear ketogenic effect of supplemental MCTs; however, it is unclear whether they independently improve time to NK and reduce symptoms of keto-induction. There is limited research on the potential for other supplements to improve time to NK and reduce symptoms of keto-induction. Few studies have specifically evaluated symptoms and adverse effects of a ketogenic diet during the induction phase. Those that have typically were not designed to evaluate these variables as primary outcomes, and thus, more research is required to elucidate the role that supplementation might play in encouraging ketogenesis, improve time to NK, and reduce symptoms associated with keto-induction.

The use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review

PubMed Central

Schofield, Grant M.; Williden, Micalla

2018-01-01

Background Adaptation to a ketogenic diet (keto-induction) can cause unpleasant symptoms, and this can reduce tolerability of the diet. Several methods have been suggested as useful for encouraging entry into nutritional ketosis (NK) and reducing symptoms of keto-induction. This paper reviews the scientific literature on the effects of these methods on time-to-NK and on symptoms during the keto-induction phase. Methods PubMed, Science Direct, CINAHL, MEDLINE, Alt Health Watch, Food Science Source and EBSCO Psychology and Behavioural Sciences Collection electronic databases were searched online. Various purported ketogenic supplements were searched along with the terms “ketogenic diet”, “ketogenic”, “ketosis” and ketonaemia (/ ketonemia). Additionally, author names and reference lists were used for further search of the selected papers for related references. Results Evidence, from one mouse study, suggests that leucine doesn’t significantly increase beta-hydroxybutyrate (BOHB) but the addition of leucine to a ketogenic diet in humans, while increasing the protein-to-fat ratio of the diet, doesn’t reduce ketosis. Animal studies indicate that the short chain fatty acids acetic acid and butyric acid, increase ketone body concentrations. However, only one study has been performed in humans. This demonstrated that butyric acid is more ketogenic than either leucine or an 8-chain monoglyceride. Medium-chain triglycerides (MCTs) increase BOHB in a linear, dose-dependent manner, and promote both ketonaemia and ketogenesis. Exogenous ketones promote ketonaemia but may inhibit ketogenesis. Conclusions There is a clear ketogenic effect of supplemental MCTs; however, it is unclear whether they independently improve time to NK and reduce symptoms of keto-induction. There is limited research on the potential for other supplements to improve time to NK and reduce symptoms of keto-induction. Few studies have specifically evaluated symptoms and adverse effects of a ketogenic diet during the induction phase. Those that have typically were not designed to evaluate these variables as primary outcomes, and thus, more research is required to elucidate the role that supplementation might play in encouraging ketogenesis, improve time to NK, and reduce symptoms associated with keto-induction. PMID:29576959

Fine tuning of the spectral properties of LH2 by single amino acid residues.

PubMed

Silber, Martina V; Gabriel, Günther; Strohmann, Brigitte; Garcia-Martin, Adela; Robert, Bruno; Braun, Paula

2008-05-01

The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 alpha-subunit. The amino acid residues, isoleucine at position -1 and serine at position -4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position -1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine -1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C13(1) keto group. In contrast, nearly all amino acid replacements of serine at position -4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position -4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.

Synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one, an intermediate in the 25-hydroxylation pathway of cholic acid biosynthesis from cholesterol.

PubMed

Dayal, B; Tint, G S; Batta, A K; Shefer, S; Salen, G; Bose, A K; Pramanik, B N

1983-02-01

This paper describes the chemical synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one via selective oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24 xi,25-pentol with silver carbonate on celite. The structure of this 24-keto bile alcohol was confirmed by gas-liquid chromatography and mass spectrometry. Synthesis of this compound via pyridinium chlorochromate oxidation of the triacetoxy derivative of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25-pentol followed by saponification further established its structure. 3 alpha,7 alpha,12 alpha,25-Tetrahydroxy-5 beta-cholestan-24-one was required for the in vivo and in vitro studies of side-chain oxidation and cleavage in the 25-hydroxylation pathway of cholic acid biosynthesis.

Lifelong Literacy: An Economic, Social and Moral Imperative

ERIC Educational Resources Information Center

Eldred, Jan

2011-01-01

Over the past year, NIACE staff have been guided and advised by a group of 11 dynamic and well-informed commissioners, led by Lord Boswell of Aynho, in conducting an independent inquiry into the state of adult literacy in England. Lord Boswell is well equipped for this role and passionate about the issue. As well as being a former minister for…

THE PRODUCTS OF THE CYCLIZING DEHYDRATION OF 1-BETA-PHENYLETHYLCYCLOHEXANOL-1 AND THE SYNTHESIS OF SPIROCYCLOHEXANE-1,1-INDANONE-3.

PubMed

Levitz, M; Perlman, D; Bogert, M T

1939-08-04

(1) Spirocyclohexane-1,1-indanone (VI) has been found among the oxidation products of the hydrocarbon mixture which results when 1-beta-phenylethylcyclohexanol-1 is dehydrated, or when 1-beta-phenylethylcyclohexene is cyclized by aluminum trichloride, and its constitution has been proved by synthesis (2) Its oxime melts at 137-137.8 degrees (corr.). The oxime of m.p. 187.5 degrees , reported by Cook et al. therefore must be derived from some other ketone, perhaps the trans-ketoöctahydrophenanthrene, since we were unable to isolate any oxime of m.p. 177 degrees , the figure which they reported for this compound.

Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

PubMed Central

Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

2017-01-01

D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

Preadipocyte 11beta-hydroxysteroid dehydrogenase type 1 is a keto-reductase and contributes to diet-induced visceral obesity in vivo.

PubMed

De Sousa Peixoto, R A; Turban, S; Battle, J H; Chapman, K E; Seckl, J R; Morton, N M

2008-04-01

Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.

Micropropagation and non-steroidal anti-inflammatory and anti-arthritic agent boswellic acid production in callus cultures of Boswellia serrata Roxb.

PubMed

Nikam, Tukaram D; Ghorpade, Ravi P; Nitnaware, Kirti M; Ahire, Mahendra L; Lokhande, Vinayak H; Chopra, Arvind

2013-01-01

Micropropagation through cotyledonary and leaf node and boswellic acid production in stem callus of a woody medicinal endangered tree species Boswellia serrata Roxb. is reported. The response for shoots, roots and callus formation were varied in cotyledonary and leafy nodal explants from in vitro germinated seeds, if inoculated on Murshige and Skoog's (MS) medium fortified with cytokinins and auxins alone or together. A maximum of 8.0 ± 0.1 shoots/cotyledonary node explant and 6.9 ± 0.1 shoots/leafy node explants were produced in 91 and 88 % cultures respectively on medium with 2.5 μM 6-benzyladenine (BA) and 200 mg l(-1) polyvinylpyrrolidone (PVP). Shoots treated with 2.5 μM IBA showed the highest average root number (4.5) and the highest percentage of rooting (89 %). Well rooted plantlets were acclimatized and 76.5 % of the plantlets showed survival upon transfer to field conditions. Randomly amplified polymorphic DNA (RAPD) analysis of the micropropagated plants compared with mother plant revealed true-to-type nature. The four major boswellic acid components in calluses raised from root, stem, cotyledon and leaf explants were analyzed using HPLC. The total content of four boswellic acid components was higher in stem callus obtained on MS with 15.0 μM IAA, 5.0 μM BA and 200 mg l(-1) PVP. The protocol reported can be used for conservation and exploitation of in vitro production of medicinally important non-steroidal anti-inflammatory metabolites of B. serrata.

Constituents of ophiuroidea. 1. Isolation and structure of three ganglioside molecular species from the brittle star Ophiocoma scolopendrina.

PubMed

Inagaki, M; Shibai, M; Isobe, R; Higuchi, R

2001-12-01

Three ganglioside molecular species, OSG-0 (1), OSG-1 (2), and OSG-2 (3) have been obtained from the polar lipid fraction of the chloroform/methanol extract of the brittle star Ophiocoma scolopendrina. The structures of these gangliosides have been determined on the basis of chemical and spectroscopic evidence as 1-O-[(N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (1), 1-O-[8-O-sulfo-(N-acetyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyll-ceramide (2) and 1-O-[(N-glycolyl-alpha-D-neuraminosyl)-(2-->8)-(N-acetyl- and N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (3). The ceramide moieties were composed of heterogeneous unsubstituted fatty acid, 2-hydroxy fatty acid and phytosphingosine units. Compounds 2 and 3 represent new ganglioside molecular species.

Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions.

PubMed Central

Jaakkola, O.; Nikkari, T.

1990-01-01

Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL. Images Figure 2 PMID:2201201

Biotechnological production of alpha-keto acids: Current status and perspectives.

PubMed

Song, Yang; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

2016-11-01

Alpha-keto (α-keto) acids are used widely in feeds, food additives, pharmaceuticals, and in chemical synthesis processes. Although most α-keto acids are currently produced by chemical synthesis, their biotechnological production from renewable carbohydrates is a promising new approach. In this mini-review, we first present the different types of α-keto acids as well as their applications; next, we summarize the recent progresses in the biotechnological production of some important α-keto acids; namely, pyruvate, α-ketoglutarate, α-ketoisovalerate, α-ketoisocaproate, phenylpyruvate, α-keto-γ-methylthiobutyrate, and 2,5-diketo-d-gluconate. Finally, we discuss the future prospects as well as favorable directions for the biotechnological production of keto acids that ultimately would be more environment-friendly and simpler compared with the production by chemical synthesis. Copyright © 2016. Published by Elsevier Ltd.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

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

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Biosynthesis of trichothecenes and apotrichothecenes.

PubMed

Zamir, L O; Nikolakakis, A; Sauriol, F; Mamer, O

1999-05-01

Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.

Biotransformation of β-keto nitriles to chiral (S)-β-amino acids using nitrilase and ω-transaminase.

PubMed

Mathew, Sam; Nadarajan, Saravanan Prabhu; Sundaramoorthy, Uthayasuriya; Jeon, Hyunwoo; Chung, Taeowan; Yun, Hyungdon

2017-04-01

To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase. An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-β-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards β-keto acids. A novel enzymatic route to generate enantiomerically pure aromatic (S)-β-amino acids from β-keto nitriles is demonstrated for the first time.

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

PubMed

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

2010-01-01

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

Chemical conversion of corticosteroids to 3 alpha,5 alpha-tetrahydro derivatives. Synthesis of 5 alpha-cortol 3-glucuronides and 5 alpha-cortolone 3-glucuronides.

PubMed

Hosoda, H; Osanai, K; Nambara, T

1991-12-01

The synthesis of the 3-glucuronides of 5 alpha-cortol-20 alpha, 5 alpha-cortolone-20 alpha and their 20 beta-epimers is described. The 5 alpha-cortol 20,21-diacetates (12, 17) and 5 alpha-cortolone 20,21-diacetates (14, 19) were the key intermediates. Sodium borohydride reduction of the carbonyl group at C-20 in 5 alpha-tetrahydrocortisol 3-tert-butyldimethylsilyl ether 17,21-acetonide (8) gave the 20 alpha-hydroxy-acetonide (9). Selective removal of the acetonide ring was successful when the 20 alpha-acetoxy-17 alpha,21-acetonide (10) was treated with 50% acetic acid. Subsequent acetylation with acetic anhydride in pyridine, followed by removal of the protecting group at C-3 in the silyl ether-acetate (11) gave the desired 20 alpha-intermediate (12). The 11-ketone (14) was prepared from 11 by oxidation with pyridinium chlorochromate, followed by desilylation. The 20 beta-acetates (17, 19) were synthesized from 21-acetoxy-3 alpha,11 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one 3-tert-butyldimethylsilyl ether (15). Introduction of the glucuronyl residue at C-3 was carried out by means of the Koenigs-Knorr reaction.

C-nor-9,11-secoestranes as modified estrogens and fertility regulation.

PubMed

Lal, K; Sharma, I; Agarwal, A K; Agnihotri, A; Ray, S

1988-06-01

The synthesis of C-nor-9,11-secoestradiol (4) has been achieved from 17 beta-acetoxy-11-chloro-3-methoxy-C-nor-9,11-secoestra-1,3,5(10)-tr ien-9-one (1) through a sequence of reactions without affecting the stereochemistry of estradiol-17 beta. Removal of the 9-keto function of 1 by hydrogenolysis and its subsequent treatment with Na/NH3 gives C-nor-9,11-secoestradiol 3-(methyl ether) (3), which has been demethylated under alkaline conditions to furnish C-nor-9,11-secoestradiol (4). Pyridinium chlorochromate oxidation of 3 gives the corresponding 17-ketone 6. Jones' oxidation of 4 to the ketone 5 and reaction of 5 and 6 with lithium acetylide gives corresponding 17 alpha-ethynyl derivatives 7 and 8. Relative binding affinity to estradiol-17 beta receptors and uterotropic, antiuterotrophic, and antiimplantation activities of compounds 3-8 have been studied. The effect of conformational flexibility on ligand-receptor interaction of these compounds is discussed.

The activity of hydrolases of larval stages of Anisakis simplex (Nematoda).

PubMed

Lopieńska-Biernat, Elzbieta; Zółtowska, Krystyna; Rokicki, Jerzy

2004-01-01

Activity of hydrolases during the third and fourth larval stage of Anisakis simplex was identified by applying the API ZYM test method. In A. simplex larvae the activity of phosphatases was high, particularly that of acid phosphatase (40 nmol/mg(-1)). Among esterases lack of activity of lipase (C14) is worth noticing while the activity of esterases (C4) and (C8) was high. The activity of those later two enzymes was higher in L3 larvae than in L4 larvae. The highest activity in the subclass of glucosidases was recorded for beta-fucosidase and N-acetyl-beta-glucosaminidase. A higher activity in L3 larvae than in L4 larvae was recorded for: beta-glucuronidase and N-acetyl-beta-glucosaminidase (2-fold) and beta-fucosidase (3-fold). Differently the activity of beta-galactosidase and beta-glucosidase was higher in L4 larvae than in L3 larvae. The tests did not show activity of alpha-galactosidase, beta-glucosidase and alpha-mannosidase on both larval forms.

Substituent Effects on Keto-Enol Equilibria Using NMR Spectroscopy

ERIC Educational Resources Information Center

Manbeck, Kimberly A.; Boaz, Nicholas C.; Bair, Nathaniel C.; Sanders, Allix M. S.; Marsh, Anderson L.

2011-01-01

In this extension to a classic physical chemistry experiment, students record the proton nuclear magnetic resonance spectra of the [beta]-diketones 2,4-pentanedione, 3-methyl-2,4-pentanedione, and 3-chloro-2,4-pentanedione to investigate the effect of substituents on keto-enol tautomerization equilibria. From the integrated intensities of keto and…

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

PubMed Central

Ramsden, Christopher E.; Domenichiello, Anthony F.; Yuan, Zhi-Xin; Sapio, Matthew R.; Keyes, Gregory S.; Mishra, Santosh K.; Gross, Jacklyn R.; Majchrzak-Hong, Sharon; Zamora, Daisy; Horowitz, Mark S.; Davis, John M.; Sorokin, Alexander V.; Dey, Amit; LaPaglia, Danielle M.; Wheeler, Joshua J.; Vasko, Michael R.; Mehta, Nehal N.; Mannes, Andrew J.; Iadarola, Michael J.

2018-01-01

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy-or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene–related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber–mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch. PMID:28831021

An evaluation of beta-hydroxybutyrate in milk and blood for prediction of subclinical ketosis in dairy cows.

PubMed

Samiei, A; Liang, J B; Ghorbani, G R; Hirooka, H; Yaakub, H; Tabatabaei, M

2010-01-01

The first objective of this study was to investigate the relationship between concentrations of beta-hydroxybutyrate (BHBA) in milk and blood to assess the reliability of the BHBA concentrations in milk measured by a semi quantitative keto-test paper to detect subclinical ketosis (SCK) in 50 fresh high-producing Iranian Holstein cows in Golestan Province, Iran. The second objective was the effects of SCK on milk yield and components. Concentrations of nonesterified fatty acids (NEFA) and BHBA were analyzed quantitatively in blood plasma and commercial keto-test paper was used for semi quantitative determination of BHBA concentration in milk. Milk yield was measured until 60 d after calving but milk compositions were measured until 30 d after calving. The mean plasma BHBA, milk BHBA, plasma NEFA, milk yield, milk fat percentage and milk fat: protein ratio were 1,234 micromol/L, 145 micromol/L, 0.482 mEq/L, 29.5 kg, 3.9% and 1.4, respectively. Fifty eight percent of the cows had SCK during the first month of lactation. High correlation coefficients were observed between blood BHBA and blood NEFA, and between blood and milk BHBA. The milk yield of cattle with SCK decreased (P < 0.01) but the fat percentage and milk fat: protein ratio increased (P < 0.01). The commercial keto-test paper used had a low false positive result at a cut-off point of 200 fmol of BHBA/L of milk. The results showed that the best time to assess SCK using the commercial keto-test paper was d 10, 14 and 17 after calving.

Two tautomeric forms of 2-amino-5,6-dimethylpyrimidin-4-one.

PubMed

Hall, Victoria M; Bertke, Jeffery A; Swift, Jennifer A

2016-06-01

Derivatives of 4-hydroxypyrimidine are an important class of biomolecules. These compounds can undergo keto-enol tautomerization in solution, though a search of the Cambridge Structural Database shows a strong bias toward the 3H-keto tautomer in the solid state. Recrystallization of 2-amino-5,6-dimethyl-4-hydroxypyrimidine, C6H9N3O, from aqueous solution yielded triclinic crystals of the 1H-keto tautomer, denoted form (I). Though not apparent in the X-ray data, the IR spectrum suggests that small amounts of the 4-hydroxy tautomer are also present in the crystal. Monoclinic crystals of form (II), comprised of a 1:1 ratio of both the 1H-keto and the 3H-keto tautomers, were obtained from aqueous solutions containing uric acid. Forms (I) and (II) exhibit one-dimensional and three-dimensional hydrogen-bonding motifs, respectively.

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis

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

Goto, Tsuyoshi, E-mail: tgoto@kais.kyoto-u.ac.jp; Research Unit for Physiological Chemistry, The Center for the Promotion of Interdisciplinary Education and Research, Kyoto University; Kim, Young-Il

2015-04-17

Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increasedmore » adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism. - Highlights: • Most LA-derived fatty acids from gut lactic acid bacteria potently activated PPARα. • Among tested fatty acids, KetoA and KetoC significantly activated PPARγ. • KetoA induced adipocyte differentiation via the activation of PPARγ. • KetoA enhanced adiponectin production and glucose uptake during adipogenesis.« less

Phenolic compounds from Nymphaea odorata.

PubMed

Zhang, Zhizhen; ElSohly, Hala N; Li, Xing-Cong; Khan, Shabana I; Broedel, Sheldon E; Raulli, Robert E; Cihlar, Ronald L; Burandt, Charles; Walker, Larry A

2003-04-01

Assay-guided fractionation of the ethanol extract of Nymphaea odorata resulted in the identification of two lignans, one new (1) and one known (2), together with six known flavonol glycosides (3-8). The structures of 1-8 were established by spectroscopic analysis as nymphaeoside A (1), icariside E(4) (2), kaempferol 3-O-alpha-l-rhamnopyranoside (afzelin, 3), quercetin 3-O-alpha-l-rhamnopyranoside (4), myricetin 3-O-alpha-l-rhamnopyranoside (myricitrin, 5), quercetin 3-O-(6' '-O-acetyl)-beta-d-galactopyranoside (6), myricetin 3-O-beta-d-galactopyranoside (7), and myricetin 3-O-(6' '-O-acetyl)-beta-d-galactopyranoside (8). Compounds 3, 4, and 7 showed marginal inhibitory effect against fatty acid synthase with IC(50) values of 45, 50, and 25 microg/mL, respectively.

[Effects of keto/amino acids and a low-protein diet on the nutritional status of patients with Stages 3B-4 chronic kidney disease].

PubMed

Milovanova, S Yu; Milovanov, Yu S; Taranova, M V; Dobrosmyslov, I A

To evaluate the efficacy of keto/amino acids in maintaining protein balance and preventing mineral metabolic disturbances and the development of uremic hyperparathyroidism in the long-term use of a low-protein diet (LPD) in patients with Stages 3B-4 chronic kidney disease (CKD). Ninety patients with CKD caused by chronic latent glomerulonephritis in 65 patients and chronic tubulointerstitial nephritis of various etiologies (gout, drug-induced, and infection) in 25 were examined. The investigators conducted clinical, laboratory, and instrumental examinations, including bioelectrical impedance analysis (body mass index (BMI), the percentages of lean and fat mass), echocardiography and radiography of the abdominal aorta in the lateral projection (the presence of cardiac valvular and aortic calcification), and pulse wave velocity measurements using a Sphygmocor apparatus (vessel stiffness estimation). The stages of CKD were defined according to the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) criteria; glomerular filtration rate was calculated using the CKD EPI equation. According to the diet used, all the patients were divided into 3 groups: 1) 30 patients who took LPD (0.6 g of protein per kg of body weight/day) in combination with the keto/amino acid ketosteril (1 tablet per 5 kg of body weight/day; Diet One); 2) 30 patients who used LPD in combination with the other keto/amino acid ketoaminol at the same dose (Diet Two); 3) 30 patients had LPD without using the keto/amino acids (Diet Three) (a control group). During a follow-up, there were no signs of malnutrition in Groups 1 and 2 patients receiving LPD (0.6 g protein per kg/day) in combination with the keto/amino acids ketosteril and ketaminol, respectively. At the same time, 11 (36.6%) patients in Group 3 (a control group) who did not take the keto/amino acids showed a BMI decrease from 24 (23; 26) kg/m2 to 18.5 (17; 19.2) kg/m2 (p < 0.05), including that of lean body mass from 37.4 (36; 38.8) to 30 (29.1; 34.7)% in the men (p<0.05) and from 29.8 (26.8; 31) to 23.9 (22; 25.7)% in the women (p<0.01). In addition, at the end of the study, there were elevated serum phosphorus levels (p<0.05) and mainly higher parathyroid hormone concentrations in Group 3 patients who received LPD without using the amino/keto acids than in Groups 1 and 2. As compared to Group 3, Groups 1 and 2 displayed no differences in the quantity of cardiac and aortic calcification and in the augmentation index (arterial stiffness). The ketosteril and ketaminol groups versus the control group had also higher s-Klotho levels (p<0.01) that were inversely correlated with glomerular filtration rate (r =-0.467; p<0.01). The keto/amino acids ketosteril or ketoaminol are an important component of LPD, which prevents malnutrition and an additional source of calcium that inhibits hyperphosphatemia and slows the development of uremic hyperparathyroidism. Incorporation of keto/amino acids into LPD leads to a less pronounced reduction in s-Klotho protein in relation to the degree of renal failure than does LPD without keto/amino acids.

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.

75 FR 10314 - Manufacturer of Controlled Substances; Notice of Registration

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-05

... Acid (2010) I Methaqualone (2565) I Alpha-ethyltryptamine (7249) I Lysergic acid diethylamide (7315) I...-Methoxyamphetamine (7411) I Alpha-methyltryptamine (7432) I Bufotenine (7433) I Diethyltryptamine (7434) I...-Methylfentanyl (9813) I Alpha-Methylfentanyl (9814) I Acetyl-alpha-methylfentanyl (9815) I Beta-hydroxyfentanyl...

Metal Complexes of New Bioactive Pyrazolone Phenylhydrazones; Crystal Structure of 4-Acetyl-3-methyl-1-phenyl-2-pyrazoline-5-one phenylhydrazone Ampp-Ph

PubMed Central

Idemudia, Omoruyi G.; Sadimenko, Alexander P.; Hosten, Eric C.

2016-01-01

The condensation reaction of phenylhydrazine and dinitrophenylhydrazine with 4-acetyl and 4-benzoyl pyrazolone precipitated air-stable acetyldinitrophenylhydrazone Ampp-Dh, benzoylphenylhydrazone Bmpp-Ph and benzoyldinitrophenylhydrazone Bmpp-Dh in their keto imine form; a study inspired by the burning interest for the development of new bioactive materials with novel properties that may become alternative therapeutic agents. Elemental analysis, FTIR, 1H, and 13C NMR, and mass spectroscopy have been used to justify their proposed chemical structures, which were in agreement with the single crystal structure of Bmpp-Dh earlier reported according to X-ray crystallography. The single crystal structure of 4-acetyl-3-methyl-1-phenyl--pyrazoline-5-one phenylhydrazone Ampp-Ph, which crystallizes in a triclinic crystal system with a P-1 (No. 2) space group is presented. Octahedral Mn(II), Ni(II), Co(II), and Cu(II) complexes of these respective ligands with two molecules each of the bidentate Schiff base, coordinating to the metal ion through the azomethine nitrogen C=N and the keto oxygen C=O, which were afforded by the reaction of aqueous solutions of the corresponding metal salts with the ligands are also reported. Their identity and proposed structures were according to elemental analysis, FTIR spectroscopy, UV-VIS spectrophotometry (electronic spectra) and Bohr magnetic moments, as well as thermogravimetric analysis (TGA) results. A look at the antibacterial and antioxidant activities of synthesized compounds using the methods of the disc diffusion against some selected bacterial isolates and 1,1-diphenyl-2-picryl-hydrazil (DPPH) respectively, showed biological activities in relation to employed standard medicinal drugs. PMID:27213342

Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme

NASA Technical Reports Server (NTRS)

Leznicki, A. J.; Bandurski, R. S.

1988-01-01

The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

Environmental Processing of Lipids Driven by Aqueous Photochemistry of α-Keto Acids

PubMed Central

2018-01-01

Sunlight can initiate photochemical reactions of organic molecules though direct photolysis, photosensitization, and indirect processes, often leading to complex radical chemistry that can increase molecular complexity in the environment. α-Keto acids act as photoinitiators for organic species that are not themselves photoactive. Here, we demonstrate this capability through the reaction of two α-keto acids, pyruvic acid and 2-oxooctanoic acid, with a series of fatty acids and fatty alcohols. We show for five different cases that a cross-product between the photoinitiated α-keto acid and non-photoactive species is formed during photolysis in aqueous solution. Fatty acids and alcohols are relatively unreactive species, which suggests that α-keto acids are able to act as radical initiators for many atmospherically relevant molecules found in the sea surface microlayer and on atmospheric aerosol particles. PMID:29806009

Urinary mutagenicity and N-acetylation phenotype in textile industry workers exposed to arylamines

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

Sinues, B.; Perez, J.; Bernal, M.L.

1992-09-15

Primary aromatic amines have been identified epidemiologically as human carcinogens. It has been suggested that the target organ affected by aromatic amines is dependent on the rate of metabolic activation. Epidemiological studies have shown an association between low acetyl transferase activity and bladder cancer risk. On this basis, our working hypothesis was that the slow acetylators could follow in a higher extent the metabolic pathway independent of N-acetylation, leading to the excretion of conjugates of electrophyles with glucuronic acid. The instability of these glucuronides could be responsible for the association between arylamine-induced bladder cancer and slow acetylator phenotype. A totalmore » of 153 individuals were included in this study: 70 exposed to arylamines (working in textile industry) and 83 nonexposed. The following parameters were determined in urine: mutagenic index in the absence of metabolic activation, S9; mutagenic index in the presence of S9; and the mutagenic index after incubation of the urine with beta-glucuronidase. All individuals were phenotyped according to their capacity of N-acetylation by using isoniazid as drug test. The results show that the mutagenic index after incubation of the urine with beta-glucuronidase is statistically higher in exposed subjects when compared with nonexposed individuals (P less than 0.001), this parameter being statistically higher among exposed subjects who were slow acetylators than among rapid metabolizers, independent of the fact that they were smokers or nonsmokers. There were no significant differences between groups for the mutagenicity in urine not incubated with beta-glucuronidase.« less

Anti-aggregatory effect of boswellic acid in high-fat fed rats: involvement of redox and inflammatory cascades

PubMed Central

2016-01-01

Introduction A high-fat diet is one of the main dietary factors promoting platelet aggregation. The present study was conducted to elucidate the involvement of boswellic acid (BA) on the platelet hyperaggregability in HFD-fed rats. As platelet hyperaggregability in HFD rats is closely linked to inflammation and enhanced free radical production, the present study was extended to evaluate the anti-inflammatory and anti-oxidative effect of BA on HFD-promoted platelet aggregation. Material and methods Rats were assigned to normal, HFD-fed, aspirin-treated (30 mg/kg), and BA-treated (250 and 500 mg/kg) groups. Results Boswellic acid administration in a high dose was effective in attenuating the severity of hyperlipidemia and platelet aggregation, indicated by lower collagen/epinephrine-induced platelet aggregation, as evidenced by the significant increase (p < 0.05) in the circulating platelet count and reduction in the number of thrombi in the lungs. Moreover, it attenuated the oxidative stress and the intensity of inflammatory mediators associated with platelet hyperaggregability, as evidenced by the inhibitory effects on interlukin-1β, COX-2 and tumor necrosis factor-α, indicating that the antiplatelet activity of BA is likely a consequence of controlling oxidative stress and inflammation. Conclusions The present data suggest that BA shows a promising anti-aggregatory effect by attenuating the enhanced hyperlipidemia, oxidative stress and inflammation associated with HFD. PMID:27904529

2-Keto acids based biosynthesis pathways for renewable fuels and chemicals.

PubMed

Tashiro, Yohei; Rodriguez, Gabriel M; Atsumi, Shota

2015-03-01

Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

Antioxidant chalcone glycosides and flavanones from Maclura (Chlorophora) tinctoria.

PubMed

Cioffi, Giuseppina; Morales Escobar, Luis; Braca, Alessandra; De Tommasi, Nunziatina

2003-08-01

Four chalcone glycosides (1-4), including three new natural products, and three flavanones (5-7) were isolated from the methanol extract of stem bark of Maclura tinctoria. The new compounds have been characterized as 4'-O-beta-D-(2' '-p-coumaroyl)glucopyranosyl-4,2',3'-trihydroxychalcone (1), 4'-O-beta-D-(2' '-p-coumaroyl-6' '-acetyl)glucopyranosyl-4,2',3'-trihydroxychalcone (2), and 3'-(3-methyl-2-butenyl)-4'-O-beta-D-glucopyranosyl-4,2'-dihydroxychalcone (3); the known derivatives were elucidated as 4'-O-beta-D-(2' '-acetyl-6' '-cinnamoyl)glucopyranosyl-4,2',3'-trihydroxychalcone (4), eriodictyol 7-O-beta-D-glucopyranoside (5), naringenin (6), and naringenin 4'-O-beta-D-glucopyranoside (7). Their structures were determined by 1D and 2D NMR and ESIMS. The antioxidant activity of all the isolated compounds was determined by measuring free-radical-scavenging effects using two different assays, namely, the Trolox Equivalent Antioxidant Capacity (TEAC) assay and the coupled oxidation of beta-carotene and linoleic acid (autoxidation assay). The results showed that compound 3 was the most active in both antioxidant assays.

Progesterone biotransformation by plant cell suspension cultures.

PubMed Central

Yagen, B; Gallili, G E; Mateles, R I

1978-01-01

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid. PMID:697360

Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

DOEpatents

Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

2005-09-13

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

11. DETAIL VIEW OF WELDED DATES 1896/1941 (S.E. CORNER) ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. DETAIL VIEW OF WELDED DATES - 1896/1941 (S.E. CORNER) 3 Photocopies of drawings labeled 'Bridge Rating Program by Boswell Engineering Company - Abbett Avenue Bridge, Spanning Whippany River at Abbett Avenue, Morristown, Morris County, NJ

Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species.

PubMed Central

Pine, L; Hoffman, P S; Malcolm, G B; Benson, R F; Franzus, M J

1986-01-01

Keto acids and reduced-oxygen-scavenging enzymes were examined for their roles in supporting the growth of Legionella species and for their potential reactions between the chemical components of the media. When grown in an experimental ACES (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid)-buffered chemically defined (ABCD) broth, the presence of keto acids shortened the lag periods, increased the rates of growth, and gave maximum cell yields. In addition, keto acids affected the specific activities of reduced-oxygen-scavenging enzymes determined during growth. The specific activities of superoxide dismutase of Legionella pneumophila (Knoxville) and L. dumoffii (TEX-KL) were increased three- to eightfold, while that of L. bozemanii (WIGA) was not affected. All strains appeared to be equally sensitive to the effects of superoxide anion (O2-) generated by light-activated riboflavin, and all were equally protected by the presence of keto acids in the ABCD broth. Production of trace amounts of acetate and succinate in pyruvate- and alpha-ketoglutarate-containing media exposed to light suggested that hydrogen peroxide was formed. Pyruvate and alpha-ketoglutarate were products of growth on amino acids, and there was no quantitative evidence that these keto acids were metabolized when they were added to the medium. The rate of cysteine oxidation in ABCD broth was increased by the presence of ferric ion or by exposure to light or by both, and keto acids reduced the rate of this oxidation. ACES buffer was a substrate for the production of O2- in the presence of light, and the combined addition of Fe2+ ions, cysteine, and either keto acid to the medium strongly inhibited the production of O2-. Thus, keto acids inhibited the rate of cysteine oxidation, they stimulated rapid growth by an unknown process, and, in combination with added Fe2+ ions and cysteine, they reversed the toxic effects of light by inhibiting O2- production. PMID:3009529

Encapsulation of boswellic acid with β- and hydroxypropyl-β-cyclodextrin: Synthesis, characterization, in vitro drug release and molecular modelling studies

NASA Astrophysics Data System (ADS)

Tambe, Amruta; Pandita, Nancy; Kharkar, Prashant; Sahu, Niteshkumar

2018-02-01

Boswellic acids (BAs) are a group of pentacyclic triterpenes present in gum-resin of Boswellia serrata. They are well known for their anti-inflammatory, hypolipidemic, immunomodulatory and anti-tumor activity, but they have poor aqueous solubility and limited bioavailability. In order to enhance their aqueous solubility, inclusion complexes of BAs with β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) were synthesized and their drug release profiles were studied. Molecular associations of β-CD and HP-β-CD with BAs were investigated by phase solubility studies. The stability constants were found to be 380.2 and 145.9 M-1 for BA: β-CD and BA: HP-β-CD inclusion complexes, respectively with AN- type curve. BA: β-CD and BA: HP-β-CD inclusion complexes were synthesized using kneading (KN), co-precipitation (CP) and solvent evaporation (SE) methods in 1:1 as well as 1:2 ratios. Further these were characterized by Fourier transform infrared (FTIR) spectrophotometry, Powder X-ray Diffraction (P-XRD) and Differential scanning calorimetric (DSC) analysis. FTIR analysis showed shifting of frequencies in complexes as compared to CDs and BAs. P-XRD data obtained for BA: β-CD complexes synthesized by CP and SE methods showed amorphous pattern. Also, DSC analysis showed a change in thermal behaviour for synthesized complexes. In vitro drug release studies of BA: β-CD complexes showed enhanced release with 1:2 complexes than 1:1 complexes at pH 1.2 and pH 6.8. Similarly, drug release enhancement was observed more with BA: HP-β-CD complexes in 1:2 ratio than 1:1. To understand the interaction of BAs with CD cavity molecular modelling studies were performed which favored 1:2 complex formation over 1:1 complexes. The study thus highlights that CDs can be used for solubility and dissolution enhancement of BAs.

Stereocontrolled reduction of alpha- and beta-keto esters with micro green algae, Chlorella strains.

PubMed

Ishihara, K; Yamaguchi, H; Adachi, N; Hamada, H; Nakajima, N

2000-10-01

The stereocontrolled reduction of alpha- and beta-keto esters using micro green algae was accomplished by a combination of the cultivation method and the introduction of an additive. The reduction of ethyl pyruvate and ethyl benzoylformate by the photoautotrophically cultivated Chlorella sorokiniana gave the corresponding alcohol in high e.e. (>99% e.e. (S) and >99% e.e. (R), respectively). In the presence of glucose as an additive, the reduction of ethyl 3-methyl-2-oxobutanoate by the heterotrophically cultivated C. sorokiniana afforded the corresponding (R)-alcohol. On the other hand, the reduction in the presence of ethyl propionate gave the (S)-alcohol. Ethyl 2-methyl-3-oxobutanoate was reduced in the presence of glycerol by the photoautotrophically cultivated C. sorokiniana or the heterotrophically cultivated C. sorokiniana to the corresponding syn-(2R,3S)-hydroxy ester with high diastereo- and enantiomeric excess (e.e.). Some additives altered the stereochemical course in the reduction of alpha- and beta-keto esters.

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

PubMed Central

Condemine, G; Hugouvieux-Cotte-Pattat, N; Robert-Baudouy, J

1986-01-01

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis. PMID:3949717

Preparation and investigation of acetyl salicylic acid-caffeine complex for rectal administration.

PubMed

Fouad, Ehab A; El-Badry, Mahmoud; Alanazi, Fars K; Arafah, Maha M; Al-Ashban, Riyadh; Alsarra, Ibrahim A

2010-06-01

An acetyl salicylic acid-caffeine complex was prepared and evaluated for the potential use in rectal administration. The results revealed the formation of a complex between acetyl salicylic acid and caffeine in a 1:1 molar ratio by a charge transfer mechanism. The effects of acetyl salicylic acid and complex on the rectal tissues showed destruction in the mucosal epithelium in case of acetyl salicylic acid; however, no change in the rectal tissues was noticed upon the administration of the complex. The effect of suppository bases on the release of the complex was studied using Witepsol H15 as fatty base and polyethylene glycols (PEG) 1000 and 4000 as a water soluble suppository base. The release profiles of acetyl salicylic acid and the complex were faster from PEG than from that of Witepsol H15. The percent release for the complex and acetyl salicylic acid from PEG base were 45.8, and 34.9%, respectively. However, it was 8.7 and 7.8%, respectively, from Witepsol H15 fatty base. The release kinetic was found to follow the non-Fickian diffusion model for complex from the suppository bases. It was concluded that acetyl salicylic acid caffeine complex can be used safely for rectal administration.

Preparation and investigation of acetyl salicylic acid-caffeine complex for rectal administration.

PubMed

Fouad, Ehab A; El-Badry, Mahmoud; Alanazi, Fars K; Arafah, Maha M; Al-Ashban, Riyadh; Alsarra, Ibrahim A

2009-07-30

An acetyl salicylic acid-caffeine complex was prepared and evaluated for the potential use in rectal administration. The results revealed the formation of a complex between acetyl salicylic acid and caffeine in a 1:1 molar ratio by a charge transfer mechanism. The effects of acetyl salicylic acid and complex on the rectal tissues showed destruction in the mucosal epithelium in case of acetyl salicylic acid; however, no change in the rectal tissues was noticed upon the administration of the complex. The effect of suppository bases on the release of the complex was studied using Witepsol H15 as fatty base and polyethylene glycols (PEG) 1000 and 4000 as a water soluble suppository base. The release profiles of acetyl salicylic acid and the complex were faster from PEG than from that of Witepsol H15. The percent release for the complex and acetyl salicylic acid from PEG base were 45.8, and 34.9%, respectively. However, it was 8.7 and 7.8%, respectively, from Witepsol H15 fatty base. The release kinetic was found to follow the non-Fickian diffusion model for complex from the suppository bases. It was concluded that acetyl salicylic acid caffeine complex can be used safely for rectal administration.

Synthesis of novel ganglioside GM4 analogues containing N-deacetylated and lactamized sialic acid: probes for searching new ligand structures for human L-selectin.

PubMed

Otsubo, N; Ishida, H; Kiso, M

2001-01-15

Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual N-acetylneuraminic acid, were synthesized in a highly efficient manner. (Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate)-(2-->3)-4,6-di-O-acetyl-2-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired beta-glycoside in high yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Me2SO into the lactamized sialic acid-containing ganglioside GM4 analogue.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies.

PubMed

Kumar, Rajeev; Wyman, Charles E

2009-09-01

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and beta-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase-xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Comparison of Values in 5th, 6th, 7th and 8th Grade Primary Education Music Class Students'? Workbooks According to Rokeach?s and Akbas's Value Classifications

ERIC Educational Resources Information Center

Çakirer, H. Serdar

2014-01-01

The aim of the present study is to compare the values in the songs of 5th, 6th, 7th and 8th grade primary education music classes students? workbooks according to the value categorizations proposed by Rockeach and Akbas and which values among the categories mentioned are taught to the students in the 5th, 6th, 7th and 8th grade primary education…

Involvement of an octose ketoreductase and two acyltransferases in the biosynthesis of paulomycins

NASA Astrophysics Data System (ADS)

Li, Jine; Wang, Min; Ding, Yong; Tang, Yue; Zhang, Zhiguo; Chen, Yihua

2016-02-01

C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4‧ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7‧-keto of PAU E (1) to give the C-4‧ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4‧ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7‧-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-03-22

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-12-06

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

RAPID TEST FOR CHITINASE ACTIVITY THAT USES 4-METHYLUMBELLIFERYL-NU-ACETYL-BETA-D-GLUCOSAMINIDE

EPA Science Inventory

One hundred and one strains of bacteria from environmental and clinical sources, most of which were Gram negative, were tested for n-acetyl-Beta-D-glucosaminidase activity using a filter paper spot test with 4-methylumbelliferyl-N-acetyl-Beta-D-glucosaminide (4-MNABetaG) as subst...

Transcriptome sequencing and metabolic pathways of astaxanthin accumulated in Haematococcus pluvialis mutant under 15% CO2.

PubMed

Cheng, Jun; Li, Ke; Zhu, Yanxia; Yang, Weijuan; Zhou, Junhu; Cen, Kefa

2017-03-01

Transcriptome sequencing and annotation was performed on Haematococcus pluvialis mutant red cells induced with high light under 15% CO 2 to demonstrate why astaxanthin yield of the mutant was 1.7 times higher than that of a wild strain. It was found that 56% of 1947 differentially expressed genes were upregulated in mutant cells. Most significant differences were found in unigenes related to photosynthesis, carotenoid biosynthesis and fatty acid biosynthesis pathways. The pyruvate kinase increased by 3.5-fold in mutant cells. Thus, more pyruvate, which was beneficial to carotenoids and fatty acid biosynthesis, was generated. Phytoene synthase, zeta-carotene desaturase, lycopene beta-cyclase involved in β-carotene biosynthesis in mutant cells were upregulated by 10.4-, 4.4-, and 5.8-fold, respectively. Beta-carotene 3-hydroxylase catalyzing conversion of β-carotene into astaxanthin was upregulated by 18.4-fold. The fatty acid biosynthesis was promoted because of the upregulation of acetyl-CoA synthetase and acetyl-CoA carboxylase, thus increasing astaxanthin esterification and accumulation in mutant cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of N-acetylated forms of alpha-MSH and beta-endorphin in the intermediate pituitary of the holostean fishes, Lepisosteus spatula, Lepisosteus osseus, and Amia calva.

PubMed

Dores, R M; Keller, H; White, Y; Marra, L E; Youson, J H

1994-01-01

Acid extracts of the intermediate pituitaries of the gars, L. spatula and L. osseus, were fractionated by Sephadex G-50 column chromatography and analyzed by radioimmunoassay. This procedure revealed that immunoreactive forms of N-acetylated beta-endorphin- and alpha-MSH-sized material were present in equimolar amounts and represented the major end products of the POMC biosynthetic pathway in these species. Cation-exchange chromatography indicated that multiple N-acetylated forms of beta-endorphin were present in the intermediate pituitaries of the two species of gar, and that these forms differed in their net positive charge and in their apparent molecular weight. Reversed-phase HPLC analysis of the alpha-MSH-related material indicated that up to 90% of the total MSH in the pituitary of the gar was N-acetylated. Furthermore, the predominant form of alpha-MSH in both species of gar was N,O-diacetyl-ACTH(1-13)-NH2. Nearly identical results were obtained following the analysis of alpha-MSH-related peptides in the intermediate pituitary of the bowfin, A. calva. The pattern of posttranslational processing of POMC observed in the intermediate pituitaries of holostean fishes is very similar to the processing events observed in lungfishes, turtles, and mammals; hence, the processing of POMC has been remarkably conserved during vertebrate evolution.

A historical sketch of the discovery and development of HIV-1 integrase inhibitors.

PubMed

Savarino, Andrea

2006-12-01

The long process of HIV-1 integrase inhibitor discovery and development can be attributed to both the complexity of HIV-1 integration and poor 'integration' of these researches into mainstream investigations on antiretroviral therapy in the mid-1990s. Of note, some fungal extracts investigated during this period contain the beta-hydroxyketo group, later recognised to be a key structural requirement for keto-enol acids (also referred to as diketo acids) and other integrase inhibitors. This review reconstructs (in the general context of the history of AIDS research) the principal steps that led to the integrase inhibitors currently in clinical trials, and discusses possible future directions.

The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells.

PubMed

Abasi, M; Massumi, M; Riazi, G; Amini, H

2012-10-11

Parkinson's disease (PD) is a neurodegenerative disorder in which the nigro-striatal dopaminergic (DAergic) neurons have been selectively lost. Due to side effects of levodopa, a dopamine precursor drug, recently cell replacement therapy for PD has been considered. Lack of sufficient amounts of, embryos and ethical problems regarding the use of dopamine-rich embryonic neural cells have limited the application of these cells for PD cell therapy. Therefore, many investigators have focused on using the pluripotent stem cells to generate DAergic neurons. This study is aimed first to establish a mouse embryonic stem (mES) cell line that can stably co-express Nurr1 (Nuclear receptor subfamily 4, group A, member 2) transcription factor in order to efficiently generate DAergic neurons, and glutathione peroxidase-1 (GPX-1) to protect the differentiated DAergic-like cells against oxidative stress. In addition to genetic engineering of ES cells, the effect of Beta-boswellic acid (BBA) on DAergic differentiation course of mES cells was sought in the present study. To that end, the feeder-independent CGR8 mouse embryonic stem cells were transduced by Nurr1- and GPX-1-harboring Lentiviruses and the generated Nurr1/GPX-1-expresssing ES clones were characterized and verified. Gene expression analyses demonstrated that BBA treatment and overexpression of Nurr1 has a synergistic effect on derivation of DAergic neurons from Nurr1/GPX-1-expressing ES cells. The differentiated cells could exclusively synthesize and secrete dopamine in response to stimuli. Overexpression of GPX-1 in genetically engineered Nurr1/GPX-1-ES cells increased the viability of these cells during their differentiation into CNS stem cells. In conclusion, the results demonstrated that Nurr1-overexpressing feeder-independent ES cells like the feeder-dependent ES cells, can be efficiently programmed into functional DAergic neurons and additional treatment of cells by BBA can even augment this efficiency. GPX-1 overexpression in Nurr1/GPX-1-ES cells increases the viability of differentiated CNS stem-like cells. The result of this study may have impact on future stem cell therapy of PD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Synthesis of 19-noraldosterone, a potent mineralocorticoid.

PubMed

Harnik, M; Kashman, Y; Cojocaru, M; Rosenthal, T; Morris, D J

1986-06-01

19-Noraldosterone has been prepared for biological re-evaluation through an extension of a recent synthesis of 19-hydroxyaldosterone: 21-hydroxy-6 beta,19-epoxy-4-pregnene-3,20-dion-20-ethylene ketal-18,11 beta-lactone (1a) was acetylated and then reduced with zinc-acetic acid-isopropanol to the 19-ol 2b. Treatment with sodium acetate transposed the double bond into conjugation, and 2a thus obtained was oxidized with pyridinium chlorochromate to the 19-oxo compound 3. Decarbonylation to the 19-nor lactone 4 was effected by heating with alkali. Protection of the C-3 carbonyl was achieved by ketalization and the resulting mixture of the 5-ene and 5(10)-ene ketals 5 was reduced with DIBAH to the corresponding mixture of the hemiacetals 6. Acid hydrolysis of the latter afforded 19-noraldosterone (7), accompanied by the 18,21-anhydro ketal 8. 19-Noraldosterone in the solid state exists in the cyclic form 7b, which appears to be also the predominant isomer present under conditions of mass spectrometry. [1H]NMR indicates that in chloroform 19-noraldosterone exists mostly as an equilibrium mixture of structures 7a and 7b. Sodium periodate oxidation furnished the gamma-etiolactone 9, confirming the 17 beta configuration in 7.

Kinetic and Mechanistic Studies of the Deuterium Exchange in Classical Keto-Enol Tautomeric Equilibrium Reactions

ERIC Educational Resources Information Center

Nichols, Michael A.; Waner, Mark J.

2010-01-01

An extension of the classic keto-enol tautomerization of beta-dicarbonyl compounds into a kinetic analysis of deuterium exchange is presented. It is shown that acetylacetone and ethyl acetoacetate undergo nearly complete deuterium exchange of the alpha-methylene carbon when dissolved in methanol-d[subscript 4]. The extent of deuteration may be…

[A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

PubMed

Kim, Yong Kyu; Kim, Tae Ue; Joung, In Sil; Im, Kyung Il

1988-06-01

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta-N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80% of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

PubMed Central

Neat, C E; Thomassen, M S; Osmundsen, H

1981-01-01

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids. PMID:6272750

Smad Acetylation: A New Level of Regulation in TGF-Beta Signaling

DTIC Science & Technology

2007-07-01

of Smad2 and Smad3 , resulting in their oligomerization with the common mediator Smad4 (10-11). This Smad2/ Smad3 /Smad4 complex can then translocate...Smad2 and Smad3 , enabling oligomerization with Smad4 and translocation of the entire Smad complex into the nucleus. Once in the nucleus, the...performed prior to DOD funding determined that Smad2 but not Smad3 is efficiently acetylated in a p300 depend manner both in in vivo and in vitro models

1. GENERAL VIEW OF MAIN (SOUTH AND EAST) FACADES OF ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. GENERAL VIEW OF MAIN (SOUTH AND EAST) FACADES OF SCHOOL, LOOKING NORTHWEST (For a brief history of Boswell and a description of St. Stanislaus Catholic Church see TOWN OF BOSWELL, HAER PA-367) - Saint Stanislaus Roman Catholic Church School, Stonycreek Street & Hower Avenue, Boswell, Somerset County, PA

Synthesis of 19-hydroxyaldosterone and the 3 beta-hydroxy-5-ene analog of aldosterone, active mineralocorticoids.

PubMed

Harnik, M; Kashman, Y; Aharonowitz, Y; Morris, D J

1985-08-01

19-Hydroxyaldosterone (20) and the 3 beta-hydroxy-5-ene analog of aldosterone (HAA) (8) were synthesized from 21-acetoxy-4-pregnene-3,20-dion-20-ethylene ketal-18, 11 beta-lactone (2) as follows: the double bond was transposed from the 4,5 to the 5,6-position by enol acetylation to 3, followed by sodium borohydride reduction. Further reduction of the resulting lactone 4a with diisobutylaluminum hydride (DIBAH) furnished the 20-ketal of HAA 6, from which free HAA (8) and the 18,21-anhydro compound 7 were obtained by acid treatment. The [1H]NMR spectrum of 8 in CDCl3 showed it to be a mixture of two isomeric forms. Correlation with the known aldosterone-gamma-etiolactone (10) was established by periodate oxidation of HAA to the corresponding etiolactone 9 followed by chromic acid oxidation. The preparation of 20 was next effected in the following manner: the diacetate 4b was converted into the 6 beta, 19-oxido compound 13b by addition of hypobromous acid followed by the hypoiodite reaction of the bromohydrin 11. Mild saponification of 13b lead to the corresponding diol 13a, and was followed by selective oxidation to the 3-one 14, readily dehydrobrominated to 15a. Reductive ring opening furnished a mixture of the 19,21-diol 16a and its 5-ene isomer 16b, which was directly converted to the diketal 17. Reduction with DIBAH gave the hemiacetal 18, and hydrolysis of the latter 19-hydroxyaldosterone (20) as a water-soluble solid, accompanied by the 18,21-anhydro compound 19. 19-Hydroxyaldosterone exists in CHCl3 and water as a mixture of mainly two isomers. Periodate oxidation furnished the etiolactone 21. Preliminary results indicate that HAA and 19-hydroxyaldosterone are active mineralocorticoids in the Kagawa bioassay and short-circuit current measurements.

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

PubMed

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

2007-06-01

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

Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

PubMed

Nunn, Charlotte E M; Johnsen, Ulrike; Schönheit, Peter; Fuhrer, Tobias; Sauer, Uwe; Hough, David W; Danson, Michael J

2010-10-29

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

Synthesis and biological activity of novel series of 4-methoxy, and 4,9-dimethoxy-5-substituted furo[2,3-g]-1,2,3-benzoxathiazine-7,7-dioxide derivatives

PubMed Central

El-Sawy, Eslam R.; Ebaid, Manal S.; Abo-Salem, Heba M.; El-Hallouty, Salwa; Kassem, Emad M.; Mandour, Adel H.

2013-01-01

A novel series of 4-methoxy, and 4,9-dimethoxy-5-substituted furo[2,3-g]-1,2,3-benzoxathiazine-7,7-dioxide derivatives 3a,b, 10a–g and 11a–g were prepared in good yields via the reaction of 4-methoxy (1a) and 4,7-dimethoxy-5-acetyl-6-hydroxybenzofurans (1b) and their α,β-unsaturated keto derivatives 6a–g and 7a–g with chlorosulfonyl isocyanate (CSI). On the other hand, N-chlorosulfonyl carbamate derivatives 4a,b, 12a,b and 13a,b were prepared and allowed to react with piperidine to give the corresponding N-piperidinosulfonyl carbamate derivatives 5a,b, 14a,b and 15a,b, respectively. Sixteen new target compounds 3a,b, 10a–g, and 11a–g were tested for their DPPH radical-scavenging, and in vitro antiproliferative activity against A-549, MCF7 and HCT-116 cancer cell lines. Compounds 10a, 11c, 11e, and 11g showed moderate DPPH radical-scavenging activity compared to ascorbic acid at 100 μg/mL. 4,9-Dimethoxy-5-substituted styrylfuro[3,2-g]-1,2,3-benzoxathiazine-7,7-dioxides 11a, 11b, and 11c were found to be highly active against A-549 and HCT-116 cancer cell lines with IC50 values ranging from 0.02 to 0.08 μmol/mL compared to doxorubicin with IC50 = 0.04 and 0.06 μmol/mL, respectively. PMID:25685501

Synthesis and biological activity of novel series of 4-methoxy, and 4,9-dimethoxy-5-substituted furo[2,3-g]-1,2,3-benzoxathiazine-7,7-dioxide derivatives.

PubMed

El-Sawy, Eslam R; Ebaid, Manal S; Abo-Salem, Heba M; El-Hallouty, Salwa; Kassem, Emad M; Mandour, Adel H

2014-05-01

A novel series of 4-methoxy, and 4,9-dimethoxy-5-substituted furo[2,3-g]-1,2,3-benzoxathiazine-7,7-dioxide derivatives 3a,b, 10a-g and 11a-g were prepared in good yields via the reaction of 4-methoxy (1a) and 4,7-dimethoxy-5-acetyl-6-hydroxybenzofurans (1b) and their α,β-unsaturated keto derivatives 6a-g and 7a-g with chlorosulfonyl isocyanate (CSI). On the other hand, N-chlorosulfonyl carbamate derivatives 4a,b, 12a,b and 13a,b were prepared and allowed to react with piperidine to give the corresponding N-piperidinosulfonyl carbamate derivatives 5a,b, 14a,b and 15a,b, respectively. Sixteen new target compounds 3a,b, 10a-g, and 11a-g were tested for their DPPH radical-scavenging, and in vitro antiproliferative activity against A-549, MCF7 and HCT-116 cancer cell lines. Compounds 10a, 11c, 11e, and 11g showed moderate DPPH radical-scavenging activity compared to ascorbic acid at 100 μg/mL. 4,9-Dimethoxy-5-substituted styrylfuro[3,2-g]-1,2,3-benzoxathiazine-7,7-dioxides 11a, 11b, and 11c were found to be highly active against A-549 and HCT-116 cancer cell lines with IC50 values ranging from 0.02 to 0.08 μmol/mL compared to doxorubicin with IC50 = 0.04 and 0.06 μmol/mL, respectively.

Major urinary metabolites of 6-keto-prostaglandin F2α in mice[S

PubMed Central

Kuklev, Dmitry V.; Hankin, Joseph A.; Uhlson, Charis L.; Hong, Yu H.; Murphy, Robert C.; Smith, William L.

2013-01-01

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF2α urinary metabolites included dinor-6-keto-PGF2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases. PMID:23644380

Alleviative effects from boswellic acid on acetaminophen-induced hepatic injury - Corrected and republished from: Biomedicine (Taipei). 2016 Jun; 6 (2): 9. doi: 10.7603/s40681-016-0009-1PMCID: PMC4864770.

PubMed

Chen, Lung-Che; Hu, Li-Hong; Yin, Mei-Chin

2017-06-01

Protective effects of boswellic acid (BA) against acetaminophen (APAP)-induced hepatotoxicity in Balb/ cA mice were examined. BA, at 0.05 or 0.1%, was supplied for 4 weeks. Acute liver injury was induced by APAP treatment. Results showed that BA intake increased hepatic BA bioavailability. APAP treatment decreased glutathione (GSH) level, increased reactive oxygen species (ROS) and oxidized glutathione (GSSG) production; and lowered activity and protein expression of glutathione reductase (GR) and heme oxygenase (HO)-1 in liver. BA intake at both doses alleviated subsequent APAP-induced oxidative stress by retaining GSH content, decreasing ROS and GSSG formations, reserving activity and expression of GR and HO-1 in liver, and lowering hepatic cytochrome P450 2E1 activity and expression. APAP treatment enhanced hepatic levels of interleukin-6, tumor necrosis factor-alpha and monocyte chemoattractant protein-1. BA pre-intake diminished APAP-induced release of those inflammatory cytokines and chemokines. APAP up-regulated hepatic protein expression of toll-like receptor (TLR)-3, TLR-4, MyD88, nuclear factor kappa B (NF-κB) p50, NF-κB p65 and JNK. BA pre-intake at both doses suppressed the expression of NF-κB p65 and p-JNK, and only at 0.1% down-regulated hepatic TLR-3, TLR-4 and MyD88 expression. APAP led to obvious foci of inflammatory cell infiltration in liver, determined by H&E stain. BA intake at both doses attenuated hepatic inflammatory infiltration. These findings support that boswellic acid is a potent hepato-protective agent. © Author(s) 2017. This article is published with open access by China Medical University.

Plasma concentrations of 15-keto-13,14-dihydro-PGF-2 alpha, oestrone sulphate, oestradiol-17 beta and progesterone in pregnant guinea-pigs treated with polychlorinated biphenyls.

PubMed

Lundkvist, U; Kindahl, H

1989-09-01

Guinea-pigs treated by gavage with a total dose of 100 mg polychlorinated biphenyls (PCB: Clophen A50) during Days 17-61 of gestation had higher plasma concentrations of 15-keto-13,14-dihydroprostaglandin F-2 alpha, oestrone sulphate and oestradiol-17 beta during the later stages of gestation than did vehicle-treated guinea-pigs. No changes were observed in plasma progesterone concentrations. Our results provide no support for the hypothesis that an enzyme-induced decrease in progesterone concentrations is the main cause of the fetal death observed in PCB-treated guinea-pigs.

1. GENERAL VIEW OF MAIN (SOUTH) FACADE OF CHURCH, LOOKING ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. GENERAL VIEW OF MAIN (SOUTH) FACADE OF CHURCH, LOOKING NORTH (For a brief history of Boswell and a description of Sts. Peter and Paul Russian Orthodox Greek Catholic Church see TOWN OF BOSWELL, HAER PA-367) - Saints Peter & Paul Russian Orthodox Greek Catholic Church, Quemahoning Street, Boswell, Somerset County, PA

Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

PubMed

Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

2007-12-27

A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells.

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

Nikolau, Basil J; Wurtele, Eve S; Oliver, David J

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method ofmore » producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.« less

Preliminary study on health-related lipid components of bakery products.

PubMed

Cercaci, Luisito; Conchillo, Ana; Rodriguez-Estrada, Maria Teresa; Ansorena, Diana; Astiasarán, Iciar; Lercker, Giovanni

2006-06-01

The purpose of this study was to evaluate the presence of health-related lipid components, in particular trans fatty acids and sterol oxidation products, in four bakery products. Both types of components are known for their adverse biological effects, especially the increase of atherogenic risk, and therefore it is advisable to monitor their presence in food products. Trans fatty acids were determined by silver-ion thin-layer chromatography-gas chromatography, whereas sterol oxidation was assessed by gas chromatography and gas chromatography-mass spectrometry determination of 7-keto derivatives (tracers of sterol oxidation). The amount of trans fatty acids (0.02 to 3.13 g/100 g of product), sterols (34.9 to 128.3 mg/100 g of product), and 7-keto derivatives of sterols (1.88 to 3.14 mg/kg of product) varied considerably among samples. The supply of phytosterols (22.5 to 64.0 mg/100 g of product) was not significant, and the extent of oxidation of most phytosterols to its corresponding 7-keto derivative was low (0.29 to 0.84%), except for that of brassicasterol (2.01 to 3.11%). The quality of ingredients and raw materials seems to have greatly influenced the fatty acid profile, stability, safety, and quality of the final product; these ingredients should be chosen with extreme care to decrease their potential negative health effects and to increase safety of these products.

In Vitro Biosynthesis and Chemical Identification of UDP-N-acetyl-d-quinovosamine (UDP-d-QuiNAc)*

PubMed Central

Li, Tiezheng; Simonds, Laurie; Kovrigin, Evgenii L.; Noel, K. Dale

2014-01-01

N-acetyl-d-quinovosamine (2-acetamido-2,6-dideoxy-d-glucose, QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-QuiNAc is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) to UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose followed by reduction of this 4-keto intermediate to UDP-QuiNAc. Several specific dehydratases are known to catalyze the first proposed step. A specific reductase for the last step has not been demonstrated in vitro, but previous mutant analysis suggested that Rhizobium etli gene wreQ might encode this reductase. Therefore, this gene was cloned and expressed in Escherichia coli, and the resulting His6-tagged WreQ protein was purified. It was tested for 4-reductase activity by adding it and NAD(P)H to reaction mixtures in which 4,6-dehydratase WbpM had acted on the precursor substrate UDP-GlcNAc. Thin layer chromatography of the nucleotide sugars in the mixture at various stages of the reaction showed that WbpM converted UDP-GlcNAc completely to what was shown to be its 4-keto-6-deoxy derivative by NMR and that addition of WreQ and NADH led to formation of a third compound. Combined gas chromatography-mass spectrometry analysis of acid hydrolysates of the final reaction mixture showed that a quinovosamine moiety had been synthesized after WreQ addition. The two-step reaction progress also was monitored in real time by NMR. The final UDP-sugar product after WreQ addition was purified and determined to be UDP-d-QuiNAc by one-dimensional and two-dimensional NMR experiments. These results confirmed that WreQ has UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose 4-reductase activity, completing a pathway for UDP-d-QuiNAc synthesis in vitro. PMID:24817117

[Effects of low-protein diet plus alpha-keto acid on micro-inflammation and the relationship between micro-inflammation and nutritional status in patients performing continuous ambulatory peritoneal dialysis: a randomized controlled trial].

PubMed

Chen, Wei; Guo, Zhi-Yong; Wu, Hao; Sun, Li-Jing; Cai, Li-Li; Xu, Hai-Yan

2008-05-01

To investigate the effects of the combination of alpha-keto acid and low-protein diet on the levels of serum cytokines in patients performing continuous ambulatory peritoneal dialysis (CAPD) and to explore the relationship between inflammation and malnutrition in CAPD patients. Eighty-nine CAPD patients were randomized into three groups, and 78 cases completed a one-year follow-up and with complete data. There were 31 cases in low-protein diet plus alpha-keto acid group, 26 cases in low-protein diet group and 21 cases in routine-protein diet group. The levels of serum albumin (Alb), prealbumin (PA), retinol-binding protein (RBP), transferrin (TRF), cholesterol (TC), triglycerides (TG), leptin, and triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC), body mass index (BMI) were measured. The changes of serum interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were also detected. Compared with low-protein diet group, serum levels of PA, RBP and TRF were significantly increased both in low-protein diet plus alpha-keto acid and routine-protein diet groups ( P<0.01), however, there was no significant difference in the levels of PA, RBP and TRF between low-protein diet plus alpha-keto acid group and routine-protein diet group. There was an increased tendency in the content of Alb, TC, TG, BMI, TSF and MAMC, but there were no significant differences. The plasma levels of IL-1alpha, IL-6 and TNF-alpha in low-protein diet plus alpha-keto acid group were decreased as compared with the routine-protein diet group, but there were no significant differences. The plasma level of CRP in low-protein diet plus alpha-keto acid group was lower than that in the routine-protein diet group ( P<0.01). The combination of alpha-keto acid and low-protein diet can ameliorate malnutrition and micro-inflammation in CAPD patients.

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

PubMed

Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

2004-08-01

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Methyl Salicylate: A Reactive Chemical Warfare Agent Surrogate to Detect Reaction with Hypochlorite (POSTPRINT)

DTIC Science & Technology

2011-05-01

DMeS11,12 (tR = 14.83 min). The 3,5- DMeS crystallized spontaneously11 as colorless needles, melting point 143 146 C; 1HNMRdata: δ 7.785, 1Hd, J = 2.6 Hz, H...dichloro-phenol. Figure 2 shows two possible mecha- nisms, both promoted by the phenolic oxygen center— tautomerization to a β- keto acid and β

Identification of keto- and hydroxy-dicarboxylic acids in remote marine aerosols from the western North Pacific: GC and GC/TOF-MS measurements

NASA Astrophysics Data System (ADS)

Vani, D.; Kawamura, K.; Tachibana, E.; Boreddy, S. K. R.

2015-12-01

Dicarboxylic acids (diacids) are dominant components of organic aerosols in the atmosphere. They contribute significantly to the total aerosol mass and have a serious impacts on global climate changes. However, studies on keto- and hydroxy-diacids in marine aerosols are limited. Compare to diacids, keto- and hydroxy-diacids are more hygroscopic due to the additional polar groups (OH and CO) and, hence, acts as cloud condensation nuclei (CCN). Molecular characterization of these compounds provides insight into organic aerosols sources and transformation pathways. We collected marine aerosols from remote Chichijima Island in the western North Pacific from December 2010 to November 2011 and studied for water-soluble keto- and hydroxy-diacids. Carboxyl groups were derivatized to dibutyl esters with 14% boron trifluoride/n-butanol, whereas hydroxyl groups were derivatized to trimethylsilyl ethers using N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA). After two-step derivatization, samples were injected to GC, GC/MS and GC/TOF-MS. In the GC chromatogram, we detected several new peaks after BSTFA derivatization of dibutyl ester fraction. Based on mass spectral interpretation, we found these peaks as homologues series of hydroxy-diacids and keto-diacids. Some of these hydroxy-diacids have been individually reported in literature in the laboratory photo-oxidation experiments and forest environments samples. But, there are no evidences to prove their sources and formation mechanism in the atmosphere. Here, we report for the first time homologous series of hydroxy-diacids (hC3di-hC6di) and keto-diacid (oxaloacetic acid, enol and keto forms) in remote marine atmosphere. Molecular distributions of hydroxy-diacids generally showed the predominance of malic acid followed by tartronic acid. Both hydroxy- and keto-diacids show significant positive correlation with oxalic acid and SO42-, suggesting that they are generated in the atmosphere and play an important role in the formation of smaller diacids through aqueous phase photo-oxidation.

Three new fatty acids from the roots of Boehmeria nivea (L.) Gaudich and their antifungal activities.

PubMed

Xu, Qiong-Ming; Liu, Yan-Li; Li, Xiao-Ran; Li, Xia; Yang, Shi-Lin

2011-03-01

Three new unsaturated fatty acids, (Z)-9,10,11-trihydroxy-12-octadecenoic acid (1), (Z)-7,8,9-trihydroxy-10-hexadecenoic acid (2) and (Z)-12-keto-7,8,9-trihydroxy-10-hexadecenoic acid (3) were isolated from the roots of Boehmeria nivea (L.) Gaudich, along with a known unsaturated fatty acid, (E)-8,11,12-trihydroxy-9-octadecenoic acid (4). The structures of the new compounds were established by HR ESI-MS, (1)H, (13)C, 2D ((1)H-(1)H COSY, HSQC, HMBC) NMR experiments. The known compound was identified by a comparison of its spectral data with published references. The three new compounds showed some antifungal activities by agar assay.

Stereochemical analysis of the elimination reaction catalyzed by D-amino-acid oxidase.

PubMed

Cheung, Y F; Walsh, C

1976-06-01

The stereochemistry of the intramolecular proton transfer catalyzed by the flavoenzyme, D-amino-acid oxidase, during the elimination reaction of beta-chloro-alpha-amino acid substrates (Walsh et al. (1973), J. Biol. Chem. 248, 1964) has been established. Both D-erythro- and D-threo-2-amino-3-chloro(2-3H) butyrate have been shown to yield (3R)-2-keto (3-3H)-2- butyrate predominantly. Tritium kinetic isotope effects on the rate of the reaction (4.7 for the D-erythro, and 3.8 for the D-threo compound) and percentages of intramolecular triton transfer (7.2% for the D-erythro- and 2.6% for the D-threo compound) have been measured. Their implications on the mechanism of this unusual elimination reaction are discussed.

GC-MS analysis of the antioxidant active fractions of Micromeria juliana with anticholinesterase activity.

PubMed

Oztürk, Mehmet; Kolak, Ufuk; Duru, Mehmet Emin; Harmandar, Mansur

2009-09-01

The aerial parts of Micromeria juliana (L.) Bentham ex Reichb. were extracted with light petroleum, acetone and methanol, successively. The antioxidant activity of different concentrations of the extracts was evaluated using different antioxidant tests, namely total antioxidant (lipid peroxidation inhibition activity), DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging, ferric reducing power, and metal chelating. Total antioxidant activity was determined using the beta-carotene-linoleic acid assay. Unexpectedly, the light petroleum extract exhibited strong lipid peroxidation inhibition activity. The extract was fractionated on a silica gel column and the antioxidant activity of the fractions was determined by the beta-carotene-linoleic assay at 25 microg/mL concentration. The fractions that exhibited more than 50% inhibition activity were analysed by GC and GC/MS; thus, the structure of fourteen compounds were elucidated. In addition, acetyl- and butyryl-cholinesterase inhibitory activities of the extracts were also determined in vitro. The light petroleum and acetone extracts were found to have mild butyrylcholinesterase inhibitory activity.

Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

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

Svensson, Jan, E-mail: jan.svensson@ki.se; Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm; Bergman, Ann-Charlotte

Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirinmore » in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.« less

Crystal Structure of TDP-Fucosamine Acetyl Transferase (WECD) from Escherichia Coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

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

Hung,M.; Rangarajan, E.; Munger, C.

2006-01-01

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence {yields}(3)-{alpha}-D-Fuc4NAc-(1{yields}4)-{beta}-D-ManNAcA-(1{yields}4)-{alpha}-D-GlcNAc-(1{yields}). While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-D-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structuremore » of WecD in apo form at a 1.95-Angstroms resolution and bound to acetyl-CoA at a 1.66-Angstroms resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.« less

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle.

PubMed

Vavvas, D; Apazidis, A; Saha, A K; Gamble, J; Patel, A; Kemp, B E; Witters, L A; Ruderman, N B

1997-05-16

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

Forms of immunoreactive beta-endorphin in the intermediate pituitary of the holostean fish, Amia calva.

PubMed

Dores, R M; Sei, C A; Morrissey, M A; Crim, J W; Kawauchi, H

1988-01-01

Acid extracts of the intermediate pituitary of the holostean fish, Amia calva, were fractionated by gel filtration chromatography and analyzed with radioimmunoassays specific for N-acetylated beta-endorphin and C-terminally amidated alpha-MSH. In these extracts beta-endorphin-related immunoreactive material and alpha-MSH-related immunoreactive material were present in roughly equimolar amounts. The immunoreactive beta-endorphin-sized material was tested for opiate receptor binding activity using a beta-endorphin radioreceptor assay. The results of these studies were negative. The immunoreactive beta-endorphin-sized material was further analyzed by cation exchange chromatography at pH 2.5. Two major and three minor peaks of immunoreactive material were isolated. Peak 5 exhibited a net charge of +7 at pH 2.5 and represented 53% of the total immunoreactivity recovered. Peak 2 with a net charge of +3 at this pH represented 38% of the total immunoreactivity recovered. The minor forms, Peaks 1, 3 and 4, exhibited net charges of +2, +4 and +6, respectively. The apparent molecular weights of Peaks 2 and 5 were determined on a Sephadex G-50 column. Peak 2 had an apparent molecular weight of 2.7 Kd and Peak 5 had an apparent molecular weight of 3.5 Kd. Reverse phase HPLC analysis of Peak 5 indicates that this form of Amia beta-endorphin had chromatographic properties similar to salmon beta-endorphin II. These results would suggest that N-terminal acetylation and C-terminal proteolytic cleavage are important post-translational modifications of the forms of Amia beta-endorphin.

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

Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method ofmore » producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.« less

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

USDA-ARS?s Scientific Manuscript database

Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

PubMed

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Factors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration, organelle integrity and ATP.

PubMed Central

Thomas, J; Debeer, L J; De Schepper, P J; Mannaerts, G P

1980-01-01

1. The first dehydrogenation step of peroxisomal beta-oxidation involves the reduction of O2 to H2O2. Production rates of H2O2 and acetyl units by purified rat liver peroxisomes oxidizing palmitoyl-CoA were equal, indicating that H2O2 production is a reliable index for the release of acetyl units during peroxisomal fatty-acid oxidation. 2. Measurements of H2O2 and acid-soluble oxidation products during [1-14C]palmitoyl-CoA oxidation by purified peroxisomes revealed that the number of acetyl units released per molecule of palmitoyl-CoA oxidized rapidly decreased with increasing unbound palmitoyl-CoA concentrations. Structural damage to the peroxisomes caused by detergents or other treatments also decreased the number of acetyl units released. Under conditions where oxidation proceeded linearly with time the theoretical maximum of 5 acetyl units released per molecule of palmitoyl-CoA oxidized [Lazarow (1978) J. Biol. Chem. 253, 1522--1528] was never reached. 3. Expressed in terms of acetyl units produced and measured at low unbound-palmitoyl-CoA concentrations, mitochondrial oxidation was 10--20-fold higher than peroxisomal oxidation. 4. ATP stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. The ATP effect required the presence of Mg2+ and was lost when peroxisomal membranes were disrupted by Triton X-100 or high concentrations of unbound palmitoyl-CoA. 5. Disruption of peroxisomes by detergents, freeze--thawing, osmotic or mechanical treatment did not stimulate palmitoyl-CoA oxidation in the presence of ATP, indicating that peroxisomal fatty-acid-CoA oxidation was not latent. In the absence of ATP, Triton X-100 stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. PMID:7470063

[Studies on chemical constituents from rhizome of Anemone flaccida].

PubMed

Zhang, Lan-tian; Takaishi, Yoshihisa; Zhang, Yan-wen; Duan, Hong-quan

2008-07-01

To study the chemical constituents from Anemone flaccida. Chemical constituents were isolated by repeated column chromatography (silica gel, Toyopearl HW-40C and preparative HPLC). The structures were elucidated on the basis of spectral data analysis. Twelve triterpenes were isolated and their structures were identified as follow: oleanolic acid (1), oleanolic acid 3-O-beta-D-glccopyranosyl-(1-->2)-beta-D-xylopyranoside (2), eleutheroside K (3), oleanolic acid 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranoside (4), oleanolic acid 3-O-beta-D-glccopyranosyl-(1-->2)-alpha-L-arabinofurnoside (5), oleanolic acid 3-O-beta-D-glccuronopyranose (6), oleanolic acid 3-O-beta-D-glccuronopyranose methyl ester (7), oleanolic acid 28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glccopyranosyl (1-->6)-beta-D-glccopyranosyl (8), oleanolic acid 3-O-beta-D-glccuronopyranose 28-O-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glccopyranosyl (1-->6)-beta-D-glccopyranoside (9), oleanolic acid 3-O-beta-D-glccopyranosyl methyl ester 28-O-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glccopyranosyl (1-->6)-beta-D-glccopyranoside (10), oleanolic acid 3-O-beta-D-glccopyranosyl-(1-->2)-beta-D-xylopyranosyl-28-O-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glccopyranosyl (1-->6)-beta-D-glccopyranoside (11), oleanolic acid 3-O-alpha-L-rh-amnopyranosyl-(1-->2)-alpha-L-arabinopyrnosyl-28-O-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glccopyranosyl (1-->6)-beta-D-glccopyranoside (12). compounds 5-8, 10, 12 were isolated from this plant for the first time. Compounds 2, 5 and 11 showed positive anti-tumor activities.

Glycosidases in Brachionus plicatilis (Rotifera).

PubMed

Kühle, K; Kleinow, W

1990-01-01

1. Tests for glycosidases were performed in homogenates of Brachionus plicatilis. 2. Hydrolytic activity was detected with the following substrates: (a) with synthetic substrates (NP = 4-nitrophenyl): NP-alpha- and NP-beta-D-glucopyranoside, NP-alpha- and NP-beta-D-galactopyranoside, NP-N-acetyl-beta-D-glucosaminide, NP-N-acetyl-beta-D-galactosaminide, NP-alpha- and NP-beta-D-mannopyranoside and NP-alpha-L-fucopyranoside; (b) with disaccharides: sucrose, maltose, trehalose, isomaltose, cellobiose, gentiobiose and lactose; (c) with polysaccharides: laminarine, carboxymethyl-cellulose, avicel, Micrococcus luteus (for lysozyme) and 4-nitrophenyl-alpha-D-maltoheptaoside (for amylase). 3. The pH dependence of the glycosidase activities was determined. 4. The distribution of enzyme activities within fractions from the homogenate was studied in order to localize them within the cell. 5. Proteins from Brachionus homogenate were separated by SDS-gel electrophoresis and the positions of the following glycosidase activities were detected by assays performed on the gels (estimated molecular weights in parentheses): alpha-glucosidase (250,000); beta-glucosidase (200,000); beta-galactosidase (70,000); N-acetyl-beta-glucosaminidase (60,000).

Synthesis and biological activity of hydrazide hydrazones and their corresponding 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles

USDA-ARS?s Scientific Manuscript database

Various new 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles (11-20) were prepared by the reaction of aryl substituted hydrazones of 4-fluorobenzoic acid hydrazide (1-10) with acetic anhydride. The structures of the newly synthesized compounds 11-20, were confirmed by UV, IR and 1H NMR spec...

Boswellic Acid Improves Cognitive Function in a Rat Model Through Its Antioxidant Activity

PubMed Central

Ebrahimpour, Saeedeh; Fazeli, Mehdi; Mehri, Soghra; Taherianfard, Mahnaz; Hosseinzadeh, Hossein

2017-01-01

Objectives: Boswellic acid (BA), a compound isolated from the gum-resin of Boswellia carterii, is a pentacyclic terpenoid that is active against many inflammatory diseases, including cancer, arthritis, chronic colitis, ulcerative colitis, Crohn's disease, and memory impairment, but the mechanism is poorly understood. This study investigated the effects of boswellic acid on spatial learning and memory impairment induced by trimethyltin (TMT) in Wistar rats. Methods: Forty male Wistar rats were randomly divided into 5 groups: Normal group, TMT-administrated rats (8.0 mg/kg, Intraperitoneally, i.p.) and TMT + BA (40, 80 and 160 mg/kg, i.p.)-administrated rats. BA was used daily for 21 days. To evaluate the cognitive improving of BA, we performed the Morris water maze test. Moreover, to investigate the neuroprotective effect of BA, we determined the acetylcholinesterase (AchE) activity, the malondialdehyde (MDA) level as a marker of lipid peroxidation, and the glutathione (GSH) content in the cerebral cortex. Results: Treatment with TMT impaired learning and memory, and treatment with BA at a dose of 160 mg/kg produced a significant improvement in learning and memory abilities in the water maze tasks. Consistent with behavioral data, the activity of AChE was significantly increased in the TMT-injected rats compared to the control group (P < 0.01) whereas all groups treated with BA presented a more significant inhibitory effect against AChE than the TMT-injected animals. In addition, TMT reduced the GSH content and increased the MDA level in the cerebral cortex as compared to the control group) P < 0.01). On the other hand, treatment with BA at 160 mg/kg slightly increased the GSH content and reduced the MDA level in comparison to the TMT-administered group (P < 0.01). Conclusion: The above results suggest that the effect of BA in improving the cognitive function may be mediated through its antioxidant activity. PMID:28392957

Hydrolyses of alpha-naphthyl acetate, beta-naphthyl acetate, and acetyl-DL-phenylalanine beta-naphthyl ester.

PubMed

Kirkeby, S; Moe, D

1983-01-01

Using simultaneous coupling azo dye techniques kidney enzymes active against alpha-naphthyl acetate, beta-naphthyl acetate, and acetyl-DL-phenylalanine beta-naphthyl ester are characterized. The enzymes show identical distribution in the section. The banding patterns in zymograms are the same after incubation with the different substrates. The enzymes might, however, be separated by difference in pH optimum, initial velocity and sensitivity to inhibitors and activators.

Biologically active cannabinoids from high-potency Cannabis sativa.

PubMed

Radwan, Mohamed M; Elsohly, Mahmoud A; Slade, Desmond; Ahmed, Safwat A; Khan, Ikhlas A; Ross, Samir A

2009-05-22

Nine new cannabinoids (1-9) were isolated from a high-potency variety of Cannabis sativa. Their structures were identified as (+/-)-4-acetoxycannabichromene (1), (+/-)-3''-hydroxy-Delta((4'',5''))-cannabichromene (2), (-)-7-hydroxycannabichromane (3), (-)-7R-cannabicoumarononic acid A (4), 5-acetyl-4-hydroxycannabigerol (5), 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenol (6), 8-hydroxycannabinol (7), 8-hydroxycannabinolic acid A (8), and 2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone (9) through 1D and 2D NMR spectroscopy, GC-MS, and HRESIMS. The known sterol beta-sitosterol-3-O-beta-d-glucopyranosyl-6'-acetate was isolated for the first time from cannabis. Compounds 6 and 7 displayed significant antibacterial and antifungal activities, respectively, while 5 displayed strong antileishmanial activity.

Novel oligorhamnosides from the stem bark of Cleistopholis glauca.

PubMed

Seidel, V; Bailleul, F; Waterman, P G

2000-01-01

A reinvestigation of the stem bark of Cleistopholis glauca yielded 14 compounds, of which seven were either novel or had not been previously reported from this species. These were identified as the farnesane sesquiterpene methyl-(2E,6E)-10-oxo-3,7, 11-trimethyl-dodeca-2,6-dienoate (1); the azaanthracene alkaloid cleistopholine (4); two partially acetylated oligorhamnoside derivatives, 1-O-dodecanyl-2,3, 4-tri-O-acetyl-alpha-rhamnopyranosyl-(1-->3)-2, 4-di-O-acetyl-alpha-rhamnopyranosyl-(1-->3)-4-O-acetyl-alpha-rhamnopy ranosyl-(1-->4)-alpha-rhamnopyranoside (6) and 1-O-dodecanyl-2, 4-di-O-acetyl-alpha-rhamnopyranosyl-(1-->3)-2, 4-di-O-acetyl-alpha-rhamnopyranosyl-(1-->3)-4-O-acetyl-alpha-rhamnopy ranosyl-(1-->4)-alpha-rhamnopyranoside (8), for which the trivial names cleistetroside-7 and cleistetroside-6 were assigned, respectively; the dihydrobenzofuran neolignan rel-(2alpha, 3beta)-7-O-methylcedrusin (12); and the flavonoids dihydroquercetin (13) and quercetin (14). Structure assignments of all compounds were established by spectroscopic methods and comparison with published data. The chemosystematic significance of the occurrence of the isolated components is mentioned. Compounds 1, 6, and 8 are novel natural products.

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

PubMed

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

2008-06-01

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

The aldo-keto reductase superfamily homepage.

PubMed

Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

2003-02-01

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

2-keto-4-(methylthio)butyric acid (keto analog of methionine) is a safe and efficacious precursor of L-methionine in chicks.

PubMed

Dilger, Ryan N; Kobler, Christoph; Weckbecker, Christoph; Hoehler, Dirk; Baker, David H

2007-08-01

Relative bioefficacy and toxicity of Met precursor compounds were investigated in young chicks. The effectiveness of DL-Met and 2-keto-4-(methylthio)butyric acid (Keto-Met) to serve as L-Met precursors was quantified using Met-deficient diets of differing composition. Efficacy was based on slope-ratio and standard-curve methodology. Using L-Met as a standard Met source added to a purified diet, DL-Met and Keto-Met were assigned relative bioefficacy values of 98.5 and 92.5%, respectively, based on weight gain. Relative bioefficacy values of 98.5 and 89.3% were assigned to DL-Met and Keto-Met, respectively, when chicks were fed a Met-deficient, corn-soybean meal-peanut meal diet. Thus, both DL-Met and Keto-Met are effective Met precursor compounds in chicks. Additionally, growth-depressing effects of L-Met, DL-Met, and Keto-Met were compared using a nutritionally adequate corn-soybean meal diet supplemented with 15 or 30 g/kg of each compound. Similar reductions in weight gain, food intake, and gain:food ratio were observed for each compound. Subjective spleen color scores, indicative of splenic hemosiderosis, increased linearly (P < 0.01) with increasing intakes of each compound, suggesting a similarity in overall toxicity among these compounds. Because conversion of Keto-Met to L-Met in vivo merely requires transamination, Keto-Met may prove to be a useful supplement not only in food animal production, but also as a component of enteral and parenteral formulas for humans suffering from renal insufficiency.

Sesquiterpene lactones from Taraxacum obovatum.

PubMed

Michalska, Klaudia; Kisiel, Wanda

2003-02-01

Two new guaianolide glucosides, deacetylmatricarin 8-O-beta-glucopyranoside and 11beta-hydroxyleukodin 11-O-beta-glucopyranoside, were isolated from roots of Taraxacum obovatum, along with four known sesquiterpene lactones, deacetylmatricarin, sonchuside A, taraxinic acid beta-glucopyranosyl ester and its 11beta,13-dihydro derivative. Their structures were established by spectral methods.

Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

PubMed

Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

2000-10-27

The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p < 0.02), isoleucine (p < 0.03), and valine (p < 0.02) while their renal fractional excretion declined (p < 0.02, p < 0.01 resp.). Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p < 0.05) and a hyperbolic relationship between inulin clearance and fractional excretion of BCAA (p < 0.01) were seen. Moreover, a significant decrease in proteinuria (p < 0.02), plasma urea concentration and renal urea excretion and a rise in albumin level (p < 0.03) were noted. We conclude that in patients with CRI on a low protein diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

Urinary 11-dehydro-thromboxane B₂ and 2,3-dinor-6-keto-prostaglandin-F₁α in healthy post-menopausal and pre-menopausal women receiving aspirin 100 mg.

PubMed

Hartanto, Marcia Dewi; Arieselia, Zita; Setiabudy, Rianto; Setiawati, Arini; Baziad, Ali

2012-07-01

The prevalence of cardiovascular diseases in women increases sharply after menopause. In postmenopausal women, thromboxane production increases while prostacyclin decreases. Low dose aspirin reduces the production of both thromboxane and prostacyclin. The present study was an open-label clinical trial with two parallel groups of 15 premenopausal women and 15 postmenopausal women. Twenty-four hours urine was collected from each subject before and after aspirin 100 mg daily for 7 days. The concentration of thromboxane and prostacyclin was measured as their metabolites (11-dehydro-thromboxane B(2) and 2,3-dinor-6-keto-prostaglandin-F(1α)) in urine using enzyme immunoassay methods. This study showed that aspirin significantly reduced thromboxane in both groups with significantly larger percentage reduction in postmenopausal women compared to premenopausal women (73.32 vs. 61.13%, p = 0.021). This study also showed that aspirin reduced prostacyclin significantly in both groups, but the percentage reduction between the groups was not significantly different. The decrease in the ratio of 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) should be compared to assess aspirin efficacy as an antithrombotic. Calculation of the ratio of 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) before aspirin consumption was higher in postmenopausal women than in premenopausal women. The decrease in 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) ratio by aspirin was greater in postmenopausal women than in premenopausal women (1.91 vs. 0.17; p = 0.022). It was concluded that aspirin reduced thromboxane and prostacyclin significantly in each group with significant 11-dTXB(2) percentage reduction between groups and non-significant 2,3-dinor-6-keto-PGF(1α) percentage reduction between groups, but reduced the 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) ratio much larger in postmenopausal women compared to that in premenopausal women.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

PubMed Central

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-01-01

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

PubMed

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-12-25

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Transformation of Unsaturated Fatty Acids/Esters to Corresponding Keto Fatty Acids/Esters by Aerobic Oxidation with Pd(II)/Lewis Acid Catalyst.

PubMed

Senan, Ahmed M; Zhang, Sicheng; Zeng, Miao; Chen, Zhuqi; Yin, Guochuan

2017-08-16

Utilization of renewable biomass to partly replace the fossil resources in industrial applications has attracted attention due to the limited fossil feedstock with the increased environmental concerns. This work introduced a modified Wacker-type oxidation for transformation of unsaturated fatty acids/esters to the corresponding keto fatty acids/esters, in which Cu 2+ cation was replaced with common nonredox metal ions, that is, a novel Pd(II)/Lewis acid (LA) catalyst. It was found that adding nonredox metal ions can effectively promote Pd(II)-catalyzed oxidation of unsaturated fatty acids/esters to the corresponding keto fatty acids/esters, even much better than Cu 2+ , and the promotional effect is highly dependent on the Lewis acidity of added nonredox metal ions. The improved catalytic efficiency is attributed to the formation of heterobimetallic Pd(II)/LA species, and the oxidation mechanism of this Pd(II)/LA catalyst is also briefly discussed.

The origin of free brain malonate

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

Riley, K.M.; Dickson, A.C.; Koeppen, A.H.

Rat brain contains substantial concentrations of free malonate (192 nmol/g wet weight) but origin and biological importance of the dicarboxylic acid are poorly understood. A dietary source has been excluded. A recently described malonyl-CoA decarboxylase deficiency is associated with malonic aciduria and clinical manifestations, including mental retardation. In an effort to study the metabolic origin of free malonate, several labeled acetyl-CoA precursors were administered by intracerebral injection. (2-14C)pyruvate or (1,5-14C)citrate produced radioactive glutamate but failed to label malonate. In contrast, (1-14C)acetate, (2-14C)acetate, and (1-14C)butyrate were converted to labeled glutamate and malonate after the same route of administration. The intracerebral injectionmore » of (1-14C)-beta-alanine as a precursor of malonic semialdehyde and possibly free malonate did not give rise to radioactivity in the dicarboxylate. The labeling pattern of malonic acid is compatible with the reaction sequence: acetyl-CoA----malonyl-CoA----malonate. The final step is thought to occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate. Labeling of malonate from acetate is most effective at the age of 7 days when the net concentration of the dicarboxylic acid in rat brain is still very low. At this age, butyrate was a better precursor of malonate than acetate. It is proposed that fatty acid oxidation provides the acetyl-CoA which functions as the precursor of free brain malonate. Compartmentation of malonate biosynthesis is likely because the acetyl-CoA precursors citrate and pyruvate are ineffective.« less

Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action.

PubMed

Muñoz-Barroso, I; García-Sastre, A; Villar, E; Manuguerra, J C; Hannoun, C; Cabezas, J A

1992-09-01

Influenza virus type C (Johannesburg/1/66) was used as a source for the enzyme O-acetylesterase (EC 3.1.1.53) with several natural sialoglycoconjugates as substrates. The resulting products were immediately employed as substrates using influenza virus type A [(Singapore/6/86) (H1N1) or Shanghai/11/87 (H3N2)] as a source for sialidase (neuraminidase, EC 3.2.1.18). A significant increase in the percentage of sialic acid released was found when the O-acetyl group was cleaved by O-acetylesterase activity from certain substrates (bovine submandibular gland mucin, rat serum glycoproteins, human saliva glycoproteins, mouse erythrocyte stroma, chick embryonic brain gangliosides and bovine brain gangliosides). A common feature of all these substrates is that they contain N-acetyl-9-O-acetylneuraminic acid residues. By contrast, no significant increase in the release of sialic acid was detected when certain other substrates could not be de-O-acetylated by the action of influenza C esterase, either because they lacked O-acetylsialic acid (human glycophorin A, alpha 1-acid glycoprotein from human serum, fetuin and porcine submandibular gland mucin) or because the 4-O-acetyl group was scarcely cleaved by the viral O-acetylesterase (equine submandibular gland mucin). The biological significance of these facts is discussed, relative to the infective capacity of influenza C virus.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

PubMed

Cioci, Gianluca; Mitchell, Edward P; Chazalet, Valerie; Debray, Henri; Oscarson, Stefan; Lahmann, Martina; Gautier, Catherine; Breton, Christelle; Perez, Serge; Imberty, Anne

2006-04-14

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

Metabolism of indole-3-acetic acid by orange (Citrus sinensis) flavedo tissue during fruit development.

PubMed

Chamarro, J; Ostin, A; Sandberg, G

2001-05-01

[5-3H, 1'-14C, 13C6, 12C] Indole-3-acetic acid (IAA), was applied to the flavedo (epicarp) of intact orange fruits at different stages of development. After incubation in the dark, at 25 degrees C, the tissue was extracted with MeOH and the partially purified extracts were analyzed by reversed phase HPLC-RC. Six major metabolite peaks were detected and subsequently analyzed by combined HPLC-frit-FAB MS. The metabolite peak 6 contained oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp) and also indole-3-acetyl-N-glutamic acid (IAGlu). The nature of metabolite 5 remains unknown. Metabolites 3 and 4 were diastereomers of oxindole-3-acetyl-N-aspartic acid (OxIAAsp). Metabolite 2 was identified as dioxindole-3-acetic acid and metabolite 1 as a DiOx-IAA linked in position three to a hexose, which is suggested to be 3-(-O-beta-glucosyl) dioxindole-3-acetic acid (DiOxIAGlc). Identification work as well as feeding experiments with the [5-3H]IAA labeled metabolites suggest that IAA is metabolized in flavedo tissue mainly through two pathways, namely IAA-OxIAA-DiOxIAA-DiOxIAGlc and IAA-IAAsp-OxIAAsp. The flavedo of citrus fruit has a high capacity for IAA catabolism until the beginning of fruit senescence, with the major route having DiOxIAGlc as end product. This capacity is operative even at high IAA concentrations and is accelerated by pretreatment with the synthetic auxins 2,4-D, NAA and the gibberellin GA3.

Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. I. Partial purification and characterization of the enzyme from Zea mays

NASA Technical Reports Server (NTRS)

Leznicki, A. J.; Bandurski, R. S.

1988-01-01

The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-beta-D-glucose from uridine-5'-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss.

Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

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

Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.

{beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rankmore » order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.« less

Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose.

PubMed

Ozaki, Aiko; Konishi, Rie; Otomo, Chisako; Kishida, Mayumi; Takayama, Seiya; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

2017-12-01

Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

Two rhamnogalacturonide tetrasaccharides isolated from semi-retted flax fibers are signaling molecules in Rubus fruticosus L. cells.

PubMed Central

Dinand, E; Excoffier, G; Liénart, Y; Vignon, M R

1997-01-01

Water extraction of semi-retted flax (Linum usitatissimum L.) fiber bundles yielded a mixture of pectic oligosaccharides and two acidic rhamnogalacturonide tetrasaccharides that were separated by size-exclusion chromatography. One- and two-dimensional nuclear magnetic resonance studies and fast atom bombardment-mass spectrometry experiments indicated that the two tetrasaccharides have a common primary structure, i.e. alpha-D-delta GalpA(1-->2)-alpha-L- Rhap(1-->4)-alpha-D-GalpA-(1-->2)-L-alpha,beta-Rhap, with a rhamnopyranose as terminal reducing end, and a 4-deoxy-beta-L-threo-hex-4-enopyranosiduronic acid at the nonreducing end. However, the two tetrasaccharides differ by an acetyl group located at the O-3 position of the internal galacturonic acid residue. These two tetrasaccharides induce the activation of D-glycohydrolases of Rubus fructicosus L. cells or protoplasts within minutes. PMID:9342877

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

PubMed Central

Kunz, Daniel A.; Chen, Jui-Lin; Pan, Guangliang

1998-01-01

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min−1 ml of culture fluid−1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role. PMID:9797306

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

PubMed

Puchner, Claudia; Eixelsberger, Thomas; Nidetzky, Bernd; Brecker, Lothar

2017-01-02

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1 H and 13 C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala 79 . In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site. Copyright © 2016. Published by Elsevier Ltd.

Free energy landscapes of peptides by enhanced conformational sampling.

PubMed

Nakajima, N; Higo, J; Kidera, A; Nakamura, H

2000-02-11

The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors. Copyright 2000 Academic Press.

pH regulation of mitochondrial branch chain alpha-keto acid transport and oxidation in rat heart mitochondria.

PubMed

Hutson, S M

1987-07-15

The kinetics of branched chain alpha-keto acid uptake and efflux were studied as a function of varied external and matrix pH. Matrix pH was determined by the distribution of 5,5'-dimethyloxazolidine-2,4-dione. When rat heart mitochondria were incubated under transport conditions at pH 7.0 with succinate as respiratory substrate, the matrix pH was significantly greater than 8.0. Matrix pH remained greater than or equal to 8.0 when the medium pH was varied from 6.3 to 8.3, and it was lowered below 8.0 by addition of 5 mM phosphate or uncoupler. No pH gradient was detectable when mitochondria were incubated in the presence of valinomycin and uncoupler. Efflux of alpha-ketoisocaproate or alpha-ketoisovalerate from rat heart mitochondria obeyed first order kinetics. Varying the external pH from 6.6 to 8.3 had no significant effect on efflux, and at an external pH of 7.0, the first order rate constant for efflux was not affected by decreasing the matrix pH. On the other hand, exchange was sensitive to changes in medium but not matrix pH. The K0.5 for external branched chain alpha-keto acid was lowered by changing the medium pH from 7.6 to 6.3. At medium pH values greater than or equal to 8.0 both K0.5 and Vmax were affected. Uptake was determined either by measuring initial rates or was calculated after measuring the first order approach to a final equilibrium value. Unlike efflux, uptake was sensitive to changes in both external and matrix pH. The rate of branched chain alpha-keto acid uptake was stimulated by decreasing the medium pH from 8.3 to 6.3 and by alkalinization of the mitochondrial matrix. The estimated external pK for proton binding was 6.9. The data indicate that the branched chain alpha-keto acid transporter is asymmetric, that is, binding sites for substrate on the inside and outside of the mitochondrial membrane are not identical. alpha-Ketoisocaproate oxidation was measured at 37 degrees C in isolated mitochondria over the pH range of 6.6 to 8.1. Changes in the rate of branched chain alpha-keto acid oxidation, particularly when ATP was added (increase delta pH), were found to parallel the pH effects observed on branched chain alpha-keto acid uptake. Therefore, transport, and by implication oxidation, can be regulated by pH changes within the physiological range. Furthermore, intracellular pH may affect the degree of compartmentation between the cytosolic and mitochondrial branched chain alpha-keto acid pools.

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

PubMed

Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

2005-02-01

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

Prostacyclin production in rabbit arteries in situ: inhibition by arachidonic acid-induced endothelial cell damage or by low-dose aspirin.

PubMed

Ingerman-Wojenski, C; Silver, M J; Smith, J B; Nissenbaum, M; Sedar, A W

1981-04-01

The central artery of the rabbit ear was perfused in situ and effluent fractions from the artery were assayed for 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha) and thromboxane B2 (TxB2), the stable metabolites of prostacyclin (PGI2) and TxA2, using specific radioimmunoassays. These metabolites of arachidonic acid (AA) were not detected in the effluent during infusion of Tyrode's solution but both metabolites were detected when small amounts of AA were infused into the artery. Examination of the arteries by scanning electron microscopy revealed that high concentrations of AA which caused a short burst of 6-K-PGF1 alpha and TxB2 production damaged the endothelial cells while lower concentrations which stimulated continuous production did not cause damage. When a non-damaging concentration of AA was infused into an artery that had previously received a damaging concentration, PG production was greatly reduced. Pretreatment of the rabbits with 4 mg/kg acetyl-salicylic acid (ASA) inhibited 6-K-PGF1 alpha production by the rabbit ear artery in response to AA and 70% inhibition was still evident 18 hours after ASA.

Therapeutic efficacy of DL-alpha-lipoic acid on cyclosporine A induced renal alterations.

PubMed

Amudha, Ganapathy; Josephine, Anthony; Mythili, Yenjerla; Sundarapandiyan, Rajaguru; Varalakshmi, Palaninathan

2007-10-01

The present study was designed to evaluate the possible beneficial effect of lipoic acid in preventing the renal damage induced by cyclosporine A in rats. Male albino rats of Wistar strain were divided into four groups and treated as follows. Two groups received cyclosporine A by oral gavage (25 mg/kg/body weight) for 21 days to induce nephrotoxicity, one of which simultaneously received lipoic acid treatment (20 mg/kg body weight) for 21 days. A vehicle (olive oil) and a lipoic acid drug control were also included. Cyclosporine A induced renal damage was evident from the decreased activities of tissue marker enzymes (alkaline phosphatase, acid phosphatase, lactate dehydrogenase, aspartate transaminase and alanine transaminase) and decreased activities of ATPases (Na+, K+-ATPase, Ca2+-ATPase and Mg2+ ATPase). An apparent increase in the levels of serum constituents (urea, uric acid and creatinine) and urinary marker enzymes (N-acetyl-beta-D-glucosaminidase, beta-glucosidase, beta-galactosidase, cathepsin-D and gamma-glutamyl transpeptidase) along with significant decline in creatinine clearance were seen in the cyclosporine treated rats, which was reversed upon treatment with lipoic acid. Ultrastructural observations were also in agreement with the above abnormal changes. Lipoic acid effectively reverted these abnormal biochemical changes and minimized the morphological lesions in renal tissue. Hence, this study clearly exemplifies that lipoic acid might be an ideal choice against cyclosporine A induced cellular abnormalities.

Effect of low-protein diet supplemented with keto acids on progression of chronic kidney disease.

PubMed

Garneata, Liliana; Mircescu, Gabriel

2013-05-01

Hypoproteic diets are most often discussed for patients with chronic kidney disease (CKD) who do not receive dialysis. A very low-protein diet supplemented with ketoanalogues of essential amino acids (keto-diet) proved effective in ameliorating metabolic disturbances of advanced CKD and delaying the initiation of dialysis without deleterious effects on nutritional status. Several recent studies report that the keto-diet could also slow down the rate of decline in renal function, with better outcomes after the initiation of dialysis. Results of a single-center randomized controlled trial addressing the rate of CKD progression revealed a 57% slower decline in renal function with the keto-diet compared with a conventional low-protein diet (LPD). The keto-diet allowed the safe management of selected patients with stage 4-5 CKD, delaying dialysis for almost 1 year, with a major impact on patient quality of life and health expenditures. Therefore, the keto-diet could be a link in the integrated care model. Careful selection of patients, nutritional monitoring, and dietary counseling are required. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Combined untargeted and targeted fingerprinting by comprehensive two-dimensional gas chromatography: revealing fructose-induced changes in mice urinary metabolic signatures.

PubMed

Bressanello, Davide; Liberto, Erica; Collino, Massimo; Chiazza, Fausto; Mastrocola, Raffaella; Reichenbach, Stephen E; Bicchi, Carlo; Cordero, Chiara

2018-04-01

This study exploits the information potential of comprehensive two-dimensional gas chromatography configured with a parallel dual secondary column-dual detection by mass spectrometry and flame ionization (GC×2GC-MS/FID) to study changes in urinary metabolic signatures of mice subjected to high-fructose diets. Samples are taken from mice fed with normal or fructose-enriched diets provided either in aqueous solution or in solid form and analyzed at three stages of the dietary intervention (1, 6, and 12 weeks). Automated Untargeted and Targeted fingerprinting for 2D data elaboration is adopted for the most inclusive data mining of GC×GC patterns. The UT fingerprinting strategy performs a fully automated peak-region features fingerprinting and combines results from pre-targeted compounds and unknowns across the sample-set. The most informative metabolites, with statistically relevant differences between sample groups, are obtained by unsupervised multivariate analysis (MVA) and cross-validated by multi-factor analysis (MFA) with external standard quantitation by GC-MS. Results indicate coherent clustering of mice urine signatures according to dietary manipulation. Notably, the metabolite fingerprints of mice fed with liquid fructose exhibited greater derangement in fructose, glucose, citric, pyruvic, malic, malonic, gluconic, cis-aconitic, succinic and 2-keto glutaric acids, glycine acyl derivatives (N-carboxy glycine, N-butyrylglycine, N-isovaleroylglycine, N-phenylacetylglycine), and hippuric acid. Untargeted fingerprinting indicates some analytes which were not a priori pre-targeted which provide additional insights: N-acetyl glucosamine, N-acetyl glutamine, malonyl glycine, methyl malonyl glycine, and glutaric acid. Visual features fingerprinting is used to track individual variations during experiments, thereby extending the panorama of possible data elaboration tools. Graphical abstract ᅟ.

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

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

Thoden, James B.; Holden, Hazel M.

2010-09-08

2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to usemore » {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.« less

Two new triterpenoid saponins from Dianthus superbus L.

PubMed

Chen, Xia; Luo, Jian-Guang; Kong, Ling-Yi

2010-06-01

Two new triterpenoid saponins (1 and 2) were isolated from the dried aerial parts of Dianthus superbus L. (Caryophyllaceae). Their structures were elucidated on the basis of spectral data to be 3-O-beta-D-glucopyranosyl olean-9(11),12-diene-23,28-dioic acid 28-O-beta-D-glucopyranoside (1) and 3-O-beta-D-glucopyranosyl olean-11,13(18)-diene-23,28-dioic acid 28-O-beta-D-glucopyranoside (2).

The formation of 3 alpha- and 3 beta-acetoxytropanes by Datura stramonium transformed root cultures involves two acetyl-CoA-dependent acyltransferases.

PubMed

Robins, R J; Bachmann, P; Robinson, T; Rhodes, M J; Yamada, Y

1991-11-04

Tropine (tropan-3 alpha-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in transformed root cultures of Datura stramonium. A further acyltransferase activity that acetylates pseudotropine (tropan-3 beta-ol) with acetyl-coenzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in forming the observed alkaloid spectrum of D. stramonium roots discussed.

Beta-ketothiolase deficiency: An unusual cause of recurrent ketoacidosis.

PubMed

Kılıç-Yıldırım, Gonca; Durmuş-Aydoğdu, Sultan; Ceylaner, Serdar; Sass, Jörn Oliver

2017-01-01

Kılıç-Yıldırım G, Durmuş-Aydoğdu S, Ceylaner S, Sass JO. Beta-ketothiolase deficiency: An unusual cause of recurrent ketoacidosis. Turk J Pediatr 2017; 59: 471-474. Beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase, MAT or T2 deficiency) is a rare autosomal recessive disorder of isoleucine and ketone body metabolism due to acetyl-CoA acetyltransferase-1 (ACAT1) gene mutations. The disease is characterized by recurrent episodes of ketoasidosis which starts with vomiting and followed by dehydration and tachypnea. Here, we present a patient who was admitted to the hospital with severe acidosis and dehydration because of vomiting induced by protein rich nutrient and was diagnosed with MAT deficiency. 3-hydroxy-butyric acid, acetoacetic acid and 3-hydroxy-iso-valeric acid levels were significantly increased and tiglyglycine as trace amount in the urine organic acid analysis of the patient. Genetic analysis for ACAT-1 showed compound heterozygosity for the mutations c.949G > A (p.D317N) and c.951C > T (p.D317D), which both are known to cause exon 10 skipping and to be pathogenic missense mutations.

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

PubMed

Siddiqui, A U; Wilson, W K; Ruecker, K E; Pinkerton, F D; Schroepfer, G J

1992-11-01

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.

Histochemistry of glycoconjugates in the skin of the bovine muzzle, with special reference to glandular structures.

PubMed

Meyer, W; Tsukise, A

1989-01-01

The distribution of glycoconjugates in the muzzle of young adult Holstein cows has been studied by means of selected light-microscopic histochemical methods, including lectin histochemistry. In the skin layers, strong reactions were confined to intercellular substances in between the cells of the vital epidermis, exhibiting neutral glycoconjugates mainly with alpha-D-galactosyl and N-acetyl-D-galactosaminyl residues. In the nasolabial glands, distinctly positive staining for neutral glycoproteins with various saccharide residues (alpha-D-galactose, alpha-N-acetylgalactosamine, D-galactose-beta(1----3)D-N-acetylgalactosamine, beta-D-galactose), and for smaller amounts of acidic glycoconjugates, was found in the secretory cells and the luminal secretion. The cells of the excretory duct system showed weak to moderate reactions (alpha-D-galactose, beta-D-galactose), only the collecting ducts reacted positively for acidic glycoproteins with sialyl residues. The results obtained are discussed in view of muzzle function, with special reference to the salivary nature of the secretion of bovine nasolabial glands.

Effect of short-term low-protein diet supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis patients.

PubMed

Li, Haiming; Long, Quan; Shao, Chunhai; Fan, Hong; Yuan, Li; Huang, Bihong; Gu, Yong; Lin, Shanyan; Hao, Chuanming; Chen, Jing

2011-01-01

To evaluate the effects of short-term restriction of dietary protein intake (DPI) supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis (MHD) patients. Forty MHD patients with uncontrolled hyperphosphatemia were randomized to either low DPI with keto acid-supplemented (sLP) or normal DPI (NP) group for 8 weeks. After 8 weeks, the sLP group was shifted to NP for another 8 weeks. Low-protein diet (LPD) was individualized with total caloric intake 30-35 kcal/kg/day, protein intake of 0.8 g/kg/day and phosphate intake of 500 mg/day. Keto acids were supplied in a dosage of 12 pills per day. Calcium phosphorous metabolism index and nutritional index (serum albumin, total protein, somatometric measurements, 3-day diaries and Mini-Nutritional Assessment score) were recorded. C-reactive protein, CO(2) combining power and Kt/V were measured to evaluate the inflammation, metabolic acidosis and dialysis adequacy, respectively. Serum phosphorus level and calcium-phosphate product were significantly decreased at the end of the first 8 weeks in the sLP group compared to the basal value and the NP group (p < 0.001). No difference was observed in C-reactive protein, Kt/V and nutritional index, while CO(2) combining power was significantly higher at week 8 in the sLP group (p < 0.001). Short-term restriction of DPI supplemented with keto acids could decrease hyperphosphatemia and calcium-phosphate product, while keeping stable nutritional status among MHD patients. Copyright © 2010 S. Karger AG, Basel.

Investigating Metabolic Control of Persister Formation in Biofilms

DTIC Science & Technology

2013-10-01

References Albe, K.R., Butler, M.H., and Wright, B.E. (1990). Cellular concentrations of enzymes and their substrates. Journal of theoretical biology 143 ...0.00 1,2- Propanediol 0.00±0.00 Tween 40 0.25±0.04 α- Keto - Glutaric Acid 0.26±0.07 α- Keto -Butyric Acid 0.18±0.03 α-Methyl-D

Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

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

Thoden, James B.; Holden, Hazel M.

2010-09-08

The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimericmore » quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.« less

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

PubMed Central

Rosa, J. C.; De Oliveira, P. S.; Garratt, R.; Beltramini, L.; Resing, K.; Roque-Barreira, M. C.; Greene, L. J.

1999-01-01

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature. PMID:10210179

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

PubMed Central

Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

2015-01-01

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

PubMed

Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

2015-10-27

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

[Herbalism, botany and components analysis study on original plants of frankincense].

PubMed

Sun, Lei; Xu, Jimin; Jin, Hongyu; Tian, Jingai; Lin, Ruichao

2011-01-01

In order to clarify original plants of traditional Chinese medicine (TCM) frankincense, a GC method for determination essential oils and a HPLC method for determination boswellic acids were carried out together with analysis of herbalism, botany, components and pharmacology papers of frankincense. It was concluded that original plants of TCM frankincense include at least Boswellia sacra, B. papyrifera and B. serrata.

Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis.

PubMed

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

2004-04-01

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

Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

PubMed Central

Sanchez, Isabelle; Dequin, Sylvie; Camarasa, Carole

2015-01-01

Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD+/NADH or, to a lesser extent, NADP+/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation. PMID:26475113

Silver-catalyzed double-decarboxylative cross-coupling of α-keto acids with cinnamic acids in water: a strategy for the preparation of chalcones.

PubMed

Zhang, Ning; Yang, Daoshan; Wei, Wei; Yuan, Li; Nie, Fafa; Tian, Laijin; Wang, Hua

2015-03-20

A silver-catalyzed double-decarboxylative protocol has been proposed for the construction of chalcone derivatives via cascade coupling of substituted α-keto acids with cinnamic acids under the mild aqueous conditions. The developed method for constructing C-C bonds via double-decarboxylative reactions is efficient, practical, and environmentally benign by using the readily available starting materials. It should provide a promising synthesis candidate for the formation of diverse and useful chalcone derivatives in the fields of synthetic and pharmaceutical chemistry.

Fimasartan Ameliorates Nonalcoholic Fatty Liver Disease through PPARδ Regulation in Hyperlipidemic and Hypertensive Conditions

PubMed Central

Jang, Yoo-Na; Han, Yoon-Mi; Kim, Hyun-Min; Jeong, Jong-Min

2017-01-01

To investigate the effects of fimasartan on nonalcoholic fatty liver disease in hyperlipidemic and hypertensive conditions, the levels of biomarkers related to fatty acid metabolism were determined in HepG2 and differentiated 3T3-L1 cells treated by high fatty acid and liver and visceral fat tissue samples of spontaneously hypertensive rats (SHRs) given high-fat diet. In HepG2 cells and liver tissues, fimasartan was shown to increase the protein levels of peroxisome proliferator-activated receptor delta (PPARδ), phosphorylated 5′ adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylated acetyl-CoA carboxylase (p-ACC), malonyl-CoA decarboxylase (MCD), medium chain acyl-CoA dehydrogenase (MCAD), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and it led to a decrease in the protein levels of 11 beta-hydroxysteroid dehydrogenase 1 (11β-HSDH1), fatty acid synthase (FAS), and tumor necrosis factor-alpha (TNF-α). Fimasartan decreased lipid contents in HepG2 and differentiated 3T3-L1 cells and liver tissues. In addition, fimasartan increased the adiponectin level in visceral fat tissues. The antiadipogenic effects of fimasartan were offset by PPARδ antagonist (GSK0660). Consequently, fimasartan ameliorates nonalcoholic fatty liver disease mainly through the activation of oxidative metabolism represented by PPARδ-AMPK-PGC-1α pathway. PMID:28386270

Mycobacterium intermedium sp. nov.

PubMed

Meier, A; Kirschner, P; Schröder, K H; Wolters, J; Kroppenstedt, R M; Böttger, E C

1993-04-01

Strains of a new type of slowly growing mycobacterium were repeatedly isolated from sputum from a patient with pulmonary disease. This photochromogenic organism grew at 22, 31, 37, and 41 degrees C, possessed catalase, acid phosphatase, esterase, beta-galactosidase, and arylsulfatase activities, and hydrolyzed Tween. It did not produce nicotinic acid or have nitrate reductase, acetamidase, benzamidase, isonicotinamidase, nicotinamidase, pyrazinamidase, succinidamidase, and acid phosphatase activities. Urease activity was variable. The organism is susceptible to ethambutol and resistant to isoniazid and streptomycin. A mycolic acid analysis revealed the presence of alpha-mycolates, alpha'-mycolates, and keto-mycolates. The results of comparative 16S rRNA sequencing placed this organism at an intermediate position between the rapidly and slowly growing mycobacteria. On the basis of the pattern of enzymatic activities and metabolic properties, the results of fatty acid analyses, and the unique 16S rRNA sequence, we propose that this organism represents a new species, for which we propose the name Mycobacterium intermedium. The type strain is strain 1669/91; a culture of this strain has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as strain DSM 44049.

Pretreated Starch Solutions for Low Environmental Impact Aircraft Deicing

DTIC Science & Technology

2010-09-01

modified monomeric organic molecules, have been the focus of recent study. Several organic acids and keto acids, such as succinate and acetate, have...1992), but the authors noted low economic feasibility without additional recovered products. Ganjyal et al. (2007) produced levulinate, a keto acid...534 -23.4 25 -22.7 52 569 -25.3 5 -25.8 104 585 -28.0 Sigma Aldrich DARCO 12-20 Mesh 0 -28.0 143 634 -28.0 50 -26.2 100 626 -27.4 Calgon F-300 9

Heterogeneous enantioselective hydrogenation of beta-keto esters using chirally modified supported Ni nanoparticles

NASA Astrophysics Data System (ADS)

Acharya, Sushma

Enantioselective heterogeneous catalysis is an important and rapidly expanding research area. The two most heavily researched examples of this type of catalysis are the enantioselective hydrogenation of α-keto-esters over Pt-based catalysts and the enantioselective hydrogenation of β-keto-esters over Ni-based catalysts. These enantioselective surface reactions are controlled by the presence of adsorbed chiral molecules i.e. tartaric acid on the surface of the metal component of the catalyst. The work presented in this thesis focuses on two parts, the synthesis of pure nickel nanoparticles and enantioselective behavior of the modified nickel nanoparticles. The works on the synthesis of pure nickel nanoparticles were carried out using two methods, the reverse microemulsion and the reduction method. It was discovered that the reverse microemulsion method produced nickel oxide nanoparticles, whereas the reduction method produced pure nickel nanoparticles. Chiral modifications of Raney nickel (RNi) and C-supported catalysts were studied. The catalysts were employed in enantioselective hydrogenation of methyl acetoacetate (MAA) to (R) - and (S)-enantiomers of methyl 3-hydroxybutyrate (MHB). The effects of modification and hydrogenation parameters such as concentration of modifier temperature, pressure and solvent on the enantioselectivity of MAA hydrogenation were discussed. For RNi methanol was found to be the best solvent, with tartaric acid concentration 0.2 mol/L for achieving the highest enantiomeric excess under 8 bar at 70 oC. Characteristic features of the in-situ modification of Raney nickel and C-supported Ni were also evaluated and the results obtained were compared with the conventional (pre-modification) approach. Parameters for the conventional and in-situ methods were optimised in a series of experiments for both types of catalysts. The in-situ modified catalyst was found more active for both RNi and C-supported catalysts with 98 % and 42% enantiomeric excess, respectively.

Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

NASA Technical Reports Server (NTRS)

Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

2004-01-01

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

PubMed

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase.

PubMed

Brodelius, P; Nilsson, K; Mosbach, K

1981-12-01

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca(2+)-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a " trickle-bed " reactor.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

HDAC and Ku70 axis- an effective target for apoptosis induction by a new 2-cyano-3-oxo-1,9-dien glycyrrhetinic acid analogue.

PubMed

Gong, Ping; Li, Kun; Li, Ying; Liu, Dan; Zhao, Linxiang; Jing, Yongkui

2018-05-24

Methyl 2-cyano-3,12-dioxo-18β-olean-1,9(11)-dien-30-oate (CDODO-Me, 10d) derived from glycyrrhetinic acid and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO-Me) derived from oleanoic acid are potent apoptosis inducers developed to clinical trials. Both compounds have high affinity for reduced  glutathione (GSH), which needs to be overcome to improve their target selectivity. We generated a new 10d analogue methyl 2-cyano-3-oxo-18β-olean-1,9(11), 12-trien-30-oate (COOTO, 10e), which retains high apoptosis inducing ability, while displaying decreased affinity for GSH, and explored the acting targets. We found that it induces Noxa level, reduces c-Flip level and causes Bax/Bak activation. Silencing of either Noxa or Bak significantly attenuated apoptosis induction of 10e. We linked these events due to targeting HDAC3/HDAC6 and Ku70 axis. 10e treatment reduced the levels of HDAC3 and HDAC6 with increased DNA damage/repair marker gamma-H2AX (γ-H2AX) and acetylated Ku70. c-Flip dissociates from acetylated Ku70 undergoing degradation, while Bax dissociates from acetylated Ku70 undergoing activation. Silencing of either HDAC3 or HDAC6 enhanced 10e-induced apoptosis. We reveal a new action cascade of this category of compounds that involves targeting of HADC3/6 proteins and Ku70 acetylation.

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria.

PubMed

Cescutti, Paola; Scussolin, Silvia; Herasimenka, Yury; Impallomeni, Giuseppe; Bicego, Massimiliano; Rizzo, Roberto

2006-01-20

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.

Branched-Chain Amino and Keto Acid Biochemistry and Cellular Biology in Central Nervous System Diseases

DTIC Science & Technology

2009-05-21

pyruvate dehydrogenase complex (PDC) and 2-oxo- glutarate dehydrogenase complex. These dehydrogenase complexes share the same basic structure, perform the...Science 312 (2006) 927-930. [20] J. Dancis, M. Levitz, R.G. Westall, Maple syrup urine disease: branched- chain keto- aciduria , Pediatrics 25 (1960...2127 2128 Dancis J, Levitz M, Westall RG. 1960. Maple syrup urine disease: branched-chain keto- aciduria . Pediatrics 25:72-9. Danner DJ, Lemmon

FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world.

PubMed

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of α-hydroxy acids and α-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized α-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476.

The role of keto acids in the supportive treatment of children with chronic renal failure.

PubMed

Mir, Sevgi; Ozkayin, Nese; Akgun, Aysegul

2005-07-01

According to the hyperfiltration theory of renal diseases characterized by a decrease in the number of functional nephrons, increased arterial blood pressure, excessive protein intake in the diet, high levels of calcium (Ca) and phosphorus (P), secondary hyperparathyroidism, hypertriglyceridemia and/or hypercholesterolemia, proteinuria and metabolic acidosis are some factors that impair the prognosis of the disease. The amount of protein in the diet is the most important of these factors. A protein-restricted diet administered to patients with chronic renal failure results in the risk of inadequate amino acid intake. To overcome this problem, the use of dysaminated alpha-keto analogues has been considered to reduce the risk of nitrogenemia resulting from the continuous intake of essential amino acids. Currently, the necessity of essential amino acids even in adult patients with chronic renal failure is controversial; besides, trials on the use of these amino acids in pediatric patients are scarce. The aim of this study is to investigate the efficacy and applicability of conservative therapy with a protein-restricted diet supplemented with keto acids in the management of chronic renal insufficiency or failure.

Synthesis of 3 alpha, 5 alpha-tetrahydroaldosterone.

PubMed

Harnik, M; Kashman, Y; Morris, D J

1984-06-01

3 alpha, 5 alpha-Tetrahydroaldosterone (12a), a metabolite of aldosterone, has been synthesized from the lactone 2a or, preferably 11 beta, 21-dihydroxy-5-pregnene-3,20-dione-18-oic acid 3,20-di-(ethylene glycol)-ketal (18----11) lactone 21-acetate (6b), via 11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione-18-oic acid 3,20-di-(ethylene glycol) ketal (18----11) lactone 21-acetate (4b). Selective hydrolysis of the latter at C-3 furnished the monoketal 5 which, on reduction with potassium tri-sec-butylborohydride, yielded predominantly 3 alpha, 11 beta, 21-trihydroxy-5 alpha-pregnan-20-one-18-oic acid 20-(ethylene glycol)-ketal (18----11) lactone (8a) and its acetate 8b. Further reduction with diisobutylaluminum hydride afforded 3 alpha, 5 alpha-tetrahydroaldosterone-20-ketal (10a), which was directly hydrolyzed to 12a with dilute acid in tetrahydrofuran-dioxan. Periodate oxidation led to the gamma-etiolactone 14a, which was then further converted into 5 alpha-dihydroaldosterone-gamma-etiolactone (14c).

Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

PubMed

Gupta, S C; Dekker, E E

1980-02-10

Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

Keto acid-supplemented low-protein diet for treatment of adult patients with hepatitis B virus infection and chronic glomerulonephritis.

PubMed

Mou, Shan; Li, Jialin; Yu, Zanzhe; Wang, Qin; Ni, Zhaohui

2013-02-01

An open-label, randomized, controlled, single-centre clinical trial to evaluate the effects of low-protein intake, with or without keto acid supplementation, on nutritional status and proteinuria, in patients with hepatitis B virus (HBV) and early stage chronic glomerulonephritis. Patients with chronic glomerulonephritis and HBV infection were randomized to receive a low-protein diet (0.6-0.8 g/kg ideal body weight [IBW] per day) either without (LP group) or with (sLP group) keto acid supplementation (0.1 g/kg IBW per day), for 12 months. Nutritional, clinical and safety parameters were recorded. The study included 17 patients (LP group n = 9; sLP group n = 8). Proteinuria and microalbuminuria were significantly lower in the sLP group at 6 and 12 months compared with baseline, and at 12 months compared with the LP group. There were no significant differences in serum creatinine level or estimated glomerular filtration rate. Nutritional parameters (serum albumin and prealbumin) were significantly improved at 12 months, compared with baseline, in the sLP group. Restriction of dietary protein intake to 0.6-0.8 g/kg IBW per day appears to have an acceptable safety profile. Supplementation with keto acids is associated with decreased urine protein excretion.

Low-protein diet supplemented with keto acids is associated with suppression of small-solute peritoneal transport rate in peritoneal dialysis patients.

PubMed

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

2011-01-01

Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6-0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6-0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0-1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/P(cr) (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0(glu) (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/P(cr) in group sLP was lower, and D/D0(glu) was higher than those in the other two groups (P < 0.05) at 12th month. D/D0(glu) increased (P < 0.05), and D/P(cr) tended to decrease, (P = 0.071) in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

PubMed

Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

2016-03-25

We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular and Enantiomeric Analysis of Organic Compounds in Carbonaceous Meteorites

NASA Technical Reports Server (NTRS)

Cooper, George

2003-01-01

Carbonaceous meteorites are relatively enriched in carbon. Much of this carbon is in the form of soluble organic compounds. The Murchison and Murray meteorites are the best-characterized carbonaceous meteorites with respect to organic chemistry. Their content of organic compounds has led to an initial understanding of early solar system organic chemistry as well as what compounds may have played a role in the origin of life (Cronin and Chang, 1993). Reported compounds include: amino acids, amides, carboxylic acids, sulfonic acids, and polyols. This talk will focus on the molecular and enantiomeric analysis of individual meteoritic compounds: polyol acids; and a newly identified class of meteorite compounds, keto acids, i.e., acetoacetic acid, levulinic acid, etc. Keto acids (including pyruvic) are critically important in all contemporary organisms. They are key intermediates in metabolism and processes such as the citric acid cycle. Using gas chromatography-mass spectrometry we identified individual meteoritic keto acids after derivatization to one or more of the following forms: isopropyl ester (ISP), trimethyIsiIy1 (TMS), tert-butyldimethylsilyl (BDMS). Ongoing analyses will determine if, in addition to certain amino acids from Murchison (Cronin and Pizzarello, 1997), other potentially important prebiotic compounds also contain enantiomeric excesses, i.e., excesses that could have contributed to the current homochirality of life.

Molecular cloning and expression profile of ß-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

USDA-ARS?s Scientific Manuscript database

Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a ...

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

PubMed

Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

2012-09-07

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Structural annotation of Beta-1,4-N-acetyl galactosaminyltransferase 1 (B4GALNT1) causing Hereditary Spastic Paraplegia 26.

PubMed

Dad, Rubina; Malik, Uzma; Javed, Aneela; Minassian, Berge A; Hassan, Muhammad Jawad

2017-08-30

Beta-1,4-N-acetyl galactosaminyltransferase 1, B4GALNT1, is a GM2/GD2 synthase, involved in the expression of glycosphingolipids (GSLs) containing sialic acid. Mutations in the gene B4GALNT1 cause Hereditary Spastic Paraplegia 26 (HSP26). In present study we have made attempt to predict the potential structural of the human B4GALNT1 protein. The results illustrated that the amino acid sequences of B4GALNT1 are not 100% conserved among selected twenty species. One signal peptide and one transmembrane domain predicted in human wild type B4GALNT1 protein with aliphatic index of 92.76 and theoretical (iso-electric point) pI of 8.93. It was a kind of unstable protein with Grand average of hydropathicity (GRAVY) of -0.127. Various post-translational modifications were also predicted to exist in B4GALNT1 and predicted to interact with different proteins including ST8SIA5, SLC33A1, GLB1 and others. In the final round, reported missense mutations have shown the further decrease in stability of the protein. This in-silico analysis of B4GALNT1 protein will provide the basis for the further studies on structural variations and biological pathways involving B4GALNT1 in the HSP26. Copyright © 2017. Published by Elsevier B.V.

Characterization of proflavine metabolites in rainbow trout.

PubMed

Yu, Z; Hayton, W L; Chan, K K

1997-04-01

Proflavine (3,6-diaminoacridine) has potential for use as an antiinfective in fish, and its metabolism by rainbow trout was therefore studied. Fourteen hours after intraarterial bolus administration of 10 mg/kg of proflavine, three metabolites were found in liver and bile, and one metabolite was found in plasma using reversed-phase HPLC with UV detection at 262 nm. Treatment with hydrochloric acid converted the three metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with beta-glucuronidase and saccharic acid 1,4-lactone, a specific beta-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination of UV-VIS absorption and mass spectra, HPLC-purified metabolites were isolated from liver. Data from these experiments suggested that the proflavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuronosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. When synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix-assisted, laser desorption ionization, time-of-flight MS. In addition, they coeluted on HPLC under different mobile phase conditions. Finally, the in vitro incubation with liver subcellular preparations confirmed this characterization and provided the evidence that APG can be formed by glucuronidation of AP or acetylation of PG.

Isolation, characterization, and biological properties of an endotoxin-like material from the gram-positive organism Listeria monocytogenes.

PubMed

Wexler, H; Oppenheim, J D

1979-03-01

The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from high-titer sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was beta-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2-keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exist in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins.

A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.

PubMed

Grant, Anne W; Steel, Gavin; Waugh, Hugh; Ellis, Elizabeth M

2003-01-21

A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

Molecular Mechanism by Which Retinoids Prevent Breast Cancer Development

DTIC Science & Technology

2007-06-01

10.11 AFFX-r2-Ec- bioB -M_at E. coli /GEN= bioB /DB_XREF=gb:J04423.1 /NOTE=SIF corresponding to nucleotides 2393-2682 of gb:J04423.1 /DEF=E.coli...7,8-diamino-pelargonic acid (bioA), biotin synthetase ( bioB ), 7-keto-8-amino-pelargonic acid synthetase (bioF), bioC protein, and dethiobiot 9.76...9.42 AFFX- BioB - M_at E. coli /GEN= bioB /DB_XREF=gb:J04423.1 /NOTE=SIF corresponding to nucleotides 2482-2739 of gb:J04423.1 /DEF=E.coli 7,8

A group of Populus trichocarpa DUF231 proteins exhibit differential O-acetyltransferase activities toward xylan.

PubMed

Zhong, Ruiqin; Cui, Dongtao; Ye, Zheng-Hua

2018-01-01

Wood represents the most abundant biomass produced by plants and one of its major components is acetyl xylan. Acetylation in xylan can occur at O-2 or O-3 of a xylosyl residue, at both O-2 and O-3 of a xylosyl residue, and at O-3 of a xylosyl residue substituted at O-2 with glucuronic acid. Acetyltransferases responsible for the regiospecific acetylation of xylan in tree species have not yet been characterized. Here we report the biochemical characterization of twelve Populus trichocarpa DUF231-containing proteins, named PtrXOATs, for their roles in the regiospecific acetylation of xylan. The PtrXOAT genes were found to be differentially expressed in Populus organs and among them, PtrXOAT1, PtrXOAT2, PtrXOAT9 and PtrXOAT10 exhibited the highest level of expression in stems undergoing wood formation. Activity assays of recombinant proteins demonstrated that all twelve PtrXOAT proteins were able to transfer acetyl groups from acetyl CoA onto a xylohexaose acceptor with PtrXOAT1, PtrXOAT2, PtrXOAT3, PtrXOAT11 and PtrXOAT12 having the highest activity. Structural analysis of the PtrXOAT-catalyzed reaction products using 1H NMR spectroscopy revealed that PtrXOAT1, PtrXAOT2 and PtrXOAT3 mediated 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation of xylosyl residues and PtrXOAT11 and PtrXOAT12 only catalyzed 2-O- and 3-O-monoacetylation of xylosyl residues. Of the twelve PtrXOATs, only PtrXOAT9 and PtrXOAT10 were capable of transferring acetyl groups onto the O-3 position of 2-O-glucuronic acid-substituted xylosyl residues. Furthermore, when expressed in the Arabidopsis eskimo1 mutant, PtrXOAT1, PtrXAOT2 and PtrXOAT3 were able to rescue the defects in xylan acetylation. Together, these results demonstrate that the twelve PtrXOATs are acetyltransferases with different roles in xylan acetylation in P. trichocarpa.

Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.

PubMed Central

Ohta, T; Ishikura, S; Shintani, S; Usami, N; Hara, A

2000-01-01

Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators. PMID:11104674

Isolation of an N-acetyl-DL-phenylalanine beta-naphthyl esterase from rabbit peritoneal polymorphonuclear leukocytes.

PubMed

Tsung, P; Kegeles, S W; Showell, H J; Becker, E L

1975-09-22

An N-acetyl-DL-phenylalanine beta-naphthyl esterase has been purified 26-fold from rabbit peritoneal polymorphonuclear leukocytes. The purified enzyme was inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate. The apparent Km for hydrolysis of N-acetyl-DL-phenylalanine beta-naphthyl ester is 71 muM. Optimal reaction rates were observed at pH 6-8. No divalent cation requirement for the activation of the enzyme activity was observed. The esterase activity was neither inhibited nor stimulated by bacterial factor, complement component C5a, guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) which are attractants or repellents for polymorphonuclear leukocytes. High chemotactic activity was observed in the partially purified fraction of the enzyme. The chemotactic activity, like the enzyme activity, was completely inhibited by 10(-7) M phosphonate.

Improved plasma amino acids pattern following 12 months of supplemented low-protein diet in peritoneal dialysis patients.

PubMed

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Cao, Liou; Wang, Qin; Ni, Zhaohui; Lindholm, Bengt; Axelsson, Jonas; Yao, Qiang

2010-07-01

Decreased plasma essential amino acid (EAA) levels, increased nonessential amino acid (NEAA) levels, and low EAA to NEAA ratio (E/NEAA) are common in peritoneal dialysis (PD) patients and may impact uremic complications. In the present study, we investigate the impact of keto acids-supplemented low-protein (sLP) diet on plasma amino acids (AAs) patterns in stable PD patients. This is a supplemental analysis of a previously published prospective and randomized trial. Thirty-nine PD patients selected from the original population were divided to receive either low (LP: 0.6-0.8 g/kg ideal body weight [IBW]/d, n = 13), keto acids-supplemented low- (sLP: 0.6-0.8 g/kg IBW/d + 0.12 g/kg IBW/d of keto acids, n = 12), or high- (HP: 1.0-1.2 g/kg IBW/d, n = 14) protein diets and followed for 1 year. Plasma AA patterns were assessed at baseline and 12 months using high-performance liquid chromatography. Whereas there were no significant differences between the three groups at baseline, following 12 months, the E/NEAA had increased significantly in group sLP (0.58 +/- 0.16 to 0.83 +/- 0.20, p < 0.05), but was not different in either LP (0.62 +/- 0.20 to 0.72 +/- 0.13, p = ns) or HP (0.66 +/- 0.14 to 0.74 +/- 0.12, p = ns) group. This change in E/NEAA in group sLP was due to a significant decrease in NEAA concomitantly with maintained EAA levels, whereas in the other two groups, neither EAA nor NEAA changed significantly. A low-protein diet supplemented with keto acids significantly improved the pattern of plasma AA in prevalent PD patients.

Dynamic Kinetic Asymmetric Transformations of β-Stereogenic-α-Keto Esters via Direct Aldolization

PubMed Central

Corbett, Michael T.; Johnson, Jeffrey S.

2014-01-01

Dynamic kinetic asymmetric transformations (DyKAT) of racemic β-bromo-α-keto esters via direct aldolization of nitromethane and acetone provide access to fully substituted α-glycolic acid derivatives bearing a β-stereocenter. The aldol adducts are obtained in excellent yield with high relative and absolute stereocontrol under mild reaction conditions. Mechanistic studies determined that the reactions proceed through a facile catalyst-mediated racemization of the β-bromo-α-keto esters under a DyKAT Type I manifold. PMID:24222195

Improving metabolic efficiency of the reverse beta-oxidation cycle by balancing redox cofactor requirement.

PubMed

Wu, Junjun; Zhang, Xia; Zhou, Peng; Huang, Jiaying; Xia, Xiudong; Li, Wei; Zhou, Ziyu; Chen, Yue; Liu, Yinghao; Dong, Mingsheng

2017-11-01

Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C 6 -C 10 , MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2g/L to 3.1g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites

PubMed Central

Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

2011-01-01

Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a “one pot” suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using 13C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems. PMID:21825143

Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites.

PubMed

Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

2011-08-23

Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a "one pot" suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using (13)C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems.

Serum alpha-tocopherol and ascorbic acid concentrations in Type 1 and Type 2 diabetic patients with and without angiopathy.

PubMed

Skrha, Jan; Prázný, Martin; Hilgertová, Jirina; Weiserová, Hana

2003-03-01

Alpha-tocopherol and ascorbic acid form a part of scavenger system influencing the level of oxidative stress in diabetes mellitus. The aim of this study was to evaluate serum concentrations of alpha-tocopherol and ascorbic acid in Type 1 and Type 2 diabetes mellitus and to compare them with the presence of vascular complications as well as with oxidative stress and endothelial dysfunction. A total of 38 Type 1 and 62 Type 2 diabetic patients were subdivided into those with and without angiopathy. Serum alpha-tocopherol and ascorbic acid concentrations were estimated in all patients and in 38 healthy persons. Their results were compared with diabetes control, with oxidative stress measured by plasma malondialdehyde and with endothelial dysfunction estimated by serum N-acetyl-beta-glucosaminidase activity. In addition, the differences in biochemical variables were compared between patients with and without angiopathy. Serum alpha-tocopherol related to the sum of cholesterol and triglyceride concentrations (AT/CHT ratio) was significantly lower in diabetic patients with macroangiopathy than in those without vascular changes (p<0.05). Serum ascorbic acid levels were significantly lower only in Type 2 diabetic patients with macroangiopathy as compared with healthy controls as well as with patients without vascular disease (p<0.01). Positive relationship was observed between serum alpha-tocopherol and cholesterol or triglyceride concentrations in both Type 1 and Type 2 diabetic patients. The presence of oxidative stress together with endothelial dysfunction measured by N-acetyl-beta-glucosaminidase activity was accompanied by lower AT/CHT ratio (p<0.005) in Type 2 diabetic patients. Diabetic patients with proven angiopathy or with advanced oxidative stress and endothelial dysfunction have significantly lower AT/CHT ratio and ascorbic acid concentration in serum. Their low concentrations may participate at the increased level of oxidative stress in these individuals.

Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

PubMed Central

Cole, Jeffrey T.; Sweatt, Andrew J.; Hutson, Susan M.

2012-01-01

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron. PMID:22654736

Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

PubMed Central

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

2011-01-01

Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0–1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/Pcr (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0glu (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/Pcr in group sLP was lower, and D/D0glu was higher than those in the other two groups (P < 0.05) at 12th month. D/D0glu increased (P < 0.05), and D/Pcr tended to decrease, (P = 0.071) in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate. PMID:21747999

Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver.

PubMed

Qureshi, A A; Elson, C E; Lebeck, L A

1982-11-19

The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.

Strong-acid, carboxyl-group structures in fulvic acid from the Suwannee River, Georgia. 1. Minor structures

USGS Publications Warehouse

Leenheer, J.A.; Wershaw, R. L.; Reddy, M.M.

1995-01-01

An investigation of the strong-acid characteristics (pKa 3.0 or less) of fulvic acid from the Suwannee River, Georgia, was conducted. Quantitative determinations were made for amino acid and sulfur-containing acid structures, oxalate half-ester structures, malonic acid structures, keto acid structures, and aromatic carboxyl-group structures. These determinations were made by using a variety of spectrometric (13C-nuclear magnetic resonance, infrared, and ultraviolet spectrometry) and titrimetric characterizations on fulvic acid or fulvic acid samples that were chemically derivatized to indicate certain functional groups. Only keto acid and aromatic carboxyl-group structures contributed significantly to the strong-acid characteristics of the fulvic acid; these structures accounted for 43% of the strong-acid acidity. The remaining 57% of the strong acids are aliphatic carboxyl groups in unusual and/or complex configurations for which limited model compound data are available.

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

Nadel, G.L.; Malik, K.U.; Lew, D.B.

The purpose of this study was to examine arachidonic acid (AA) metabolism and to characterize the type of adrenergic receptor (AR) involved in the production of the major metabolite of this fatty acid. ({sup 14}C)AA was incubated with GPT-rings and the radiolabelled products were extracted and separated by TLC method. The medium was also assayed for radiolabelled immunoreactive PG's (iPG's) and leukotrienes (LT) B4 and C4 by RIA or Enzyme immunoassay (EIA) after exposure to various AR agonists. ({sup 14}C)AA was incorporated into GPT-rings and metabolized mainly into iPGE2 and smaller amounts into PGF2{alpha}. Trace amounts of PGD2 and 6-keto-PGF1{alpha}more » but not LTB4 or LTC4 were detected by RIA and/or EIA. Incubation of GPT rings for 15 minutes with isoproterenol and salbutamol resulted in a significant increase of PGE2 synthesis (optimum conc: 10{sup {minus}7}, 10{sup {minus}7}M respectively). In contrast, dobutamine, norepinephrine, phenylnephrine and xylazine (up to 10{sup {minus}6}M) did not significantly increase PGE2 production. Isoproterenol-induced iPGE2 production was inhibited by a selective {beta}2 antagonist, butoxamine (70%: 10{sup {minus}7}M, 91%: 10{sup {minus}6}M) and somewhat reduced by {beta}1 antagonists practolol and metoprolol (30-64%:10{sup {minus}6}M). These data suggest that isoproterenol induced iPGE2 synthesis is primarily mediated via activation of {beta}2 adrenergic receptor.« less

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine.

PubMed Central

Savage, T. J.; Hristova, M. K.; Croteau, R.

1996-01-01

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev.& Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane. PMID:12226360

Intermediates of peroxisomal beta-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanoate.

PubMed Central

Bartlett, K; Hovik, R; Eaton, S; Watmough, N J; Osmundsen, H

1990-01-01

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation. Images Fig. 2. Fig. 3. Fig. 4. PMID:2396977

[Autophagy-lysosome pathway in skeletal muscle of diabetic nephropathy rats and the effect of low-protein diet plus α-keto acids on it].

PubMed

Huang, Juan; Yuan, Wei-jie; Wang, Jia-lin; Gu, Li-jie; Yin, Jun; Dong, Ting; Bao, Jin-fang; Tang, Zhi-huan

2013-11-26

To explore the regulation of autophagy-lysosome pathway (ALP) in skeletal muscle of diabetic nephropathy and examine the effect of low protein diet plus α-keto acid on ALP. A total of 45 24-week-old Goto-Kakizaki rats were randomized to receive normal protein (22%) diet (NPD), low-protein (6%) diet (LPD) or low-protein (5%) plus α-keto acids (1%) diet (Keto) (n = 15 each). Wistar control rats had a normal protein diet. The mRNA and protein levels of ALP markers LC3B, Bnip3, Cathepsin L in soleus muscle were evaluated at 48 weeks. Electron microscopy was used to confirm the changes of autophagy. Compared with CTL group, the mRNA levels of LC3B, Bnip3, Cathepsin L in soleus muscle of rats on NPD were higher, and protein levels of LC3B-I, LC3B-II, Bnip3, Cathepsin L in soleus muscle of rats on NPD also higher than CTL group (0.82 ± 0.33 vs 0.25 ± 0.07, 0.76 ± 0.38 vs 0.20 ± 0.12, 1.25 ± 0.30 vs 0.56 ± 0.19, 1.29 ± 0.40 vs 0.69 ± 0.20). The mRNA levels of LC3B, Bnip3 and Cathepsin L in LPD group were slightly lower, compared with NPD group. However there was no statistical significance. Similarly the protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L in LPD group were slightly lower with no statistical significance. In contrast, the mRNA levels of LC3B, Bnip3 and Cathepsin L were greatly lower in Keto group in comparison with NPD and LPD. And protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L were also greatly lower in Keto group in comparison with NPD and LPD. Additionally, autophagosome or auto-lysosome was found in NPD and LPD groups by electron microscopy. ALP is activated in skeletal muscle of diabetic nephropathy rats. And low protein plus α-keto acid decrease the activation of ALP and improve muscle wasting.

Theoretical study on keto-enol tautomerisation of glutarimide for exploration of the isomerisation reaction pathway of glutamic acid in proteins using density functional theory

NASA Astrophysics Data System (ADS)

Fukuyoshi, Shuichi; Nakayoshi, Tomoki; Takahashi, Ohgi; Oda, Akifumi

2017-03-01

In order to elucidate the reason why glutamic acid residues have lesser racemisation reactivity than asparaginic acid, we investigated the racemisation energy barrier of piperidinedione, which is the presumed intermediate of the isomerisation reaction of L-Glu to D-Glu, by density functional theory calculations. In two-water-molecule-assisted racemisation, the activation barrier for keto-enol isomerisation was 28.1 kcal/mol. The result showed that the activation barrier for the racemisation of glutamic acid residues was not different from that for the racemisation of aspartic acid residues. Thus, glutamic acid residues can possibly cause the racemisation reaction if the cyclic intermediate stably exists.

Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

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

Puddu, A., E-mail: alep100@hotmail.com; Storace, D.; Odetti, P.

2010-04-23

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation preventsmore » FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.« less

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

K Kucera; A Koblansky; L Saunders

Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa such as Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor (TLR) 11 generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long {beta}-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the interleukin (IL)-12 secretion response to TgPRF in peritoneal macrophages. Deletion ofmore » both the acidic loop and the {beta}-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and {beta}-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite Cryptosporidium parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from Plasmodium falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the {beta}-hairpin and diverges widely from the actin-binding surfaces of vertebrate profilins.« less

Domino-Fluorination-Protodefluorination Enables Decarboxylative Cross-Coupling of α-Oxocarboxylic Acids with Styrene via Photoredox Catalysis.

PubMed

Zhang, Muliang; Xi, Junwei; Ruzi, Rehanguli; Li, Nan; Wu, Zhongkai; Li, Weipeng; Zhu, Chengjian

2017-09-15

Domino-fluorination-protodefluorination decarboxylative cross-coupling of α-keto acids with styrene has been developed via photoredox catalysis. The critical part of this strategy is the formation of the carbon-fluorine (C-F) bond by the capture of a carbon-centered radical intermediate, which will overcome side reactions during the styrene radical functionalization process. Experimental studies have provided evidence indicating a domino-fluorination-protodefluorination pathway with α-keto acid initiating the photoredox cycle. The present catalytic protocol also affords a novel approach for the construction of α,β-unsaturated ketones under mild conditions.

Effect of HMB supplementation on body composition, fitness, hormonal profile and muscle damage indices.

PubMed

Portal, Shawn; Eliakim, Alon; Nemet, Dan; Halevy, Orna; Zadik, Zvi

2010-07-01

There is a huge market for ergogenic supplements for athletes. However, only a few products have been proven to have ergogenic effects and to be effective at improving muscle strength and body composition. One such supplement is beta-hydroxy beta-methylbutyrate (HMB). Derived from the amino acid leucine and its keto acid alpha-ketoisocaproate (KIC), HMB has been well documented as an oral ergogenic supplement commonly used by athletes. Several studies have shown that combining exercise training with HMB supplementation leads to increased muscle mass and strength, and there is some anecdotal evidence of aerobic improvement. However, HMB supplementation has been found to be effective mainly for untrained individuals. While previous reviews have emphasized three main pathways for HMB's mode of action: 1) enhancement of sarcolemmal integrity via cytosolic cholesterol, 2) inhibition of protein degradation via proteasomes, and 3) increased protein synthesis via the mTOR pathway, more recent studies have suggested additional possible mechanisms for its physiological effects. These include decreased cell apoptosis and enhanced cell survival, increased proliferation, differentiation and fusion via the MAPK/ERK and PI3K/Akt pathways, and enhanced IGF-I transcription. These are described here, and hormonal interactions are discussed, along with HMB dosage and safety issues.

Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid concentrations and branched-chain keto-acid dehydrogenase activity.

PubMed

Langer, S; Scislowski, P W; Brown, D S; Dewey, P; Fuller, M F

2000-01-01

The present experiment was designed to elucidate the mechanism of the methionine-sparing effect of excess branched-chain amino acids (BCAA) reported in the previous paper (Langer & Fuller, 2000). Twelve growing gilts (30-35 kg) were prepared with arterial catheters. After recovery, they received for 7 d a semipurified diet with a balanced amino acid pattern. On the 7th day blood samples were taken before (16 h postabsorptive) and after the morning meal (4 h postprandial). The animals were then divided into three groups and received for a further 7 d a methionine-limiting diet (80% of requirement) (1) without any amino acid excess; (2) with excess leucine (50% over requirement); or (3) with excesses of all three BCAA (leucine, isoleucine, valine, each 50% over the requirement). On the 7th day blood samples were taken as in the first period, after which the animals were killed and liver and muscle samples taken. Plasma amino acid and branched-chain keto acid (BCKA) concentrations in the blood and branched-chain keto-acid dehydrogenase (BCKDH; EC 1.2.4.4) activity in liver and muscle homogenates were determined. Compared with those on the balanced diet, pigs fed on methionine-limiting diets had significantly lower (P < 0.05) plasma methionine concentrations in the postprandial but not in the postabsorptive state. There was no effect of either leucine or a mixture of all three BCAA fed in excess on plasma methionine concentrations. Excess dietary leucine reduced (P < 0.05) the plasma concentrations of isoleucine and valine in both the postprandial and postabsorptive states. Plasma concentrations of the BCKA reflected the changes in the corresponding amino acids. Basal BCKDH activity in the liver and total BCKDH activity in the biceps femoris muscle were significantly (P < 0.05) increased by excesses of leucine or all BCAA.

VUV photoionization and dissociative photoionization of the prebiotic molecule acetyl cyanide: Theory and experiment

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

Bellili, A.; Hochlaf, M., E-mail: hochlaf@univ-mlv.fr, E-mail: martin.schwell@lisa.u-pec.fr; Schwell, M., E-mail: hochlaf@univ-mlv.fr, E-mail: martin.schwell@lisa.u-pec.fr

2014-10-07

The present combined theoretical and experimental investigation concerns the single photoionization of gas-phase acetyl cyanide and the fragmentation pathways of the resulting cation. Acetyl cyanide (AC) is inspired from both the chemistry of cyanoacetylene and the Strecker reaction which are thought to be at the origin of medium sized prebiotic molecules in the interstellar medium. AC can be formed by reaction from cyanoacetylene and water but also from acetaldehyde and HCN or the corresponding radicals. In view of the interpretation of vacuum ultraviolet (VUV) experimental data obtained using synchrotron radiation, we explored the ground potential energy surface (PES) of acetylmore » cyanide and of its cation using standard and recently implemented explicitly correlated methodologies. Our PES covers the regions of tautomerism (between keto and enol forms) and of the lowest fragmentation channels. This allowed us to deduce accurate thermochemical data for this astrobiologically relevant molecule. Unimolecular decomposition of the AC cation turns out to be very complex. The implications for the evolution of prebiotic molecules under VUV irradiation are discussed.« less

Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.

PubMed

Gunawardana, Subhadra C; Liu, Yi-Jia; Macdonald, Michael J; Straub, Susanne G; Sharp, Geoffrey W G

2004-11-01

Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.

Pretreatment with β-Boswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation

PubMed Central

Wang, Mingming; Chen, Minchun; Ding, Yi; Zhu, Zhihui; Zhang, Yikai; Wei, Peifeng; Wang, Jingwen; Qiao, Yi; Li, Liang; Li, Yuwen; Wen, Aidong

2015-01-01

Vascular endothelial cells play an important role in modulating anti-thrombus and maintaining the natural function of vascular by secreting many active substances. β-boswellic acid (β-BA) is an active triterpenoid compound from the extract of boswellia serrate. In this study, it is demonstrated that β-BA ameliorates plasma coagulation parameters, protects endothelium from blood stasis induced injury and prevents blood stasis induced impairment of endothelium-dependent vasodilatation. Moreover, it is found that β-BA significantly increases nitric oxide (NO) and cyclic guanosine 3’, 5’-monophosphate (cGMP) levels in carotid aortas of blood stasis rats. To stimulate blood stasis-like conditions in vitro, human umbilical vein endothelial cells (HUVECs) were exposed to transient oxygen and glucose deprivation (OGD). Treatment of β-BA significantly increased intracellular NO level. Western blot and immunofluorescence as well as immunohistochemistry reveal that β-BA increases phosphorylation of enzyme nitric oxide synthase (eNOS) at Ser1177. In addition, β-BA mediated endothelium-dependent vasodilatation can be markedly blocked by eNOS inhibitor L-NAME in blood stasis rats. In OGD treated HUEVCs, the protective effect of β-BA is attenuated by knockdown of eNOS. In conclusion, the above findings provide convincing evidence for the protective effects of β-BA on blood stasis induced endothelial dysfunction by eNOS signaling pathway. PMID:26482008

Plant-derived juvenile hormone III analogues and other sesquiterpenes from the stem bark of Cananga latifolia.

PubMed

Yang, Heejung; Kim, Hye Seong; Jeong, Eun Ju; Khiev, Piseth; Chin, Young-Won; Sung, Sang Hyun

2013-10-01

Juvenile hormone III (JH III) is a larval metamorphosis-regulating hormone present in most insect species. JH III was first isolated from the plant, Cyperus iria L., but the presence of JH III has not been reported in other plant species. In the present study, proof of the existence of JH III and its analogues from Cananga latifolia was established. From an aqueous MeOH extract of C. latifolia stem bark, six compounds were isolated along with nine known compounds. These were identified by using spectroscopic analyses as: (2E,6E,10R)-11-butoxy-10-hydroxy-3,7,11-trimethyldodeca-2,6-dienoic acid methyl ester, (2E,6E)-3,7,11-trimethyl-10-oxododeca-2,6-dienoic acid methyl ester, (2E)-3-methyl-5-[(1S,2R,6R)-1,2,6-trimethyl-3-oxocyclohexyl]-pent-2-enoic acid methyl ester, 1β-hydroxy-3-oxo-4β, 5α,7α-H-eudesmane 11-O-α-l-rhamnopyranoside, 4-epi-aubergenone 11-O-2',3'-di-O-acetyl-α-l-rhamnopyranoside and 4-epi-aubergenone 11-O-2',4'-di-O-acetyl-α-l-rhamnopyranoside. Three of the previously known compounds, (2E,6E,10R)-10-hydroxy-3,7,11-trimethyldodeca-2,6,11-trienoaic acid methyl ester, (2E,6E,10R)-10,11-dihydroxy-3,7,11-trimethyldodeca-2,6-dienoic acid and (2E,6S)-3-methyl-6-hydroxy-6-[(2R,5R)-5-(2-hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl]-hex-2-enoaic acid methyl ester have now been found in a plant species. Ultra performance liquid chromatography-quadruple time-of-flight mass spectroscopy (UPLC-QTOF/MS) analysis of the chemical constituents of C. latifolia showed that several were predominant in the sub-fractions of a C. latifolia stem bark extract. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chemical of Vitex trifolia.

PubMed

Liu, Quan-Yu; Chen, Yong-Sheng; Wang, Fei; Chen, Shi-Wu; Zhang, Yong-Hong

2014-06-01

A new steroidal ester, beta-rosaterol palmitate (1) along with ten known compounds, uvaol(2), 3-epi-ursolic acid (3), 2alpha, 3beta, 24-trihydroxyolean-12-en-28-oic acid (4), 2alpha, 3alpha, 24-trihydroxyurs-12-en-28-oic acid (5), 2alpha, 3alpha, 24-trihydroxyolean-12-en-28-oic acid (6), 2alpha, 3alpha, 24-trihydroxyolean-12-en-28-oic acid-28-O-beta-D-glucopyranosyl ester (7), (Z)-9-hexadecenoic acid (8), octacosyl alcohol (9), beta-sitosterol (10) and beta-daucosterol (11), has been isolated from the stems and leaves of Vitex trifolia. Their structures were elucidated using a combination of 1D and 2D NMR techniques (COSY, HMQC, and HMBC)and HR-ESI-MS analyses. Compounds 2-7 were isolated from this plant for the first time.

A convenient synthesis of 6-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one and related 4,6-disubstituted pyrazolopyrimidine nucleosides.

PubMed Central

Cottam, H B; Revankar, G R; Robins, R K

1983-01-01

The glycosylation of 4,6-dichloropyrazolo[3,4-d]pyrimidine and 4-chloro-6-methylthiopyrazolo[3,4-d]pyrimidine via the corresponding trimethylsilyl intermediate and tetra-O-acetyl-beta-D-ribofuranose in the presence of trimethylsilyl triflate as a catalyst, gave selective glycosylation at N1 as the only nucleoside product. The intermediates 4,6-dichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 7 and 4-chloro-6-methylthio-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 13 gave new and convenient synthetic routes to the inosine analog 1, the guanosine analog 2, the adenosine analog 3, and the isoguanosine analog 16. Glycosylation of the trimethylsilyl derivative of 6-chloropyrazolo[3,4-d]pyrimidine-4-one unexpectedly gave the N2-glycosyl isomer 20 as the major product. A number of new 4,6-disubstituted pyrazolo[3,4-d]pyrimidine nucleosides were prepared from these glycosyl intermediates. PMID:6835838

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

PubMed Central

Coffey, M D; Cole, R A; Colles, S M; Chisolm, G M

1995-01-01

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID:7560078

Keto-acids in Carbonaceous Meteorites

NASA Technical Reports Server (NTRS)

Cooper, G.; Chang, P. M.; Dugas, A.; Byrd, A.; Chang, P. M.; Washington, N.

2005-01-01

The Murchison and Murray meteorites are the best-characterized carbonaceous meteorites with respect to organic chemistry and are generally used as references for organic compounds in extraterrestrial material. Among the classes of organic compounds found in these meteorites are amino acids, carboxylic acids, hydroxy acids, purines, and pyrimidines. Such compounds, important in contemporary biochemistry, are thought to have been delivered to the early Earth in asteroids and comets and may have played a role in early life and/or the origin of life. Absent among (today's) critically important biological compounds reported in carbonaceous meteorites are keto acids, i.e., pyruvic acid, acetoacetic acid, and higher homologs. These compounds are key intermediates in such critical processes as glycolysis and the citric acid cycle. In this study several individual meteoritic keto acids were identified by gas chromatography-mass spectrometry (GC-MS) (see figure below). All compounds were identified as their trimethylsilyl (TMS), isopropyl ester (ISP), and tert-butyldimethylsilyl (tBDMS) derivatives. In general, the compounds follow the abiotic synthesis pattern of other known meteorite classes of organic compounds [1,2]: a general decrease in abundance with increasing carbon number within a class of compounds and many, if not all, possible isomers present at a given carbon number. The majority of the shown compounds was positively identified by comparison of their mass spectra to commercially available standards or synthesized standards.

In vitro effectiveness of triterpenoids and their synergistic effect with antibiotics against Staphylococcus aureus strains.

PubMed

Hamza, Muhammad; Nadir, Maha; Mehmood, Nadir; Farooq, Adeel

2016-01-01

The aim of this study is to evaluate the effect of four triterpenoids such as oleanolic acid, ursolic acid, cycloastragenol, and beta-boswellic acid alone and in combination with antibiotics against Staphylococcus aureus strains. Sixteen clinical strains of S. aureus from infected wounds were isolated. Eight were methicillin-sensitive S. aureus (MSSA), and the other eight were methicillin-resistant S. aureus (MRSA). The activity was also seen in reference S. aureus American Type Culture Collection ™ strains. The activity of all the triterpenoids and antibiotics against S. aureus was evaluated by broth microdilution method. The effectiveness was judged by comparing the minimum inhibitory concentrations (MICs) of the compounds with antibiotics. The combination of antibiotics with compounds was evaluated by their fractional inhibitory concentrations (FIC). Against both clinical and reference MSSA strains, none of the compounds exhibited comparable activity to antibiotics vancomycin or cefradine except for ursolic acid (MIC 7.8 μg/ml). Against MRSA, all compounds (MIC 16-128 μg/ml) showed lesser activity than vancomycin (MIC 5.8 μg/ml). Among triterpenoid-antibiotic combinations, the most effective were ursolic acid and vancomycin against clinical strain MSSA (FIC S 0.17). However, overall, different combinations between triterpenoids and antibiotics showed 95%-46% ( P < 0.05) reduction in MICs of antibiotics compared to when antibiotics were used alone. Cefradine, a drug not suitable for treating MRSA (MIC = 45 μg/ml), showed a remarkable decrease in its MIC (87% P< 0.01) when it was used in combination with oleanolic acid or ursolic acid in both clinical and reference strains. The tested triterpenoids are relatively weaker than antibiotics. However, when used in combination with antibiotics, they showed remarkable synergistic effect and thus can help in prolonging the viability of these antibiotics against S. aureus infections. Furthermore, reduction in MIC of cefradine with oleanolic acid indicates their potential use against MRSA.

Phenylpropanoid derivatives from edible canna, Canna edulis.

PubMed

Yun, Young Sook; Satake, Motoyoshi; Katsuki, Shigeki; Kunugi, Akira

2004-07-01

Two phenylpropanoid sucrose esters were isolated from dry rhizomes of Canna edulis Ker Gawl., along with a known phenylpropanoid sucrose ester and four known phenylpropanoids. On the basis of analysis of spectroscopic data and chemical evidence, these two phenylpropanoid sucrose esters were shown to be 3-O-p-coumaroyl-6-O-feruloyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside and 3,6-di-O-p-coumaroyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside.

Theoretical Study of the Mechanism Behind the para-Selective Nitration of Toluene in Zeolite H-Beta

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

Andersen, Amity; Govind, Niranjan; Subramanian, Lalitha

Periodic density functional theory calculations were performed to investigate the origin of the favorable para-selective nitration of toluene exhibited by zeolite H-beta with acetyl nitrate nitration agent. Energy calculations were performed for each of the 32 crystallographically unique Bronsted acid sites of a beta polymorph B zeolite unit cell with multiple Bronsted acid sites of comparable stability. However, one particular aluminum T-site with three favorable Bronsted site oxygens embedded in a straight 12-T channel wall provides multiple favorable proton transfer sites. Transition state searches around this aluminum site were performed to determine the barrier to reaction for both para andmore » ortho nitration of toluene. A three-step process was assumed for the nitration of toluene with two organic intermediates: the pi- and sigma-complexes. The rate limiting step is the proton transfer from the sigma-complex to a zeolite Bronsted site. The barrier for this step in ortho nitration is shown to be nearly 2.5 times that in para nitration. This discrepancy appears to be due to steric constraints imposed by the curvature of the large 12-T pore channels of beta and the toluene methyl group in the ortho approach that are not present in the para approach.« less

John Boswell: Posting Historical Landmarks at the Leading Edge of the Culture Wars

ERIC Educational Resources Information Center

Cisneros, Jeffrey

2013-01-01

One of the most enduring and controversial figures in the field of history is John E. Boswell. His work on homosexuality and the history of the Christian Church was published at a key time during the Stonewall Riots in the late 1960s and the removal of homosexuality from the list of diagnostic mental disorders in the mid 1970s. This social…

Further Clarification on the Hom, Mitchell, Lee, and Griffeth (2012) Model: Reply to Bergman, Payne, and Boswell (2012) and Maertz (2012)

ERIC Educational Resources Information Center

Griffeth, Rodger W.; Lee, Thomas W.; Mitchell, Terence R.; Hom, Peter W.

2012-01-01

In this article, we reply to Bergman, Payne, and Boswell (2012) and Maertz (2012), who commented on our reconceptualization of the employee turnover criterion and proximal withdrawal states (Hom, Mitchell, Lee, & Griffeth, 2012). We agree with some points (e.g., anticipated destinations) but take issue with others (e.g., turnover intentions as…

[Comparison of the effects of alpha-keto/ amino acid supplemented low protein diet and diabetes diet in patients with diabetic nephropathy].

PubMed

Qiu, Hong-yu; Liu, Fang; Zhao, Li-jun; Huang, Song-min; Zuo, Chuan; Zhong, Hui; Chen, Feng

2012-05-01

To investigate if a-keto/amino acid supplemented low protein diet can slow down the progression of diabetic nephrophathy in comparison with non-supplemented diabetes diet. A prospective, randomized, controlled clinical study was conducted. Twenty three cases of type 2 diabetic nephropathy in IV stage were randomly divided into alpha-keto/amino acid supplemented diet group (trial group) and conventional diabetes diet group (control group), The treatment duration was 52 weeks. 24 h urine protein was measured at 0, 12, 20, 36 and 52 weeks. Before and after the 52 weeks treatment, all the patients received the measurement of glomerular filtration rate (GFR), blood glucose, blood lipids, inflammatory markers, as well as nutritional status. After the treatment for 20, 36, 52 weeks, mean 24 h urine protein decreased significantly in trial groups (P < 0.05), and 24 h urine protein in trial group were significantly decreased (P < 0.05) compared with control group in 20 weeks after treatment. Either in trial group or in control group, GFR remained relatively stable during the observation period. Nutrition status, inflammatory markers, and serum calcium, phosphorus levels between the two groups were no significantly difference. The adverse events experienced by the patients in trial group were similar and consistent with the patients underlying renal diseases. Alpha-keto/amino acid can reduce proteinuria more effectively, while improve renal function and nutritional status in diabetic nephropathy patients with well-toleration.

The Baker's Yeast Reduction of Keto-Esters in Organic Solvents: A One Week Research Project for Undergraduate Students

NASA Astrophysics Data System (ADS)

North, Michael

1998-05-01

An experiment has been designed which allows final year undergraduate students to carry out a mini-research project in one week and thus get a flavour of the joys and tribulations of conducting chemical research before they undertake a major research project. The experiment is an investigation into the reduction of alpha- or beta-keto esters using non-fermenting Baker's yeast in petroleum ether. There are a number of advantages to this method of using Baker's yeast, including a reduction in the amount of organic solvent used, and a much simplified purification procedure. During the course of the mini-project, the substrate specificity of the yeast is investigated, and the conditions for the optimisation of a particular keto ester are determined. Each product is analysed by a variety of analytical techniques including polarimetry, IR, NMR, and GC. In addition, the use of correct stereochemical nomenclature to describe prochiral, and chiral compounds as well as chemical reactions are discussed.

Computational Study of the Malonic Acid Tautomerization Products in Highly Concentrated Particles

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

Dick-Pérez, Marilú; Windus, Theresa L.

Knowing the tautomeric form of malonic acid (MA) in concentrated particles is critical to understanding its effect on the atmosphere. Energies and vibrational modes of hydrated MA particles were calculated using density functional theory (DFT) at the B3LYP/6-31G(d,p) level and the effective fragment potential (EFP) method. Visualization of the keto and enol isomer vibrational modes enabled the assignment of keto isomer peaks in the 1710–1750 cm –1 range, and previously unidentified experimental IR peaks in the 1690–1710 cm –1 can now be attributed to the enol isomer. Furthermore, a comparison of calculated spectra of pure hydrated enol or keto isomersmore » confirm recent experimental evidence, of a shift in the keto–enol tautomer equilibrium when MA exists as concentrated particles.« less

Computational Study of the Malonic Acid Tautomerization Products in Highly Concentrated Particles

DOE PAGES

Dick-Pérez, Marilú; Windus, Theresa L.

2017-03-09

Knowing the tautomeric form of malonic acid (MA) in concentrated particles is critical to understanding its effect on the atmosphere. Energies and vibrational modes of hydrated MA particles were calculated using density functional theory (DFT) at the B3LYP/6-31G(d,p) level and the effective fragment potential (EFP) method. Visualization of the keto and enol isomer vibrational modes enabled the assignment of keto isomer peaks in the 1710–1750 cm –1 range, and previously unidentified experimental IR peaks in the 1690–1710 cm –1 can now be attributed to the enol isomer. Furthermore, a comparison of calculated spectra of pure hydrated enol or keto isomersmore » confirm recent experimental evidence, of a shift in the keto–enol tautomer equilibrium when MA exists as concentrated particles.« less

Xanthanolides and xanthane epoxide derivatives from Xanthium strumarium.

PubMed

Mahmoud, A A

1998-12-01

From the aerial parts of Xanthium strumarium, three new xanthanolide and xanthane-type sesquiterpenoids, 11alpha,13-dihydroxanthatin, 4beta,5beta-epoxyxanthatin-1alpha,4alpha-endoperoxide, and 1beta,4beta,4alpha,5alpha-diepoxyxanth-11(13)-en-12-oic acid have been isolated, together with seven known compounds. The structures were determined by spectroscopic methods, particularly high resolution 1D, 2D NMR spectroscopy and NOE experiments.

Better preservation of residual renal function in peritoneal dialysis patients treated with a low-protein diet supplemented with keto acids: a prospective, randomized trial.

PubMed

Jiang, Na; Qian, Jiaqi; Sun, Weilan; Lin, Aiwu; Cao, Liou; Wang, Qin; Ni, Zhaohui; Wan, Yanping; Linholm, Bengt; Axelsson, Jonas; Yao, Qiang

2009-08-01

While a low-protein diet may preserve residual renal function (RRF) in chronic kidney disease (CKD) patients before the start of dialysis, a high-protein intake is usually recommended in dialysis patients to prevent protein-energy wasting. Keto acids, which were often recommended to pre-dialysis CKD patients treated with a low-protein diet, had also been reported to be associated with both RRF and nutrition maintenance. We conducted a randomized trial to test whether a low-protein diet with or without keto acids would be safe and associated with a preserved RRF during peritoneal dialysis (PD). To assess the safety of low protein, we first conducted a nitrogen balance study in 34 incident PD patients randomized to receive in-centre diets containing 1.2, 0.9 or 0.6 g of protein/kg ideal body weight (IBW)/day for 10 days. Second, 60 stable PD patients [RRF 4.04 +/- 2.30 ml/ min/1.73 m(2), urine output 1226 +/- 449 ml/day, aged 53.6 +/- 12.8 years, PD duration 8.8 (1.5-17.8) months] were randomized to receive either a low- (LP: 0.6-0.8 g/kg IBW/day), keto acid-supplemented low- (sLP: 0.6-0.8 g/kg IBW/day with 0.12 g/kg IBW/day of keto acids) or high-protein (HP: 1.0-1.2 g/kg IBW/day) diet. The groups were followed for 1 year and RRF as well as nutritional status was evaluated serially. A neutral or positive nitrogen balance was achieved in all three groups. RRF remained stable in group sLP (3.84 +/- 2.17 to 3.39 +/- 3.23 ml/min/1.73 m(2), P = ns) while it decreased in group LP (4.02 +/- 2.49 to 2.29 +/- 1.72 ml/min/1.73 m(2), P < 0.05) and HP (4.25 +/- 2.34 to 2.55 +/- 2.29 ml/min/1.73 m(2), P < 0.05). There was no change from baseline on nutritional status in any of the groups during follow-up. A diet containing 0.6-0.8 g of protein/kg IBW/day is safe and, when combined with keto acids, is associated with an improved preservation of RRF in relatively new PD patients without significant malnutrition or inflammation.

Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity.

PubMed Central

Duée, P H; Pégorier, J P; Quant, P A; Herbin, C; Kohl, C; Girard, J

1994-01-01

In newborn-pig hepatocytes, the rate of oleate oxidation is extremely low, despite a very low malonyl-CoA concentration. By contrast, the sensitivity of carnitine palmitoyltransferase (CPT) I to malonyl-CoA inhibition is high, as suggested by the very low concentration of malonyl-CoA required for 50% inhibition of CPT I (IC50). The rates of oleate oxidation and ketogenesis are respectively 70 and 80% lower in mitochondria isolated from newborn-pig liver than from starved-adult-rat liver mitochondria. Using polarographic measurements, we showed that the oxidation of oleoyl-CoA and palmitoyl-L-carnitine is very low when the acetyl-CoA produced is channelled into the hydroxymethylglutaryl-CoA (HMG-CoA) pathway by addition of malonate. In contrast, the oxidation of the same substrates is high when the acetyl-CoA produced is directed towards the citric acid cycle by addition of malate. We demonstrate that the limitation of ketogenesis in newborn-pig liver is due to a very low amount and activity of mitochondrial HMG-CoA synthase as compared with rat liver mitochondria, and suggest that this could promote the accumulation of acetyl-CoA and/or beta-oxidation products that in turn would decrease the overall rate of fatty acid oxidation in newborn- and adult-pig livers. Images Figure 1 Figure 2 PMID:7907471

Detection of occupational lead nephropathy using early renal markers.

PubMed

Kumar, B D; Krishnaswamy, K

1995-01-01

Automotive use of leaded gasoline continues to be an important source of occupational exposure to lead in India and other countries. The present study assessed the renal function and markers of early renal damage of 22 mechanics at three automobile garages. Urinary N-acetyl-3-D-glucosaminidase activity and beta-2-microglobulin levels were significantly increased in auto garage mechanics with blood leads of 30-69 micrograms/dL. A significant correlation was observed between blood lead levels and urinary N-acetyl-3-D-glucosaminidase activity but not with urine beta-2-microglobulin levels. A marginal impairment in creatinine clearance was not statistically significant. Urinary N-acetyl-3-D-glucosaminidase activity offers a sensitive monitor of blood lead and renal tubular injury.

Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation

NASA Astrophysics Data System (ADS)

Wu, Zhaoguan; Li, Henghui; Zhang, Qiwei; Liu, Xin; Zheng, Qi; Li, Jianjun

2017-04-01

O-Acetylation of sialic acid in protein N-glycans is an important modification and can occur at either 4-, 7-, 8- or 9-position in various combinations. This modification is usually labile under alkaline reaction conditions. Consequently, a permethylation-based analytical method, which has been widely used in glycomics studies, is not suitable for profiling O-acetylation of sialic acids due to the harsh reaction conditions. Alternatively, methylamidation can be used for N-glycan analysis without affecting the base-labile modification of sialic acid. In this report, we applied both permethylation and methylamidation approaches to the analysis of O-acetylation in sialic acids. It has been demonstrated that methylamidation not only stabilizes sialic acids during MALDI processing but also allow for characterization of their O-acetylation pattern. In addition, LC-MS/MS experiments were carried out to distinguish between the O-acetylated glycans with potential isomeric structures. The repeatability of methylamidation was examined to evaluate the applicability of the approach to profiling of O-acetylation in sialic acids. In conclusion, the combination of methylamidation and permethylation methodology is a powerful MALDI-TOF MS-based tool for profiling O-acetylation in sialic acids applicable to screening of N-glycans.

[Glycosilated derivatives of substituted hydroxylamine. II. Phase transfer synthesis and investigation of glycosyl transfer reaction of glucosaminides of substituted hydroxylavine].

PubMed

Kur'ianov, V O; Lushchik, A A; Chupakhina, T A

2013-01-01

1-(2-Acetamido-3,4,6,-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyloxy)-benzotriazole reacted in boiling dichloromethane, in the presence of Luis acids as a promotors with primary and secondary aliphatic and cycloaliphatic alcohols and diisopropilidene galactose with alkyl-O-1,2-trans-glucosaminides formation. It was shown that the other glucosaminides of substituted hydroxylamine are not participated in this reaction. Structures of glucosaminides were identify by 1H-NMR-spectroscopy and comparison with known compounds.

Secondary Structures in a Freeze-Dried Lignite Humic Acid Fraction Caused by Hydrogen-Bonding of Acidic Protons with Aromatic Rings.

PubMed

Cao, Xiaoyan; Drosos, Marios; Leenheer, Jerry A; Mao, Jingdong

2016-02-16

A lignite humic acid (HA) was separated from inorganic and non-HA impurities (i.e., aluminosilicates, metals) and fractionated by a combination of dialysis and XAD-8 resin. Fractionation revealed a more homogeneous structure of lignite HA. New and more specific structural information on the main lignite HA fraction is obtained by solid-state nuclear magnetic resonance (NMR) spectroscopy. Quantitative (13)C multiple cross-polarization (multiCP) NMR indicated oxidized phenyl propane structures derived from lignin. MultiCP experiments, conducted on potassium HA salts titrated to pH 10 and pH 12, revealed shifts consistent with carboxylate and phenolate formation, but structural changes associated with enolate formation from aromatic beta keto acids were not detected. Two-dimensional (1)H-(13)C heteronuclear correlation (2D HETCOR) NMR indicated aryl-aliphatic ketones, aliphatic and aromatic carboxyl groups, phenol, and methoxy phenyl ethers. Acidic protons from carboxyl groups in both the lignite HA fraction and a synthetic HA-like polycondensate were found to be hydrogen-bonded with electron-rich aromatic rings. Our results coupled with published infrared spectra provide evidence for the preferential hydrogen bonding of acidic hydrogens with electron-rich aromatic rings rather than adjacent carbonyl groups. These hydrogen-bonding interactions likely result from stereochemical arrangements in primary structures and folding.

METHOD OF PREPARING COMPLEXES OF PLUTONIUM WITH DIKETONES

DOEpatents

Dixon, J.S.; Katz, J.J.; Orlemann, E.F.

1961-06-20

A process is described for sepsrating Pu from an aqueous alkaline solution by either precipitating with a beta -diketone or extracting into a solution of the beta -dixetone in an organic water-immiscible solvent. Acetyl acetone and benzoyl acetone are the beta -diketones used.

Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated coronary microvessel endothelial cells.

PubMed Central

Gerritsen, M E; Cheli, C D

1983-01-01

Isolated microvessels and isolated and cultured microvessel endothelial cells were prepared from rabbit cardiac muscle. Pathways of arachidonic acid metabolism were determined by measurement of exogenous substrate utilization [( 1-14C]arachidonic acid incorporation and release from intact tissue and cells; [1-14C]prostaglandin H2 (PGH2) metabolism by broken cell preparations) and by quantification of endogenous products (immunoreactive 6-keto-prostaglandin F1 alpha (PGF1 alpha) and prostaglandin E (PGE) release) by selective radioimmunoassay. Rabbit coronary microvessels and derived microvascular endothelial cells (RCME cells) synthesized two major products of the cyclooxygenase pathway: 6-keto-PGF1 alpha (hydrolytic product of prostaglandin I2) and PGE2. A reduced glutathione requiring PGH-E isomerase was demonstrated in coronary microvessels and RCME cells, but not in rabbit circumflex coronary artery or aorta. In addition, a minor amount of a compound exhibiting similar characteristics to 6-keto-PGE1 was found to be produced by microvessels and RCME cells. Measurement of endogenously released prostaglandins indicated that under basal and stimulated conditions, PGE release exceeded that of 6-keto-PGF1 alpha. Microvessels and microvessel endothelial cells derived from cardiac muscle of rabbit exhibit pathways of arachidonate metabolism that are different from those of many large blood vessels and derived endothelial cells. Images PMID:6415116

Studies on the cytotoxicity of miscellaneous compounds from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae).

PubMed

Rocha, Danilo D; Dantas, Ivana N F; Albuquerque, Maria Rose Jane R; Montenegro, Raquel C; Pessoa, Cláudia; de Moraes, Manoel Odorico; Pessoa, Otília Deusdênia L; Silveira, Edilberto R; Costa-Lotufo, Letícia V

2007-12-01

A detailed study on the cytotoxic effects of five known constituents isolated from the flowers and roots of Eupatorium betonicaeforme is reported, including 2,2-dimethyl-6-vinylchroman-4-one (1), 2-senecioyl-4-vinylphenol (2), 6-acetyl-2,2-dimethylchroman-4-one (3), (4E)-8beta-angeloyloxy-9beta,10beta-dihydroxy-1-oxogermacra-4,11(13)-dien-12,6alpha-olide (4), and 3beta-hydroxyicosan-1,5beta-olide (5). The sesquiterpene lactone 4 exhibited the highest cytotoxicity, with IC50 values ranging from 3.9 to 9.9 microM, showing some degree of cell selectivity. The antiproliferative activity of 4 was examined towards HL-60 cells, and found to diminish cell viability in a dose-dependent manner. Moreover, at all concentrations tested, there was a decrease in the number of cells capable of incorporating 5-bromo-2'-deoxyuridine (BrdU), indicating disruption of DNA synthesis. The morphological changes induced by 4 were compatible with apoptotic cell death. This work, thus, corroborates the anticancer potential of Eupatorium secondary metabolites.

Lipid peroxidation products do not activate hepatic stellate cells.

PubMed

Fang, Hsun-Lang; Lin, Wen-Chuan

2008-11-20

Lipid peroxidation (LPO) is known to be associated with liver fibrosis in chronic liver injury. However, direct effects of the products of LPO on liver fibrogenesis are still not clear. In this study, we examined the LPO products, such as malondiladehyde (MDA), 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), and 15-keto-13,14-dihydro-PGF(2alpha) (15-keto-PGF(2alpha)), on the activation of hepatic stellate cells (HSCs) in vivo and in vitro. Carbon tetrachloride (CCl(4)) was given orally to rats twice a week for 8 weeks. Corn oil was given daily to rats for 8 weeks. CCl(4) induced both free-radical-medicated and cyclooxygenase-2-dependent LPO. Free radical-medicated LPO showed an increase with corn oil treatment, whereas no effect was reflected on COX-2-dependent LPO. CCl(4) induced liver fibrosis in rats, but no liver fibrosis was observed in rats treated with corn oil. In vitro studies demonstrated that MDA, 8-iso-PGF(2alpha) and 15-keto-PGF(2alpha), did not activate HSCs, which were preactivated or not preactivated by TGF-beta1. Our results clearly indicate that LPO products, such as MDA, 8-iso-PGF(2alpha) and 15-keto-PGF(2alpha), cannot directly activate HSCs.

Dual fluorescence of N-phenylanthranilic acid: Effect of solvents, pH and beta-cyclodextrin.

PubMed

Rajendiran, N; Balasubramanian, T

2007-11-01

Spectral characteristics of N-phenylanthranilic acid (NPAA) have been studied in different solvents, pH and beta-cyclodextrin (beta-CD) and compared with anthranilic acid (2-aminobenzoic acid, 2ABA). In all solvents a dual fluorescence is observed in NPAA, whereas 2ABA gives single emission. Combining the results observed in the absorption, fluorescence emission and fluorescence excitation spectra, it is found that strong intramolecular hydrogen bonding (IHB) interactions present in NPAA molecule. The inclusion complex of NPAA with beta-CD is analysed by UV-vis, fluorimetry, FT-IR, (1)H NMR, scanning electron microscope and AM 1 method. The above spectral studies show that NPAA forms a 1:1 inclusion complex with beta-CD and COOH group present in the beta-CD cavity. A mechanism is proposed to explain the inclusion process.

Screening for the anti-inflammation quality markers of Xiaojin Pills based on HPLC-MS/MS method, COX-2 inhibition test and protein interaction network.

PubMed

Xiong, Xi; He, Ya-Nan; Feng, Bi; Pan, Yuan; Zhang, Hai-Zhu; Ke, Xiu-Mei; Zhang, Yi; Yang, Ming; Han, Li; Zhang, Ding-Kun

2018-05-10

Nowadays, breast disorders seriously affect women's health in an increasing number. In China, Xiaojin Pills are commonly used in the treatment of breast diseases. Doctors have concluded that the combined use of Xiaojin Pills with conventional therapy can significantly improve the efficacy with fewer side effects. However, the prescription of Xiaojin Pills is complicated and their quality control methods cannot completely ensure the quality of Xiaojin Pills. On the basis of its mechanism, our study combined chemical evaluation and biological evaluation to identify the anti-inflammatory markers of Xiaojin Pills. In this manuscript, 13 compounds in Xiaojin Pills were quantified. At the same time, the cyclooxygenase-2 inhibition rates of different Xiaojin Pills were measured and the possible markers were screened by spectrum-effect relationship. Further, anti-inflammatory activities of markers were verified and protein interaction network was analyzed, identifying the components of Protocatechuate, Beta-Boswellic acid and Levistilide A as the anti-inflammatory quality markers of Xiaojin Pills. We hope our studies can provide a scientific theoretical basis for accurately quality control of Xiaojin Pills and reasonable suggestions for pharmaceutical companies and new ideas for the quality control of other medicines.

Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-acetyltransferase from Shewanella denitrificans¶

PubMed Central

Chantigian, Daniel P.; Thoden, James B.; Holden, Hazel M.

2014-01-01

Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to inversion of configuration about the hexose C-4' carbon. PMID:24128043

Lack of enzyme induction with oxcarbazepine (600 mg daily) in healthy subjects.

PubMed Central

Larkin, J G; McKee, P J; Forrest, G; Beastall, G H; Park, B K; Lowrie, J I; Lloyd, P; Brodie, M J

1991-01-01

1. Oxcarbazepine (OXC), the 10-keto analogue of carbamazepine (CBZ), has similar anticonvulsant efficacy and possibly improved patient tolerability. Unlike CBZ, it is metabolised by reduction and may not induce hepatic monooxygenase enzymes. 2. Serum concentrations of OXC and its active metabolite 10-OH-carbazepine (10-OH-CZ) were followed after a single 300 mg dose and during and after 300 mg OXC twice daily for 29 doses in eight healthy male volunteers. 3. Antipyrine metabolism, urinary 6-beta-hydroxycortisol excretion, sex hormone binding globulin (SHBG) levels and circulating androgens were measured as indices of hepatic enzyme induction before, during and after treatment with OXC. 4. Elimination half-lives (mean +/- s.e. mean) of 10-OH-CZ were unaltered by 2 weeks' therapy with OXC (before 11.3 +/- 1.1 h; after 13.9 +/- 3 h). Trough plasma concentrations of 10-OH-CZ at steady-state (31 +/- 2.2 mumol l-1) were higher than predicted (16.5 +/- 4 mumol l-1). 5. Antipyrine metabolism, urinary 6-beta-hydroxycortisol excretion, SHBG levels and circulating androgens were unaltered by treatment with OXC. 6. OXC (600 mg daily) does not induce hepatic monooxygenase enzymes and so is likely to have more predictable dose-concentration relationships and to produce fewer physiological and pharmacological interactions than CBZ. PMID:2015172

Sequential derivatization of polar organic compounds in cloud water using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, N,O-bis(trimethylsilyl)trifluoroacetamide, and gas-chromatography/mass spectrometry analysis.

PubMed

Sagona, Jessica A; Dukett, James E; Hawley, Harmonie A; Mazurek, Monica A

2014-10-03

Cloud water samples from Whiteface Mountain, NY were used to develop a combined sampling and gas chromatography-mass spectrometric (GCMS) protocol for evaluating the complex mixture of highly polar organic compounds (HPOC) present in this atmospheric medium. Specific HPOC of interest were mono- and di keto-acids which are thought to originate from photochemical reactions of volatile unsaturated hydrocarbons from biogenic and manmade emissions and be a major fraction of atmospheric carbon. To measure HPOC mixtures and the individual keto-acids in cloud water, samples first must be derivatized for clean elution and measurement, and second, have low overall background of the target species as validated by GCMS analysis of field and laboratory blanks. Here, we discuss a dual derivatization method with PFBHA and BSTFA which targets only organic compounds that contain functional groups reacting with both reagents. The method also reduced potential contamination by minimizing the amount of sample processing from the field through the GCMS analysis steps. Once derivatized only gas chromatographic separation and selected ion monitoring (SIM) are needed to identify and quantify the polar organic compounds of interest. Concentrations of the detected total keto-acids in individual cloud water samples ranged from 27.8 to 329.3ngmL(-1) (ppb). Method detection limits for the individual HPOC ranged from 0.17 to 4.99ngmL(-1) and the quantification limits for the compounds ranged from 0.57 to 16.64ngmL(-1). The keto-acids were compared to the total organic carbon (TOC) results for the cloud water samples with concentrations of 0.607-3.350mgL(-1) (ppm). GCMS analysis of all samples and blanks indicated good control of the entire collection and analysis steps. Selected ion monitoring by GCMS of target keto-acids was essential for screening the complex organic carbon mixtures present at low ppb levels in cloud water. It was critical for ensuring high levels of quality assurance and quality control and for the correct identification and quantification of key marker compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

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

Swinney, D.C.; Ryan, D.E.; Thomas, P.E.

1988-07-26

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6..beta..-hydroxyprogesterone and 16..cap alpha..-hydroxyprogesterone), two secondary metabolites (6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16..cap alpha..-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 ..mu..M, while the K/sub m,app/ for metabolism of 6..beta..- and 16..cap alpha..-hydroxyprogesterone was found to be 5-10 ..mu..M. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 ..mu..M, and a lag in formation of secondary metabolites was not observed inmore » 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-keto-16..cap alpha..-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6..beta..-hydroxyprogesterone or 16..cap alpha..-hydroxyprogesterone. Incubation of 6..beta..-hydroxyprogesterone with a reconstituted system in an atmosphere of /sup 18/I/sub 2/ resulted in > 90% incorporation of /sup 18/O in the 16..cap alpha..-position of 6..beta..,16..cap alpha..-dihydroxyprogesterone but no incorporation of /sup 18/O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O/sub 2/, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.« less

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells

PubMed Central

Pardridge, William M.; Casanello-Ertl, Delia; Duducgian-Vartavarian, Luiza

1980-01-01

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-14C]leucine to 14CO2 or to the [1-14C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of 14CO2 from [1-14C]leucine was saturable with a Km = 6.3 mM and a maximum oxidation rate (Vmax) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-14C]leucine (Ki = 0.85 mM) and [U-14C]isoleucine, but had no effect on the oxidation of [U-14C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-14C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of 14CO2 and had relatively little effect on the production of [1-14C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD+ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase. PMID:7400311

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

PubMed

Chen, Xiaoyan; Xu, Jingliang; Yang, Liu; Yuan, Zhenhong; Xiao, Shiyuan; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

2015-11-01

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Investigation of mitigating effect of colon-specific prodrugs of boswellic acid on 2,4,6-trinitrobenzene sulfonic acid-induced colitis in Wistar rats: Design, kinetics and biological evaluation

PubMed Central

Sarkate, Ajinkya; Dhaneshwar, Suneela S

2017-01-01

AIM To develop a colon-targeting bioreversible delivery system for β-boswellic acid (BBA) and explore utility of its prodrugs in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. METHODS Synthesis of 4 co-drugs of BBA with essential amino acids was achieved by CDI coupling, followed by their spectral characterization. In vitro kinetics were studied by HPLC in aqueous buffers, homogenates of gastrointestinal tract and fecal matter. In vivo kinetic studies were performed in Wistar rat plasma, urine and feces. The prodrugs were screened in TNBS-induced colitis modeled Wistar rats. Statistical significance was assumed at P < 0.05, P < 0.01, P < 0.001 when compared with disease controls using one-way and two-way ANOVAs. RESULTS Prodrugs were stable in 0.05 mol/L HCl buffer (pH 1.2) and stomach homogenates. Negligible hydrolysis was observed in phosphate buffer and intestinal homogenates. Substantial release (55%-72% and 68%-86%) of BBA was achieved in rat fecal matter and homogenates of colon. In vivo studies of BBA with L-tryptophan (BT) authenticated colon-specific release of BBA. But, surprisingly substantial concentration of BBA was seen to reach the systemic circulation due to probable absorption through colonic mucosa. Site-specifically enhanced bioavailability of BBA could be achieved in colon, which resulted in demonstration of significant mitigating effect on TNBS-induced colitis in rats without inducing any adverse effects on stomach, liver and pancreas. Prodrug of BT was found to be 1.7% (P < 0.001) superior than sulfasalazine in reducing the inflammation to colon among all prodrugs tested. CONCLUSION The outcome of this study strongly suggests that these prodrugs might have dual applicability to inflammatory bowel disease and chronotherapy of rheumatoid arthritis. PMID:28275295

IN0523 (Urs-12-ene-3α,24β-diol) a plant based derivative of boswellic acid protect Cisplatin induced urogenital toxicity

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

Singh, Amarinder

The limiting factor for the use of Cisplatin in the treatment of different type of cancers is its toxicity and more specifically urogenital toxicity. Oxidative stress is a well-known phenomenon associated with Cisplatin toxicity. However, in Cisplatin treated group, abnormal animal behavior, decreased body weight, cellular and sub-cellular changes in the kidney and sperm abnormality were observed. Our investigation revealed that Cisplatin when administered in combination with a natural product derivative (Urs-12-ene-3α,24β-diol, labeled as IN0523) resulted in significant restoration of body weight and protection against the pathological alteration caused by Cisplatin to kidney and testis. Sperm count and motility weremore » significantly restored near to normal. Cisplatin caused depletion of defense system i.e. glutathione peroxidase, catalase and superoxide dismutase, which were restored close to normal by treatment of IN0523. Reduction in excessive lipid peroxidation induced by Cisplatin was also found by treatment with IN0523. The result suggests that IN0523 is a potential candidate for ameliorating Cisplatin induced toxicity in the kidney and testes at a dose of 100 mg/kg p.o. via inhibiting the oxidative stress/redox status imbalance and may be improving the efflux mechanism. - Highlights: • Synthesis of a novel boswellic acid derivative (IN0523) • Counter oxidative stress induced due to Cisplatin • Protect against urogenital toxicity induced by Cisplatin.« less

Pharmacokinetic and Pharmacodynamic Interaction of Boswellic Acids and Andrographolide with Glyburide in Diabetic Rats: Including Its PK/PD Modeling.

PubMed

Samala, Sujatha; Veeresham, Ciddi

2016-03-01

The effect of boswellic acids (BA) and andrographolide (AD) on the pharmacokinetics and pharmacodynamics of glyburide in normal as well as in streptozotocin-induced diabetic rats was studied. In normal and diabetic rats, the combination of glyburide with BA or AD increased significantly (p < 0.01) all the pharmacokinetic parameters, such as Cmax, AUC0-n, AUCtotal, t1/2, and mean residence time, and decreased the clearance, Vd, markedly as compared with the control group. In rat liver, microsomes BA and AD have shown CYP3A4 inhibitory activity significantly (p < 0.01), compared with the vehicle group. The increase in hypoglycemic action by concomitant administration of glyburide with BA or AD was more in diabetic rats than when the drugs were used singly and with the control group, which suggests the enhancement of glucose reduction capacity of glyburide in diabetic rats along with BA or AD. In PK/PD modeling of BA and AD with glyburide, the predicted PK and PD parameters are in line with the observed PK and PD parameters. The results revealed that BA and AD led to the PK/PD changes because of glyburide-increased bioavailability and because of the inhibition of CYP3A4 enzyme. In conclusion, add-on preparations containing BA or AD may increase the bioavailability of glyburide, and hence the dose should be monitored. Copyright © 2016 John Wiley & Sons, Ltd.

Triterpene glycosides from the tubers of Anemone coronaria.

PubMed

Mimaki, Yoshihiro; Watanabe, Kazuki; Matsuo, Yukiko; Sakagami, Hiroshi

2009-07-01

Six new triterpene glycosides (1-6), together with 11 known ones (7-17), have been isolated from a glycoside-enriched fraction prepared from the tubers of Anemone coronaria L. (Ranunculaceae). On the basis of extensive spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage, the structures of 1-6 were determined to be 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta,23-dihydroxyolean-12-en-28-oic acid (1), 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->4)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid (2), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (3), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta,23-dihydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (4), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (5), and 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-18-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (6), respectively. Furthermore, the isolated compounds were evaluated for their cytotoxic activity against HSC-2 cells.

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

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

Higa, H.; Varki, A.

1986-05-01

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

Acetylation contributes to hypertrophy-caused maturational delay of cardiac energy metabolism.

PubMed

Fukushima, Arata; Zhang, Liyan; Huqi, Alda; Lam, Victoria H; Rawat, Sonia; Altamimi, Tariq; Wagg, Cory S; Dhaliwal, Khushmol K; Hornberger, Lisa K; Kantor, Paul F; Rebeyka, Ivan M; Lopaschuk, Gary D

2018-05-17

A dramatic increase in cardiac fatty acid oxidation occurs following birth. However, cardiac hypertrophy secondary to congenital heart diseases (CHDs) delays this process, thereby decreasing cardiac energetic capacity and function. Cardiac lysine acetylation is involved in modulating fatty acid oxidation. We thus investigated what effect cardiac hypertrophy has on protein acetylation during maturation. Eighty-four right ventricular biopsies were collected from CHD patients and stratified according to age and the absence (n = 44) or presence of hypertrophy (n = 40). A maturational increase in protein acetylation was evident in nonhypertrophied hearts but not in hypertrophied hearts. The fatty acid β-oxidation enzymes, long-chain acyl CoA dehydrogenase (LCAD) and β-hydroxyacyl CoA dehydrogenase (βHAD), were hyperacetylated and their activities positively correlated with their acetylation after birth in nonhypertrophied hearts but not hypertrophied hearts. In line with this, decreased cardiac fatty acid oxidation and reduced acetylation of LCAD and βHAD occurred in newborn rabbits subjected to cardiac hypertrophy due to an aortocaval shunt. Silencing the mRNA of general control of amino acid synthesis 5-like protein 1 reduced acetylation of LCAD and βHAD as well as fatty acid oxidation rates in cardiomyocytes. Thus, hypertrophy in CHDs prevents the postnatal increase in myocardial acetylation, resulting in a delayed maturation of cardiac fatty acid oxidation.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

PubMed

Thapa, Dharendra; Zhang, Manling; Manning, Janet R; Guimarães, Danielle A; Stoner, Michael W; O'Doherty, Robert M; Shiva, Sruti; Scott, Iain

2017-08-01

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice. Copyright © 2017 the American Physiological Society.

Rethinking fat as a fuel for endurance exercise.

PubMed

Volek, Jeff S; Noakes, Timothy; Phinney, Stephen D

2015-01-01

A key element contributing to deteriorating exercise capacity during physically demanding sport appears to be reduced carbohydrate availability coupled with an inability to effectively utilize alternative lipid fuel sources. Paradoxically, cognitive and physical decline associated with glycogen depletion occurs in the presence of an over-abundance of fuel stored as body fat that the athlete is apparently unable to access effectively. Current fuelling tactics that emphasize high-carbohydrate intakes before and during exercise inhibit fat utilization. The most efficient approach to accelerate the body's ability to oxidize fat is to lower dietary carbohydrate intake to a level that results in nutritional ketosis (i.e., circulating ketone levels >0.5 mmol/L) while increasing fat intake for a period of several weeks. The coordinated set of metabolic adaptations that ensures proper interorgan fuel supply in the face of low-carbohydrate availability is referred to as keto-adaptation. Beyond simply providing a stable source of fuel for the brain, the major circulating ketone body, beta-hydroxybutyrate, has recently been shown to act as a signalling molecule capable of altering gene expression, eliciting complementary effects of keto-adaptation that could extend human physical and mental performance beyond current expectation. In this paper, we review these new findings and propose that the shift to fatty acids and ketones as primary fuels when dietary carbohydrate is restricted could be of benefit for some athletes.

Comparative Genomics of Regulation of Fatty Acid and Branched-chain Amino Acid Utilization in Proteobacteria

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

Kazakov, Alexey E.; Rodionov, Dmitry A.; Arkin, Adam Paul

2008-10-31

Bacteria can use branched-chain amino acids (ILV, i.e. isoleucine, leucine, valine) and fatty acids (FA) as sole carbon and energy sources convering ILV into acetyl-CoA, propanoyl-CoA and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR and GntR families binding tomore » eleven distinct DNA motifs. The ILV degradation genes in gamma- and beta-proteobacteria are mainly regulated by anovel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species), in addition, the TetR-type regulator LiuQ was identified in some beta-proteobacteria (8 species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in the Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gamma-proteobacteria (34 species), PsrA in gamma- and beta-proteobacteria (45 species), FadP in beta-proteobacteria (14 species), and LiuR orthologs in alpha-proteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from the functional and evolutionary points of view.« less

Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

PubMed

Latif, S A; Sheff, M F; Ribeiro, C E; Morris, D J

1997-02-01

We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

Variable clinical spectrum of the most common inborn error of bile acid metabolism--3beta-hydroxy-Delta 5-C27-steroid dehydrogenase deficiency.

PubMed

Subramaniam, Pushpa; Clayton, Peter T; Portmann, Bernard C; Mieli-Vergani, Giorgina; Hadzić, Nedim

2010-01-01

We studied the clinical features of children with 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase (3beta-HSDH) deficiency presenting to King's College and Great Ormond Street hospitals between 1989 and 2005. The diagnosis was made biochemically by detection of sulphated dihydroxycholenoic acids and trihydroxycholenoic acids in urine by fast atom bombardment mass spectrometry or electrospray ionisation tandem mass spectrophotometry and a plasma bile acid profile showing absent or low cholic and chenodeoxycholic acid levels and high concentrations of 3beta-7 alpha-dihydroxy-5-cholenoic acid and 3beta-7 alpha-12 alpha-trihydroxy-5-cholenoic acid. Eighteen children (12 male) with 3beta-HSDH deficiency were identified and diagnosed at a median age of 1.35 years (range 8 weeks-11 years). The presenting features included neonatal cholestasis (n = 11), rickets (n = 8, 1 of whom also had hypocalcaemic tetany, seizures, and normal liver biochemical markers), hepatomegaly (n = 7), pruritus (n = 3), and steatorrhoea and failure to thrive (n = 3). Ten children had low serum 25-OH vitamin D levels, of whom 8 also had low vitamin E and 6 had low vitamin A serum levels. Liver histology showed giant cell change and hepatocyte disarray in all with added features of cholestasis in 11, bridging fibrosis in 6, micronodular cirrhosis in 1, fatty change in 1, and active lobular and portal inflammation in 1. Five patients were treated with cholic acid and chenodeoxycholic acid (7 mg x kg(-1) x day(-1) of each), 7 with chenodeoxycholic acid only (7-18 mg x kg(-1) x day(-1)), and 1 with cholic acid (8 mg x kg(-1) x day(-1)) only. Repeated liver biopsies performed in 4 patients 6 months after starting replacement therapy showed improved histological changes. Three children died untreated before 5 years of age. After a median follow-up of 5.5 years (range 1-17 years) 12 out of 13 treated children have no clinical signs of liver disease or of fat-soluble vitamin deficiency. 3beta-HSDH deficiency is a rare inborn error of metabolism with diverse clinical features. Early replacement treatment leads to clinical and biochemical control and prevents chronic liver and bone disease, at least in the medium term.

[Phenolic acid derivatives from Bauhinia glauca subsp. pernervosa].

PubMed

Zhao, Qiao-Li; Wu, Zeng-Bao; Zheng, Zhi-Hui; Lu, Xin-Hua; Liang, Hong; Cheng, Wei; Zhang, Qing-Ying; Zhao, Yu-Ying

2011-08-01

To study the chemical constituents of Bauhinia glauca subsp. pernervosa, eleven phenolic acids were isolated from a 95% ethanol extract by using a combination of various chromatographic techniques including column chromatography over silica gel, ODS, MCI, Sephadex LH-20, and semi-preparative HPLC. By spectroscopic techniques including 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS, these compounds were identified as isopropyl O-beta-(6'-O-galloyl)-glucopyranoside (1), ethyl O-beta-(6'-O-galloyl)-glucopyranoside (2), 3, 4, 5-trimethoxyphenyl-(6'-O-galloyl)-O-beta-D-glucopyranoside (3), 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside (4), gallic acid (5), methyl gallate (6), ethyl gallate (7), protocatechuic acid (8), 3, 5-dimethoxy-4-hydroxybenzoic acid (9), erigeside C (10) and glucosyringic acid (11). Among them, compound 1 is a new polyhydroxyl compound; compounds 2, 10, and 11 were isolated from the genus Bauhinia for the first time, and the other compounds were isolated from the plant for the first time. Compounds 6 and 8 showed significant protein tyrosine phosphatase1B (PTP1B) inhibitory activity in vitro with the IC50 values of 72.3 and 54.1 micromol x L(-1), respectively.

Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

PubMed

Kawashima, Nagako; Tsuji, Daisuke; Okuda, Tetsuya; Itoh, Kohji; Nakayama, Ken-ichi

2009-11-01

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

PubMed

Imabayashi, Yuki; Suzuki, Shun'ichi; Kawasaki, Hisashi; Nakamatsu, Tsuyoshi

2016-01-01

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

The AMP-activated protein kinase is involved in the regulation of ketone body production by astrocytes.

PubMed

Blázquez, C; Woods, A; de Ceballos, M L; Carling, D; Guzmán, M

1999-10-01

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.

Biotransformation of ent-kaur-16-en-19-oic acid by Psilocybe cubensis.

PubMed

Pechwang, Jaraslak; Sihanonth, Prakitsin; Pornpakakul, Surachai; Muangsin, Nongnuj; Piapukiew, Jittra; Vangnai, Alisa; Chaichit, Narongsak; Chuchawankul, Siriporn; Petsom, Amorn

2010-06-01

Biotranformation of ent-kaur-16-en-19-oic acid (1) using Psilocybe cubensis resulted in hydroxylated products. After two days of incubation, ent-16beta,17-dihydroxy-kauran-19-oic acid (2) was isolated. After further incubation for nine days, two novel metabolites, ent-12alpha,16beta,17-trihydroxy-kauran-19-oic acid (3) and ent-11alpha,16beta,17-trihydroxy-kauran-19-oic acid (4), were obtained. The metabolites were identified by spectroscopic methods and X-ray crystallography. Compounds 1-4 were evaluated for their cytotoxic properties against the human leukaemia K562 cell line; only compound 1 showed moderate activity.

LC-MS-MS-TOF analysis of oxygenated organic compounds in ambient aerosol

NASA Astrophysics Data System (ADS)

Roempp, A.; Moortgat, G.

2003-04-01

Ambient aerosol samples were taken at different sites across Europe. The fine mode aerosol was collected on quartz filters at flow rates of 160 L/min and 500 L/min. These samples were analyzed for organic acids (C>4) by an HPLC system coupled to a hybrid mass spectrometer. The mass spectrometer consists of a quadrupole mass analyzer, a quadrupole collision cell and a time-of-flight mass analyzer (TOF). Analytes were identified by standards when available or MS-MS experiments and exact mass measurements utilizing the high mass resolution of the TOF instrument. Monoterpenes (alpha-pinene, beta-pinene, sabinene, limonene, 3-carene) were ozonolyzed in the laboratory and compared with field samples. Besides the commonly measured organic acids (pinic, pinonic and norpinic acid) sabinic, caric and caronic acid were identified for the first time in ambient aerosol. In addition, nearly all samples showed significant concentrations of newly identified keto dicarboxylic acids (C9 - C12). Laboratory experiments were used to investigate the formation mechanisms of these compounds. By comparing laboratory measurements of wood combustion and field samples from the Eastern Mediterranean region, nitrocatechol was identified as a possible tracer for biomass burning. The data obtained is used to determine the role of biogenic sources in secondary organic aerosol formation.

Seasonal variations of low molecular weight hydroxy-dicarboxylic acids and oxaloacetic acid in remote marine aerosols from Chichijima Island in the western North Pacific (December 2010-November 2011)

NASA Astrophysics Data System (ADS)

Gowda, Divyavani; Kawamura, Kimitaka

2018-05-01

Concentrations of homologous hydroxy-dicarboxylic acids (diacids) (hC3-hC6) and keto-diacid (oxaloacetic acid) were measured in the atmospheric aerosols collected at Chichijima Island (27.04° N, 142.13° E) in the western North Pacific from December 2010 to November 2011. The monthly averaged concentrations of hydroxy-diacids and oxaloacetic acid were significantly higher in spring followed by winter and autumn. Molecular distributions of hydroxy-diacids demonstrated that malic acid was the most abundant species in all four seasons, followed by tartronic acid in winter and spring and 3- and 2-hydroxyglutaric acids in summer and autumn. Hydroxy-diacids and keto-diacid maximized in spring and winter when air masses originated from the Asian continent with westerly winds. The concentrations of total hydroxy-diacids and oxaloacetic acid ranged from 0.1 to 27.3 ng m-3 and <0.005 to 2 ng m-3, respectively. The enhanced concentrations of diacids and their intermediates in winter and spring are associated with a long-range atmospheric transport of pollutants from East Asia to remote Chichijima Island followed by photochemical processing of organic aerosols. Seasonal molecular distribution of hydroxy-diacids and oxaloacetic acid was found to be dependent on the source strengths and plausible photochemical processing to form smaller diacids. Moderate to strong correlations among hydroxy-diacids, oxaloacetic acid and low molecular weight (LMW) diacids suggest that hydroxy-diacids and oxaloacetic acid are the intermediates in the photochemical oxidation of LMW diacid. Hence, photochemical formation of the most abundant LMW diacids, i.e., oxalic acid, could be produced from hydroxy- and keto-diacid as intermediates.

[Clinical characteristics and analysis of mass spectrometric data in 33 patients with maple syrup urine disease].

PubMed

Yang, Nan; Han, Lian-shu; Ye, Jun; Qiu, Wen-juan; Zhang, Hui-wen; Gao, Xiao-lan; Wang, Yu; Li, Xiao-yan; Xu, Hao; Gu, Xue-fan

2012-10-30

To explore the clinical characteristics and the diagnostic method of maple syrup urine disease (MSUD). From January 2003 to December 2011, a total of 14 000 patients with suspected inherited metabolism diseases were tested. The blood levels of leucine and valine of these patients were detected by tandem mass spectrometry. The urinary level of branched-chain α-ketoacids was tested by gas chromatography-mass spectrometry. And the diagnosis was based on the elevated levels of leucine and valine in blood and branched-chain α-ketoacids in urine. Thirty-three MSUD patients were confirmed. Their median age of initial visit was 0.17 years old (range: 7 days to 30 years old). The peak onset age of them was 2-30 days old, including 28 cases of neonatal onset (84.8%). The presenting symptoms of 28 cases were feeding difficulties (n=14), poor response, lethargy and seizures. Their median blood levels of leucine and valine (1901 (458-5804) and 600 (315-1617) µmol/L) were significantly higher than their normal levels ((50-300) and (60-250) µmol/L, both P<0.01). Their urinary levels of 2-OH-isovaleric acid, 2-keto-isovaleric acid, 2-keto-3-methylvaleric acid, 2-keto-isocaproic and acetylglycine (262.5 (5.4-624.3), 35.8 (1.9-156.0), 133.8 (7.4-611.5), 518.7 (17.2-2121.2) and 280.5 (11.0-1087.9) respectively) significantly higher than their normal levels (0, <0.1, 0, 0, <0.1 respectively, all P<0.01). In 5 intermittent MSUD patients, their blood levels of leucine and valine (402 (348-958) and 556 (322-808) µmol/L) were significantly higher than their normal levels (both P<0.01). The urinary level of 2-OH-isovaleric acid was significantly higher than its normal levels (P<0.01) while the urinary levels of other α-ketoacids were normal. The confirmation of MSUD remains difficult because of a lack of specific clinical features. The detections of tandem mass spectrometry and gas chromatography-mass spectrometry may aid its early diagnosis.

[Studies on chemical constituents from herbs of Taraxacum mongolicum].

PubMed

Shi, Shu-Yun; Zhou, Chang-Xin; Xu, Yan; Tao, Qiao-Feng; Bai, Hua; Lu, Fu-Sheng; Lin, Wen-Yan; Chen, Hai-Yong; Zheng, Wei; Wang, Li-Wei; Wu, Yi-Hang; Zeng, Su; Huang, Ke-Xin; Zhao, Yu; Li, Xiao-Kun; Qu, Jia

2008-05-01

To investigate the chemical constituents of the herbs of Taraxacum mongolicum. The chemical constituents were isolated by various column chromatographic methods and their structures elucidated mainly by NMR and MS evidences. Forty-four components were obtained and identified were as artemetin (1), quercetin (2), quercetin-3', 4', 7-trime-thyl ether (3), luteolin (4), luteolin-7-O-beta-D-glucopyranoside (5), luteolin-7-O-beta-D-galactopyranoside (6), genkwanin (7), isoetin (8), hesperetin (9), genkwanin-4'-O-beta-D-lutinoside (10), hesperidin (11), quercetin-7-O-[beta-D-glucopyranosyl (1-->6) -beta-D-glucopyranoside (12), quercetin-3, 7-O-beta-D-diglucopyranoside (13), isoetin-7-O-beta-D-glucopyranosyl- 2'-O-alpha-L-arabinopyranoside (14), isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside (15), isoetin-7- O-beta-D-glucopyranosyl-2'-O-beta-D-xyloypyranoside (16), caffeic acid (17), furulic acid (18), 3-O-caffeoylquinic acid (19), 3, 5-di-O-caffeoylquinic acid (20), 3, 4-di-O-caffeoylquinic acid (21), 4, 5-di-O-caffeoylquinic acid (22), 1-hydroxymethyl-5-hydroxy-phenyl-2-O-beta-D-glucopyranoside (23), p-hydroxybenzoic acid (24), p-coumaric acid (25), 3, 5-dihydroxylbenzoic acid (26), gallic acid (27), gallicin (28), syringic acid (29), 3, 4-dihydroxybenzoic acid (30), caffeic acid ethyl ester (31), esculetin (32), rufescidride (33), mongolicumin A [6, 9, 10-trihydroxy-benzoxanthene-1, 2-dicarboxylic acid] (34), mongolicumin B [1 l-hydroxy-2-oxo-guaia-1 (10), 3, 5-trien-8, 12-lactone] (35), isodonsesquitin A (36), taraxacin (37), sesquiterpene ketolactone (38), taraxasteryl acetate (39), phi-taraxasteryl acetate (40) and lupenol acetate (41), palmitic acid (42), beta-sitosterol (43), and stigmasterol (44). Four compounds (14, 15, 34 and 35) were new compounds, compounds 1, 3, 6-13, 20-22, 30 and 31 were isolated from this genus for the first time, while compounds 18, 23-29, 32 and 37-42 were obtained from this species for the first time.

Pulmonary fatty acid synthesis. I. Mitochondrial acetyl transfer by rat lung in vitro.

PubMed

Evans, R M; Scholz, R W

1977-04-01

Incorporation of tritiated water into fatty acids by rat adipose tissue and lung tissue slices incubated with 5 mM glucose indicated a level of fatty acid synthesis in rat lung approximately 15% that observed in adipose tissue in vitro. (-)-Hydroxycitrate, and inhibitor of ATP citrate lyase, markedly reduced tritiated water incorporation into fatty acids by lung tissue slices. The effects of (-)-hydroxycitrate and n-butymalonate on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate suggested that citrate is a major acetyl carrier for de novo fatty acid synthesis in lung tissue. Alternative mechanisms to citrate as an acetyl carrier were also considered. Lung mitochondrial preparations formed significant levels of acetylcarnitine in the presence of pyruvate and carnitine. However, the effect of carnitine on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate into fatty acids by lung tissue slices indicated that acetylcarnitine may not be a significant acetyl carrier for fatty acid synthesis but may serve as an acetyl "buffer" in the control of mitochondrial acetyl-CoA levels. Additionally, it appears unlikely that either acetylaspartate or acetoacetate are of major importance in acetyl transfer in lung tissue.

[Suplemented restricted diet in old patients with chronic renal disease].

PubMed

Teplan, Vladimír

2016-01-01

In last decades was confirmed remarkable increase in number of old patients with chronic kidney disease. Despide of developments in dialysis technology and kidney transplantation there is a growing number of old patients who are not suitable for these methods. Recently were published data showing long-term effect of protein restricted diet supplemented with keto amino acids in elderly. Based on our results obtained in re-analysis of 3 000 patients we can confirm also good compliance and low risk of malnutrition.Key words: chronic kidney disease - keto amino acids - old age - restricted diet.

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa.

PubMed

Rosselli, Luciana K; Oliveira, Cristiano L P; Azzoni, Adriano R; Tada, Susely F S; Catani, Cleide F; Saraiva, Antonio M; Soares, José Sérgio M; Medrano, Francisco J; Torriani, Iris L; Souza, Anete P

2006-09-15

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.

Synthesis and characterization of the 6 alpha- and 6 beta-hydroxylated derivatives of corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol.

PubMed

Kraan, G P; van Wee, K T; Wolthers, B G; van der Molen, J C; Nagel, G T; Drayer, N M; van Leusen, D

1993-10-01

This report describes the synthesis of 6 alpha, 17,21- and 6 beta, 17,21-trihydroxypregn-4-ene-3,20-dione, 6 alpha, 7,21- and 6 beta, 11 beta, 21-trihydroxypregn-4-ene-3,20-dione, and--for the first time--that of 6 alpha, 21- and 6 beta, 21-dihydroxypregn-4-ene-3,11,20-trione. The former four compounds were prepared by 6-hydroxylation of 17,21-trihydroxypregn-4-ene-3,20-dione and 11 beta, 21-dihydroxypregn-4-ene-3,20-dione, respectively. This was achieved by autoxidation or by oxidation with 3-chloroperbenzoic acid, of the 3-methoxy-pregna-3,5-dienes of the latter two steroids. The yield of the 6 beta-hydroxylated steroids, but not of their corresponding 6 alpha-epimers, was higher using autoxidation than the peracid. The two 6-hydroxylated pregnenetriones were prepared from 6 alpha, 21-diacetoxy-11 beta-hydroxypregn-4-ene-3,20-dione and 6 beta, 21-diacetoxy-11 beta-hydroxypregn-4-ene-3,20-dione, respectively, by oxidation with pyridinium chlorochromate. The above-mentioned six steroids were identified and characterized by nuclear magnetic resonance, infrared, ultraviolet, high performance liquid chromatography, gas chromatography, and mass spectrometry.

Research in acetylene containing monomers

NASA Technical Reports Server (NTRS)

Ogliaruso, M. A.

1976-01-01

The preparation of precursor bisbenzils with pendant acetylene linkages for use in the synthesis of new aromatic poly (phenyl quinoxalines) was investigated. Attempts to condense para, para prime-dibromo benzil and potassium acetylide in liquid ammonia and in toluene, to prepare 4-phenyl acetyl phenyl ether, 4-(paraacetylphenyl) acetyl phenyl ether, 4-phenyl acetyl-4 primeacetyl phenyl acetyl phenyl ether, the reaction of 4-phenyl acetyl phenyl ether with Villsmeier reagent to prepare 4-(beta-chloro cinnamaldehyde) phenyl ether, the reaction of 4-(para-acetyl phenyl) acetyl phenyl ether with Villsmeier reagent, and the oxidation of bibenzil to prepare benzil are described. The reactions of phenyl acetylene with oxidizing agent, of phenyl acetylene with bromine, of 1,1,2,2-tetrabromo ethyl benzene with zinc and with oxidizing agent are described.

Control of Growth Within Drosophila Peripheral Nerves by Ras and Protein Kinase A

DTIC Science & Technology

2009-02-01

channel gene on behavior and axonal excitability. Genetics 124: 133– 143 . 20. Chouinard SW, Wilson GF, Schlimgen AK, Ganetzky B (1995) A potassium...channel beta subunit related to the aldo- keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci U S A 92: 6763–6767

Decarboxylation of Δ 9-tetrahydrocannabinol: Kinetics and molecular modeling

NASA Astrophysics Data System (ADS)

Perrotin-Brunel, Helene; Buijs, Wim; van Spronsen, Jaap; van Roosmalen, Maaike J. E.; Peters, Cor J.; Verpoorte, Rob; Witkamp, Geert-Jan

2011-02-01

Efficient tetrahydrocannabinol (Δ 9-THC) production from cannabis is important for its medical application and as basis for the development of production routes of other drugs from plants. This work presents one of the steps of Δ 9-THC production from cannabis plant material, the decarboxylation reaction, transforming the Δ 9-THC-acid naturally present in the plant into the psychoactive Δ 9-THC. Results of experiments showed pseudo-first order reaction kinetics, with an activation barrier of 85 kJ mol -1 and a pre-exponential factor of 3.7 × 10 8 s -1. Using molecular modeling, two options were identified for an acid catalyzed β-keto acid type mechanism for the decarboxylation of Δ 9-THC-acid. Each of these mechanisms might play a role, depending on the actual process conditions. Formic acid proved to be a good model for a catalyst of such a reaction. Also, the computational idea of catalysis by water to catalysis by an acid, put forward by Li and Brill, and Churchev and Belbruno was extended, and a new direct keto-enol route was found. A direct keto-enol mechanism catalyzed by formic acid seems to be the best explanation for the observed activation barrier and the pre-exponential factor of the decarboxylation of Δ 9-THC-acid. Evidence for this was found by performing an extraction experiment with Cannabis Flos. It revealed the presence of short chain carboxylic acids supporting this hypothesis. The presented approach is important for the development of a sustainable production of Δ 9-THC from the plant.

A Quantitative Acetylomic Analysis of Early Seed Development in Rice (Oryza sativa L.).

PubMed

Wang, Yifeng; Hou, Yuxuan; Qiu, Jiehua; Li, Zhiyong; Zhao, Juan; Tong, Xiaohong; Zhang, Jian

2017-06-27

PKA (protein lysine acetylation) is a critical post-translational modification that regulates various developmental processes, including seed development. However, the acetylation events and dynamics on a proteomic scale in this process remain largely unknown, especially in rice early seed development. We report the first quantitative acetylproteomic study focused on rice early seed development by employing a mass spectral-based (MS-based), label-free approach. A total of 1817 acetylsites on 1688 acetylpeptides from 972 acetylproteins were identified in pistils and seeds at three and seven days after pollination, including 268 acetyproteins differentially acetylated among the three stages. Motif-X analysis revealed that six significantly enriched motifs, such as (DxkK), (kH) and (kY) around the acetylsites of the identified rice seed acetylproteins. Differentially acetylated proteins among the three stages, including adenosine diphosphate (ADP) -glucose pyrophosphorylases (AGPs), PDIL1-1 (protein disulfide isomerase like 1-1), hexokinases, pyruvate dehydrogenase complex (PDC) and numerous other regulators that are extensively involved in the starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle and photosynthesis pathways during early seed development. This study greatly expanded the rice acetylome dataset, and shed novel insight into the regulatory roles of PKA in rice early seed development.

Structural Studies of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

2002-01-01

Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

The oligosaccharidic content of the glycoconjugates of the prepubertal descended and undescended testis: lectin histochemical study.

PubMed

Gheri, Gherardo; Sgambati, Eleonora; Thyrion, Giorgia D Zappoli; Vichi, Debora; Orlandini, Giovanni E

2004-01-01

The saccharidic content of the glycoconjugates has been studied in the descended the undescended testes of a 8 years old boy. For this purpose, a battery of seven HRP-conjugated lectins (SBA, DBA,PNA,WGA,UEAI, LTA and ConA) was used. D-galactose-N-acetyl-D-galactosamine and alpha-L-fucose sugar residues, which were present in the cytoplasm of the Sertoli cells of the normally positioned prepubertal testis, were not detected in the same cells of the undescended testis. The Leydig's cells of the descended testis appeared characterized by N-acetyl-D-glucosamine which was absent in the rare and atrophic Leydig's cells of the cryptorchid testis. Differences in sugar residues distribution between the descended and the undescended testis were also detected in the lamina propria of the seminiferous tubules. Peritubular myoid cells in the undescended testis only reacted with PNA, after neuraminidase digestion, thus revealing the presence of D-galactose (beta1-->3)-N-acetyl-D-galactosamine and sialic acid. In this study a complete distributional map of the sugar residues of the glycoconjugates in the descended and undescended prepubertal testis is reported.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

PubMed

Yueh, Wen-Shiun; Thomas, Peter; Chang, Ching-Fong

2005-02-01

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Structure of N-acetyl-[beta]-D-glucosaminidase (GcnA) from the Endocarditis Pathogen Streptococcus gordonii and its Complex with the Mechanism-based Inhibitor NAG-thiazoline

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

Langley, David B.; Harty, Derek W.S.; Jacques, Nicholas A.

2008-09-17

The crystal structure of GcnA, an N-acetyl-{beta}-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal {alpha}-helical domain has not been observed previously and forms a large dimerization interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical ({beta}/{alpha}){sub 8} TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a familymore » 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-{beta}-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.« less

Application of NIRS coupled with PLS regression as a rapid, non-destructive alternative method for quantification of KBA in Boswellia sacra

NASA Astrophysics Data System (ADS)

Al-Harrasi, Ahmed; Rehman, Najeeb Ur; Mabood, Fazal; Albroumi, Muhammaed; Ali, Liaqat; Hussain, Javid; Hussain, Hidayat; Csuk, René; Khan, Abdul Latif; Alam, Tanveer; Alameri, Saif

2017-09-01

In the present study, for the first time, NIR spectroscopy coupled with PLS regression as a rapid and alternative method was developed to quantify the amount of Keto-β-Boswellic Acid (KBA) in different plant parts of Boswellia sacra and the resin exudates of the trunk. NIR spectroscopy was used for the measurement of KBA standards and B. sacra samples in absorption mode in the wavelength range from 700-2500 nm. PLS regression model was built from the obtained spectral data using 70% of KBA standards (training set) in the range from 0.1 ppm to 100 ppm. The PLS regression model obtained was having R-square value of 98% with 0.99 corelationship value and having good prediction with RMSEP value 3.2 and correlation of 0.99. It was then used to quantify the amount of KBA in the samples of B. sacra. The results indicated that the MeOH extract of resin has the highest concentration of KBA (0.6%) followed by essential oil (0.1%). However, no KBA was found in the aqueous extract. The MeOH extract of the resin was subjected to column chromatography to get various sub-fractions at different polarity of organic solvents. The sub-fraction at 4% MeOH/CHCl3 (4.1% of KBA) was found to contain the highest percentage of KBA followed by another sub-fraction at 2% MeOH/CHCl3 (2.2% of KBA). The present results also indicated that KBA is only present in the gum-resin of the trunk and not in all parts of the plant. These results were further confirmed through HPLC analysis and therefore it is concluded that NIRS coupled with PLS regression is a rapid and alternate method for quantification of KBA in Boswellia sacra. It is non-destructive, rapid, sensitive and uses simple methods of sample preparation.

Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis.

PubMed

Brown, D M; Upcroft, J A; Dodd, H N; Chen, N; Upcroft, P

1999-01-25

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD.

PubMed

S Sonnet, Davis; N O'Leary, Monique; A Gutierrez, Mark; M Nguyen, Steven; Mateen, Samiha; Hsu, Yuehmei; P Mitchell, Kylie; J Lopez, Antonio; Vockley, Jerry; K Kennedy, Brian; Ramanathan, Arvind

2016-07-04

Maple Syrup Urine Disease (MSUD) is an inherited disorder caused by the dysfunction in the branched chain keto-acid dehydrogenase (BCKDH) enzyme. This leads to buildup of branched-chain keto-acids (BCKA) and branched-chain amino acids (BCAA) in body fluids (e.g. keto-isocaproic acid from the BCAA leucine), leading to numerous clinical features including a less understood skeletal muscle dysfunction in patients. KIC is an inhibitor of mitochondrial function at disease relevant concentrations. A murine model of intermediate MSUD (iMSUD) shows significant skeletal muscle dysfunction as by judged decreased muscle fiber diameter. MSUD is an orphan disease with a need for novel drug interventions. Here using a 96-well plate (liquid chromatography- mass spectrometry (LC-MS) based drug-screening platform we show that Metformin, a widely used anti-diabetic drug, reduces levels of KIC in patient-derived fibroblasts by 20-50%. This Metformin-mediated effect was conserved in vivo; Metformin-treatment significantly reduced levels of KIC in the muscle (by 69%) and serum (by 56%) isolated from iMSUD mice, and restored levels of mitochondrial metabolites (e.g. AMP and other TCA). The drug also decreased the expression of mitochondrial branched chain amino transferase (BCAT) which produces KIC in skeletal muscle. This suggests that Metformin can restore skeletal muscle homeostasis in MSUD by decreasing mitochondrial KIC production.

An unexplored pathway for degradation of cholate requires a 7α-hydroxysteroid dehydratase and contributes to a broad metabolic repertoire for the utilization of bile salts in Novosphingobium sp. strain Chol11.

PubMed

Yücel, Onur; Drees, Steffen; Jagmann, Nina; Patschkowski, Thomas; Philipp, Bodo

2016-12-01

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ 1,4 -diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ 4,6 -structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ 4,6 -metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ 1,4 -diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to the formation of a dead-end steroid with a 3-keto-7-deoxy-Δ 4,6 -diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

A new double coupling system: synthesis of citronellyl acetate via transacetylation to citronellol from acetyl coenzyme A produced from glucose and free fatty acids.

PubMed

Oda, S; Ohta, H

2001-08-01

A double coupling system, which couples metabolism of glucose and transacetylation, is a unique procedure for the production of acetic esters. In the novel coupling system described in this article, acetyl coenzyme A (acetyl-CoA) was supplied via metabolism of both glucose and exogenous saturated fatty acids. While short and middle chain fatty acids having C4-8 were very biotoxic, myristic acid (C14) was effectively used as a source of acetyl-CoA.

Dynamic mechanistic modeling of the multienzymatic one-pot reduction of dehydrocholic acid to 12-keto ursodeoxycholic acid with competing substrates and cofactors.

PubMed

Sun, Boqiao; Hartl, Florian; Castiglione, Kathrin; Weuster-Botz, Dirk

2015-01-01

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers.

Role of Aldo-Keto Reductase Family 1 (AKR1) Enzymes in Human Steroid Metabolism

PubMed Central

Rižner, Tea Lanišnik; Penning, Trevor M.

2013-01-01

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ4-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. PMID:24189185

A Core Facility for the Study of Neurotoxins of Biological Origin

DTIC Science & Technology

1992-02-15

a somewhat different approach was used. TVL was incubated with or without N-acetyl-g- glucosamine (1 x 10-1 M) for 30 min. This mixture was then...galactosamine, N-acetyl-0-galactosamine, N-acetyl-cz ,lucosamine and N-acetyl-3- glucosamine . None of these lectins was a potent antagonist of botulinum...sialic acid, whereas TVL has affinity for both N-acetyl-,6- glucosamine and N-acetyl-a-sialic acid. However, the fact that the lectin from Datora

Chemotactic activity from rabbit peritoneal neutrophils. Lack of identity with N-acetyl-DL-phenylalanine beta-napthyl esterase.

PubMed

Tsung, P K; Showell, H J; Kegeles, S W; Becker, E L

1976-08-12

The chemotactic and N-acetyl-DL-phenylalanine beta-naphthyl esterase activities of rabbit peritoneal neutrophils are separable from each other by both DEAE cellulose and Sephadex G-100 column chromatography. Partially purified esterase obtained from DEAE-cellulose chromatography had molecular weight of 70 000. However, the partially purified fraction contained chemotactic activities with major activity in molecular weight of 28000 and minor activities in the molecular weights of 45000, 21900, 14500 and 10500. Esterase activity is inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate but chemotactic activity is not.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-d-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

PubMed Central

Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

2014-01-01

d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications. PMID:24419616

Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin.

PubMed Central

Eisenreich, W; Menhard, B; Hylands, P J; Zenk, M H; Bacher, A

1996-01-01

A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524]. Images Fig. 1 Fig. 4 PMID:8692832

Human aldo-keto reductases 1B1 and 1B10: a comparative study on their enzyme activity toward electrophilic carbonyl compounds.

PubMed

Shen, Yi; Zhong, Linlin; Johnson, Stephen; Cao, Deliang

2011-05-30

Aldo-keto reductase family 1 member B1 (AKR1B1, 1B1 in brief) and aldo-keto reductase family 1 member B10 (AKR1B10, 1B10 in brief) are two proteins with high similarities in their amino acid sequences, stereo structures, and substrate specificity. However, these two proteins exhibit distinct tissue distributions; 1B10 is primarily expressed in the gastrointestinal tract and adrenal gland, whereas 1B1 is ubiquitously present in all tissues/organs, suggesting their difference in biological functions. This study evaluated in parallel the enzyme activity of 1B1 and 1B10 toward alpha, beta-unsaturated carbonyl compounds with cellular and dietary origins, including acrolein, crotonaldehyde, 4-hydroxynonenal, trans-2-hexenal, and trans-2,4-hexadienal. Our results showed that 1B10 had much better enzyme activity and turnover rates toward these chemicals than 1B1. By detecting the enzymatic products using high-performance liquid chromatography, we measured their activity to carbonyl compounds at low concentrations. Our data showed that 1B10 efficiently reduced the tested carbonyl compounds at physiological levels, but 1B1 was less effective. Ectopically expressed 1B10 in 293T cells effectively eliminated 4-hydroxynonenal at 5 μM by reducing to 1,4-dihydroxynonene, whereas endogenously expressed 1B1 did not. The 1B1 and 1B10 both showed enzyme activity to glutathione-conjugated carbonyl compounds, but 1B1 appeared more active in general. Together our data suggests that 1B10 is more effectual in eliminating free electrophilic carbonyl compounds, but 1B1 seems more important in the further detoxification of glutathione-conjugated carbonyl compounds. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

PubMed

Cescutti, P; Toffanin, R; Pollesello, P; Sutherland, I W

1999-01-31

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

Enzyme-Inspired Chiral Secondary-Phosphine-Oxide Ligand with Dual Noncovalent Interactions for Asymmetric Hydrogenation.

PubMed

Chen, Caiyou; Zhang, Zhefan; Jin, Shicheng; Fan, Xiangru; Geng, Mingyu; Zhou, Yan; Wen, Songwei; Wang, Xinrui; Chung, Lung Wa; Dong, Xiu-Qin; Zhang, Xumu

2017-06-06

Inspired by the unique character of enzymes, we developed novel chiral SPO (secondary-phosphine-oxide) ligand (SPO-Wudaphos) which can enter into both ion pair and H-bond noncovalent interactions. The novel chiral SPO-Wudaphos exhibited excellent results in the asymmetric hydrogenation of α-methylene-γ-keto carboxylic acids, affording the chiral γ-keto acids with up to over 99 % ee. A series of control experiments and DFT calculations were conducted to illustrate the critical roles of both the ion pair and H-bond noncovalent interactions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature.

PubMed

Sutiono, Samuel; Carsten, Jörg; Sieber, Volker

2018-06-28

Branched chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. It is a key enzyme for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C) hindering the production of the alcohols at high temperatures. In this study, structure-guided engineering toward improved thermostability of KdcA is outlined. Several strategies such as, stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With 7 point mutations, our mutant (7M.D) showed an increase of T501h by 14.8 °C without compromising its substrate specificity. 7M.D exhibited >400-fold improvement of half-life at 70 °C and >600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) as well as in cell-free applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292.

PubMed

Tazoe, Masaaki; Oishi, Hiromi; Kobayashi, Setsuko; Hoshino, Tatsuo

2016-08-01

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O2 or a cofactor for the conversion, but was stimulated by Mn(2+). N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

PubMed Central

Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

2005-01-01

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii.

PubMed

El-Katatny, M H; Gudelj, M; Robra, K H; Elnaghy, M A; Gübitz, G M

2001-07-01

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels.

PubMed

Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

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

Zhong, Linlin; Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001; Liu, Ziwen

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene,more » protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.« less

A new C-methylated flavonoid glycoside from Pinus densiflora.

PubMed

Jung, M J; Choi, J H; Chung, H Y; Jung, J H; Choi, J S

2001-12-01

A new C-methyl flavonol glycoside, 5,7,8,4'-tetrahydroxy-3-methoxy-6-methylflavone 8-O-beta-D-glucopyranoside (1), has been isolated from the needles of Pinus densiflora, together with kaempferol 3-O-beta-(6"-acetyl)-galactopyranoside.

Sesquiterpenes and a phenylpropanoid from Cordia trichotoma.

PubMed

de Menezes, Jane Eire S A; Machado, Francisca Elane A; Lemos, Telma Leda G; Silveira, Edilberto R; Braz Filho, Raimundo; Pessoa, Otília Deusdênia L

2004-01-01

Two new secondary metabolites, the phenylpropanoid 3-(2',4',5'-trimethoxyphenyl)propanoic acid (1) and the sesquiterpene (+)-1beta,4beta,6alpha-trihydroxyeudesmane (2) were isolated from the heartwood of Cordia trichotoma Vell., along with the known sesquiterpenes (-)-1beta,4beta,7alpha-trihydroxyeudesmane (3) and (+)-1beta,4beta,11-trihydroxyoppositane (4). Their structures were elucidated by means of spectroscopic data interpretation, mainly 1D and 2D NMR and mass spectrometry.

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.

Building blocks for the synthesis of glycosyl-myo-inositols involved in the insulin intracellular signalling process.

PubMed

Zapata, A; Martín-Lomas, M

1992-10-09

Glycosylation of (+/- )-1-O-benzyl-2,3:5,6-di-O-isopropylidene-myo-inositol (4) with 6-O-acetyl-4-O-allyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranosyl trichloroacetimidate (6) gave the 4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)- myo-inositol derivative (9) as a mixture of diastereoisomers which could be resolved by chromatography. Likewise alpha-glycosylation of 4 with 6-O-acetyl-2-azido-3-O-benzoyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta- D- galactopyranosyl)-D-glucopyranosyl trichloroacetimidate (10) gave the corresponding pseudotrisaccharide derivative 16 as a mixture of diastereomers which could be resolved partially by chromatography. alpha-Glycosylation of enantiomerically pure 2,3:5,6- (18) and 2,3:4,5-di-O-isopropylidene-1-O-menthoxycarbonyl-myo-inositol (19) with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate (20) gave the pseudodisaccharide derivatives 21 and 22, respectively. Likewise, alpha-glycosylation of 18 with 10 afforded a pseudotrisaccharide derivative (23).

Interplay between cell cycle and autophagy induced by boswellic acid analog

PubMed Central

Pathania, Anup S.; Guru, Santosh K.; Kumar, Suresh; Kumar, Ashok; Ahmad, Masroor; Bhushan, Shashi; Sharma, Parduman R.; Mahajan, Priya; Shah, Bhahwal A.; Sharma, Simmi; Nargotra, Amit; Vishwakarma, Ram; Korkaya, Hasan; Malik, Fayaz

2016-01-01

In this study, we investigated the role of autophagy induced by boswellic acid analog BA145 on cell cycle progression in pancreatic cancer cells. BA145 induced robust autophagy in pancreatic cancer cell line PANC-1 and exhibited cell proliferation inhibition by inducing cells to undergo G2/M arrest. Inhibition of G2/M progression was associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Pre-treatment of cells with autophagy inhibitors or silencing the expression of key autophagy genes abrogated BA145 induced G2/M arrest and downregulation of cell cycle regulatory proteins. It was further observed that BA145 induced autophagy by targeting mTOR kinase (IC50 1 μM), leading to reduced expression of p-mTOR, p-p70S6K (T389), p-4EBP (T37/46) and p-S6 (S240/244). Notably, inhibition of mTOR signalling by BA145 was followed by attendant activation of AKT and its membrane translocation. Inhibition of Akt through pharmacological inhibitors or siRNAs enhanced BA145 mediated autophagy, G2/M arrest and reduced expression of G2/M regulators. Further studies revealed that BA145 arbitrated inhibition of mTOR led to the activation of Akt through IGFR/PI3k/Akt feedback loop. Intervention in IGFR/PI3k/Akt loop further depreciated Akt phosphorylation and its membrane translocation that culminates in augmented autophagy with concomitant G2/M arrest and cell death. PMID:27680387

Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts.

PubMed

Sun, Boqiao; Kantzow, Christina; Bresch, Sven; Castiglione, Kathrin; Weuster-Botz, Dirk

2013-01-01

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCA-the two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens and 3α-hydroxysteroid dehydrogenase (3α-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3α-HSDH, 7β-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3α-HSDH and 7β-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12-keto-UDCA within 4.5 h in a simple batch process on a liter scale. Copyright © 2012 Wiley Periodicals, Inc.

Prevention of DNA damage by L-carnitine induced by metabolites accumulated in maple syrup urine disease in human peripheral leukocytes in vitro.

PubMed

Mescka, Caroline Paula; Wayhs, Carlos Alberto Yasin; Guerreiro, Gilian; Manfredini, Vanusa; Dutra-Filho, Carlos Severo; Vargas, Carmen Regla

2014-09-15

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of L-carnitine (L-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of L-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and L-Car was able to significantly prevent this damage, mainly that due to KIC. Copyright © 2014 Elsevier B.V. All rights reserved.

[Studies on chemical components of Lobelia chinensis].

PubMed

Jiang, Yanyan; Shi, Renbing; Liu, Bin; Wang, Qiuying; Dai, Ying

2009-02-01

To study on the chemical constituents of Lobelia chinensis. The coloumn chromatographic techniques were applied to isolate constituents, and their structures were elucidated by means of spectral data analysis. Sixteen compounds were isolated and identified as daucosterol (1), diosmetin (2), apigenin (3), chrysoeriol (4), loteolin (5), hesperidin (6), loteolin-7-O-beta-D-glucoside (7), apigenin-7-O-beta-D-glucoside (8), linarin (9), diosmin(10), 5,7-dimethoxy-8- hydroxycoumarin (11), palmitinic acid (12), lacceroic acid (13), stearic acid (14), beta-sitosterol (15), daucosterol (16). All of these compouds were obtained from L. chinensis for the first time.

Genetically Epilepsy-Prone Rats Have Increased Brain Regional Activity of an Enzyme Which Liberates Glutamate from N-acetyl-aspartyl-glutamate

DTIC Science & Technology

1992-01-01

DISTRIBUTION C OOt .APPROVED FOR PUPLIC RELEASE: DISTRIBUTION UNLIMITED Ii. A STRA T (Minls.m200oids N-Acetylated-a- 1 n (’ed acidic dip cpL,2ase (N...aspartate (NAA) and the excitatory amino acid , glutamate (CLU). Although there is evidence that NAAG might be a neurotransmitter, this dipoptide could...Genetics; Itippocampus: E-ctlsatlltmt pilepsy-, Glutamate: N-Acetylated-o-1 inked acidic dipeptidasc-: Enrniatic: IIrosz:NAAG: Aspartalc N-Acetylated-a

Androstadienetrione, a boldenone-like component, detected in cattle faeces with GC-MS(n) and LC-MS(n).

PubMed

Poelmans, S; De Wasch, K; Noppe, H; Van Hoof, N; Van de Wiele, M; Courtheyn, D; Gillis, W; Vanthemsche, P; Janssen, C R; De Brabander, H F

2005-09-01

Boldenone (1,4-androstadiene-17-ol-3-one, Bol) has been the subject of a heated debate because of ongoing confusion about its endogenous or exogenous origin when detected in one of its forms in faecal or urine samples from cattle. An expert report was recently written on the presence and metabolism of Bol in various animal species. Androstadienedione (ADD) is a direct precursor of 17beta-boldenone (betaBol). It is a 3,17-dione; ssBol is a 17-ol-3-one. Not much is published on 1,4-androstadiene-3,17-diol, which is a 3,17-diol (ADL). If animals were exposed for a longer period to one of these analytes, a metabolic pathway would be initiated to eliminate these compounds. Similar to recent testosterone metabolism studies in the aquatic invertebrate Neomysis integer, ADD, ssBol and ADL could also be eliminated as hydroxymetabolites after exposure. The presence of 11-keto-steroids or 11-hydroxy-metabolites in faecal samples can interfere with a confirmation method by gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS), after oxidation of corticosteroids with a double bond in the A-ring (e.g. prednisolone or its metabolite prednisone). The presence of androstadienetrione (ADT) in faecal samples of cattle has never been reported. The origin of its presence can be explained through different pathways, which are presented in this paper.

Amino Acid Degradations Produced by Lipid Oxidation Products.

PubMed

Hidalgo, Francisco J; Zamora, Rosario

2016-06-10

Differently to amino acid degradations produced by carbohydrate-derived reactive carbonyls, amino acid degradations produced by lipid oxidation products are lesser known in spite of being lipid oxidation a major source of reactive carbonyls in food. This article analyzes the conversion of amino acids into Strecker aldehydes, α-keto acids, and amines produced by lipid-derived free radicals and carbonyl compounds, as well as the role of lipid oxidation products on the reactions suffered by these compounds: the formation of Strecker aldehydes and other aldehydes from α-keto acids; the formation of Strecker aldehydes and olefins from amines; the formation of shorter aldehydes from Strecker aldehydes; and the addition reactions suffered by the olefins produced from the amines. The relationships among all these reactions and the effect of reaction conditions on them are discussed. This knowledge should contribute to better control food processing in order to favor the formation of desirable beneficial compounds and to inhibit the production of compounds with deleterious properties.

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

PubMed

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49.

PubMed

Tang, Xin; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Song, Yuanda; Chen, Wei

2017-06-21

Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.

Synthesis and cytotoxic activity of some 17-picolyl and 17-picolinylidene androstane derivatives.

PubMed

Djurendić, Evgenija A; Ajduković, Jovana J; Sakač, Marija N; Csanádi, János J; Kojić, Vesna V; Bogdanović, Gordana M; Penov Gaši, Katarina M

2012-08-01

New 17-picolyl and 17-picolinylidene androstane derivatives, 3-10, 15, 18, 19, 22 and 23, were synthesized starting from 17α-picolyl-androst-5-en-3β,17β-diol (1) and 17(Z)-picolinylidene-androst-5-en-3β-ol (2). Reaction of 1 with m-chloroperoxybenzoic acid gives 5α,6α-epoxy N-oxide derivative 3, or, with Jones reagent, 3,6-dione derivative 4; while 17α-picolyl-androst-5-en-3β,4α,17β-triol (5) or 3β,4β,17β-triol (6) derivatives are obtainable from 1 using SeO(2) in dioxane. Base-catalyzed tosyl group elimination from 7 or 9 affords AB conjugated derivatives 8 and 10. Oppenauer oxidation of 1 and 2 yields 4-en-3-one derivatives 11 and 12, which, with H(2)O(2) in 4 M NaOH, affords 4α,5α and 4β,5β-epoxides 13, 14, 16 and 17. New 4-methoxy-3-keto derivatives 15 and 18 were obtained from 13 and 14, or, with methanol in 4 M NaOH, from 16 and 17. Reduction of 11 with NaBH(4) gives 22, which was then acetylated to obtain 23. All new derivatives were screened for antitumor activity against human breast adenocarcinoma ER+, MCF-7; human breast adenocarcinoma ER-, MDA-MB-231; prostate cancer AR-, PC-3; human cervix carcinoma, HeLa; and colon cancer, HT-29 cells; as well as one human non-tumor cell line, MRC-5. Compounds 3, 5, 6, 8, 10, 18, 19 and 22 exhibited significant antitumor activity against MDA-MB-231 breast cancer cells; while 5, 6 and 10 also showed strong cytotoxicity against HT-29. Only compound 19 exhibited significant activity against MCF-7 breast cancer cells. No compounds displayed cytotoxicity against non-tumor MRC-5 cells. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

PubMed

Buchanan, Bob B; Sirevåg, Reidun; Fuchs, Georg; Ivanovsky, Ruslan N; Igarashi, Yasuo; Ishii, Masaharu; Tabita, F Robert; Berg, Ivan A

2017-11-01

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO 2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO 2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO 2 , reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO 2 . Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

[Studies on chemical constituents from leaves of Vaccinium bracteatum].

PubMed

Li, Zeng-Liang; Zhang, Lin; Tian, Jing-Kui; Zhou, Wen-Ming

2008-09-01

To investigate the chemical constituents from the leaves of Vaccinium bracteatum. Many column chromatographic techniques were used for the isolation and separation of chemical constituents. Their structures were elucidated on the basis of spectral analysis and chemical evidences. Twelve compounds were isolated from the plant, and they were identified as chrysoeriol (1), scopoletin (2), trans-p-hydroxycinnamic acid (3), trans-p-hydroxycinnamic acid ethyl ester (4), cafeic acid ethyl ester (5), beta-sitosterol (6), iuteolin (7), quercetin (8), esculetin (9), cafeic acid (10), isolariciresinol-9-O-beta-D-xyloside (11), 10-O-trans-p-coumaroylsandoside (12). Compounds 4, 5, 11, 12 were isolated from the genus Vaccinium for the first time, and compounds 1, 2, 9, 10 were isolated from this plant for the first time.

Effects of epidermal growth factor, interleukin 1 and nitric oxide on prostaglandin production by guinea-pig uterus.

PubMed

Keeble, J E; Poyser, N L

2002-08-01

Initial experiments in the present study investigated the effects of epidermal growth factor (EGF), interleukin 1beta (IL-1beta) and sodium nitroprusside (a nitric oxide donor) on the output of prostaglandins from guinea-pig uterus on day 7 of the oestrous cycle. Superfusion of day 7 guinea-pig uterus in vitro with either EGF or sodium nitroprusside increased the output of PGF(2alpha) and 6-keto-PGF(1alpha), but not of PGE(2). IL-1beta had no effect on the output of these three prostaglandins. EGF still increased the output of PGF(2alpha), but did not increase the output of 6-keto-PGF(1alpha) in a calcium-depleted superfusate. Subsequent experiments investigated the effect of sodium nitroprusside on contractile activity of day 7 guinea-pig uterus. Basal spontaneous activity of both the intact uterus and isolated myometrium superfused in vitro was low. Sodium nitroprusside increased the contractile activity of these tissues two- to fourfold. EGF did not affect the contractile activity of the uterus, indicating that sodium nitroprusside-induced contractions are not due to increased prostaglandin production. Overall, the findings indicate that EGF and nitric oxide may act as mediators in the mechanism by which oestradiol acting on a progesterone-primed uterus stimulates the increase in PGF(2alpha) production by the guinea-pig uterus necessary for luteolysis. Nitric oxide may increase the spontaneous activity of the uterus when this activity is low.

Transition metal-free one-pot cascade synthesis of 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones from biomass-derived levulinic acid under mild conditions.

PubMed

Jha, Amitabh; Naidu, Ajaya B; Abdelkhalik, Ashraf M

2013-11-21

An efficient, environmentally benign, transition-metal free, tandem C-N, C-O bond formation reaction is developed for the synthesis of tricyclic 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones and their homologs from easily available starting materials, including renewable levulinic acid, a keto acid. The reaction of keto acids with methyl chloroformate and variously substituted o-aminobenzyl alcohols using triethylamine as a base in toluene at room temperature gave good to excellent yields. This newly developed protocol was successfully utilized for the synthesis of a variety of polycyclic 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones and related compounds.

[Analysis of constituents of essential oil from the skin of water caltrop].

PubMed

Liang, Rui; Peng, Qi-Jun

2006-01-01

To analyze the constituents of essential oil from the skin of water caltrop. Water steam distillation and GC-MS were used. 58 componds were separated respectively. 56 componds being identified which were 96. 5% of the totle essential oil. Diethyl phthalate, acetamide, N-acetyl-N, N'-1,2-ethanediylbis-, isopropyl palmitate, hexadecanoic acid, Z-11 and octadecanoic acid are the main component of essential oil from the skin of water caltrop.

Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

PubMed

Rižner, Tea Lanišnik; Penning, Trevor M

2014-01-01

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. Copyright © 2013 Elsevier Inc. All rights reserved.

Elaboration of 2-(trifluoromethyl)indoles via a cascade coupling/condensation/deacylation process.

PubMed

Chen, Yu; Wang, Yuji; Sun, Zheming; Ma, Dawei

2008-02-21

CuI/l-proline-catalyzed coupling of 2-halotrifluoroacetanilides with beta-keto esters in anhydrous DMSO under the action of Cs2CO3 at 40-80 degrees C produces polysubstituted 2-(trifluoromethyl)indoles in good to excellent yields. This reaction is suggested to occur via a novel coupling/condensation/deacylation mechanism, and many functional groups are tolerated under these conditions.

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

NASA Technical Reports Server (NTRS)

Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.

1992-01-01

We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

A simple synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid isopropyl ester, a key sugar for the bacterial population living under metallic stress.

PubMed

Grison, Claire M; Renard, Brice-Loïc; Grison, Claude

2014-02-01

2-Keto-3-deoxy-D-erythro-hexonic acid (KDG) is the key intermediate metabolite of the Entner Doudoroff (ED) pathway. A simple, efficient and stereoselective synthesis of KDG isopropyl ester is described in five steps from 2,3-O-isopropylidene-D-threitol with an overall yield of 47%. KDG isopropyl ester is studied as an attractive marker of a functional Entner Doudoroff pathway. KDG isopropyl ester is used to promote growth of ammonium producing bacterial strains, showing interesting features in the remediation of heavy-metal polluted soils. Copyright © 2013 Elsevier Inc. All rights reserved.

Bioactivities and chemical constituents of a Vietnamese medicinal plant Che Vang, Jasminum subtriplinerve Blume (Oleaceae).

PubMed

Ngan, Dai Hue; Hoai, Ho Thi Cam; Huong, Le Mai; Hansen, Poul Erik; Vang, Ole

2008-01-01

Five crude extracts were made from leaves and stems of Jasminum subtriplinerve Blume (Oleaceae) and investigated for antimicrobial, antioxidant and cytotoxic activities. The extractions were done with petroleum ether, ethyl acetate, ethanol, methanol or water. All extracts exhibited anti-bacterial activity except the water fraction. On the other hand, all extracts exhibit antioxidant activity except the petroleum ether fraction using the DPPH radical scavenging assay. Only the petroleum ether fraction showed a cytotoxicity activity against tested cell-lines, Hep-G2 and RD with IC(50) values of 19.2 and 20 microg mL(-1), respectively. From the petroleum ether and ethyl acetate extracts, two triterpenes namely 3beta-acetyl-oleanolic acid and lup-20-en-3beta-ol and a sterol, stigmast-5-en-3beta-ol were isolated. The structure of those compounds were elucidated by spectrometric methods IR, MS, 1D-NMR, 2D-NMR and simulated ACD/NMR spectra. The data presented here indicate that J. subtriplinerve do contain compounds with interesting biological activity.

Isolation and structures of glycoprotein-derived free oligosaccharides from the unfertilized eggs of Scyliorhinus caniculus. Characterization of the sequences galactose(alpha 1-4)galactose(beta 1-3)-N-acetylglucosamine and N-acetylneuraminic acid(alpha 2-6)galactose(beta 1-3)-N-acetylglucosamine.

PubMed

Plancke, Y; Delplace, F; Wieruszeski, J M; Maes, E; Strecker, G

1996-01-15

As previously reported [Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H. & Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K. & Inoue, Y. (1989) J. Biol. Chem. 264, 18520-185261, the unfertilized eggs of two different species of fresh-water fish, Plecoglossus altivelis and Tribodolon hakonensis, contain relatively large amounts of free sialooligosaccharides. These oligosaccharides were found to derive from glycophosphoproteins, owing to the activity of a peptide - N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase [Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1992) J. Biol. Chem. 267, 24287-24296; Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114]. Here we describe a new type of free oligosaccharides, isolated from unfertilized eggs of Scyliorhinus caniculus. From the structural analysis, based upon 1H-NMR spectroscopy, the following glycan units are proposed.[Formula: see text

Effects of different ratios of monounsaturated and polyunsaturated fatty acids to saturated fatty acids on regulating body fat deposition in hamsters.

PubMed

Liao, Fang-Hsuean; Liou, Tsan-Hon; Shieh, Ming-Jer; Chien, Yi-Wen

2010-01-01

Effects of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid consumption on regulating body fat accumulation and body weight gain are controversial between animal and human studies. We designed a 2 x 2 factorial study, with two levels of MUFAs (60% and 30%) and two levels of polyunsaturated-to-saturated fatty acid (P/S) ratio (5 and 3) to prepare four kinds of experimental oils consisting of 60% MUFAs with a high or low P/S ratio (HMHR or HMLR, respectively) or 30% MUFAs with a high or low P/S ratio (LMHR or LMLR, respectively). Thirty-two male golden Syrian hamsters were randomly divided into four groups and fed the experimental diets containing 15% (w/w) fat for 12 wk. No difference was observed in the mean daily food intake. Hamsters fed the LMLR diet had increased weight gain, epididymal and retroperitoneal white adipose tissues, plasma non-esterified fatty acids, insulin, hepatic acetyl coenzyme A carboxylase and malic enzyme activities, and mRNA expressions of peroxisome proliferator-activated receptor-alpha and sterol regulatory element-binding protein-1c among all groups (P < 0.05). Hamsters fed the HMHR diet had lower plasma insulin levels and hepatic acetyl coenzyme A carboxylase activities among groups (P < 0.05) and elevated hepatic acyl coenzyme A oxidase and carnitine palmitoyltransferase-I activities compared with those fed the LMLR diet (P < 0.05). Hamsters fed the LMLR diet had increased weight gain and body fat accumulation, whereas the HMHR diet appeared to be beneficial in preventing white adipose tissue accumulation by decreasing plasma insulin levels and increasing hepatic lipolytic enzyme activities involved in beta-oxidation. 2010 Elsevier Inc. All rights reserved.

Bacterial iron transport: coordination properties of azotobactin, the highly fluorescent siderophore of Azotobacter vinelandii.

PubMed

Palanché, Tania; Blanc, Sylvie; Hennard, Christophe; Abdallah, Mohamed A; Albrecht-Gary, Anne-Marie

2004-02-09

Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin delta, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore. The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids: (L)-Asp-(D)-Ser-(L)-Hse-Gly-(D)-beta-threo-HOAsp-(L)-Ser-(D)-Cit-(L)-Hse-(L)-Hse lactone-(D)-N(delta)-Acetyl, N(delta)-HOOrn. Azotobactin delta has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (N(delta)-Acetyl, N(delta)-HOOrn), one alpha-hydroxycarboxylic function in the middle of the chain (beta-threo-hydroxyaspartic acid), and one catechol group on the chromophore. The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid-base functions of the uncomplexed free siderophore. Strongly negatively charged ferric species were observed at neutral p[H]'s corresponding to a predominant absolute configuration Lambda of the ferric complex in solution as deduced from CD measurements. The presence of an alpha-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins. The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release. Formation and dissociation kinetics of the azotobactin delta ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle. The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin delta complex. Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

PubMed

Uenishi, Yuko; Fujita, Yukiko; Kusunose, Naoto; Yano, Ikuya; Sunagawa, Makoto

2008-02-01

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

Field Observation of Heterogeneous Formation of Dicarboxylic acids, Keto-carboxylic acids, α-Dicarbonyls and Nitrate in Xi'an, China during Asian dust storm periods

NASA Astrophysics Data System (ADS)

Wang, G.; Wang, J.; Ren, Y.; Li, J.

2015-12-01

To understand the formation mechanism of secondary organic aerosols (SOA) on dust surfaces, this study investigated the concentrations and compositions of dicarboxylic acids (C2-C11), keto-carboxylic acids (C3-C7), α-dicarbonyls and inorganic ions in size-segregated aerosols (9-stages) collected in Xi'an, China during the nondust storm and dust storm periods of 2009 and 2011. During the events the ambient particulate dicarboxylic acids were 932-2240 ng m-3, which are comparable and even higher than those in nondust periods. Molecular compositions of the above SOA are similar to those in nondust periods with oxalic acid being the leading species. In the presence of the dust storms, all the above mentioned SOA species in Xi'an were predominantly enriched on the coarse particles (>2.1μm), and oxalic acid well correlated with NO3- (R2=0.72, p<0.001) rather than SO42-.This phenomenon differs greatly from the SOA in any other nondust period that is characterized by an enrichment of oxalic acid in fine particles and a strong correlation of oxalic acid with SO42-. Our results further demonstrate that NO3- in the dust periods in Xi'an was mostly derived from secondary oxidation, whereas SO42- during the events was largely derived from surface soil of Gobi deserts. We propose a formation pathway to explain these observations, in which nitric acid and/or nitrogen oxides react with dust to produce Ca(NO3)2 and form a liquid phase on the surface of dust aerosols via water vapor-absorption of Ca(NO3)2, followed by a partitioning of the gas-phase water-soluble organic precursors (e.g.,glyoxal and methylglyoxal) into the aqueous-phase and a subsequent oxidation into oxalic acid. To the best of our knowledge, we found for the first time the enrichment of glyoxal and methylglyoxal on dust surface. Our data suggest an important role of nitrate in the heterogeneous formation process of SOA on the surface of Asian dust.

Conversion of Human Steroid 5[beta]-Reductase (AKR1D1) into 3[beta]-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: Example of Perfect Enzyme Engineering

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

Chen, Mo; Drury, Jason E.; Christianson, David W.

2012-10-10

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5{beta}-reduction of {Delta}{sup 4}-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5{beta}-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5{alpha}-configuration and, unlike most of the AKR1Cmore » enzymes, shows a dominant stereochemical preference to act as a 3{beta}-HSD as opposed to a 3{alpha}-HSD. The catalytic efficiency achieved for 3{beta}-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5{beta}-dihydrotestosterone, and {Delta}{sup 4}-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the {Delta}{sup 4}-double bond and confers 3{beta}-HSD activity on the 5{beta}-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its {alpha}-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.« less

Targeting Inflammatory Pathways by Triterpenoids for Prevention and Treatment of Cancer

PubMed Central

Yadav, Vivek R.; Prasad, Sahdeo; Sung, Bokyung; Kannappan, Ramaswamy; Aggarwal, Bharat B.

2010-01-01

Traditional medicine and diet has served mankind through the ages for prevention and treatment of most chronic diseases. Mounting evidence suggests that chronic inflammation mediates most chronic diseases, including cancer. More than other transcription factors, nuclear factor-kappaB (NF-κB) and STAT3 have emerged as major regulators of inflammation, cellular transformation, and tumor cell survival, proliferation, invasion, angiogenesis, and metastasis. Thus, agents that can inhibit NF-κB and STAT3 activation pathways have the potential to both prevent and treat cancer. In this review, we examine the potential of one group of compounds called triterpenes, derived from traditional medicine and diet for their ability to suppress inflammatory pathways linked to tumorigenesis. These triterpenes include avicins, betulinic acid, boswellic acid, celastrol, diosgenin, madecassic acid, maslinic acid, momordin, saikosaponins, platycodon, pristimerin, ursolic acid, and withanolide. This review thus supports the famous adage of Hippocrates, “Let food be thy medicine and medicine be thy food”. PMID:22069560

The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

NASA Technical Reports Server (NTRS)

Morowitz, Harold; Peterson, Eta; Chang, Sherwood

1995-01-01

This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

Smad Acetylation: A New Level of Regulation in TGF-Beta Signaling

DTIC Science & Technology

2005-07-01

Our lab has determined that Smad2, but not Smad3 , can be acetylated by the acetyltransferase protein p300 in vivo and in vitro. The residues...terminal of Smad2 and Smad3 , allowing oligomerization with the common mediator Smad4 [9-10]. The Smad2/3/4 complex then translocates to the nucleus where...Smad2, but not Smad3 , could be acetylated in a p300 dependent manner. Both in vivo and in vitro data support the conclusion that only Smad2 could be

Further drimane sesquiterpenes from Drimys brasiliensis stem barks with cytotoxic potential.

PubMed

Fratoni, Eduarda; Claudino, Vanessa Duarte; Yunes, Rosendo Augusto; Franchi, Gilberto C; Nowill, Alexandre E; Filho, Valdir Cechinel; Monache, Franco Delle; Malheiros, Angela

2016-07-01

Drimys brasiliensis Miers (Winteraceae) is used in folk medicine for the treatment of cancer. Its anti-tumor activity has been demonstrated in vitro models using extracts and isolated compounds. This study investigates the cytotoxic effects of stem bark extracts of D. brasiliensis as well as isolated compounds that may be responsible for the activitys and evaluates them in leukemia cells. The stem bark extract were subjected to column chromatography, and the structures of compounds were elucidated based on spectroscopic methods by using NMR and infrared spectroscopy and GC/MS. The cytotoxicity of the isolated compounds was evaluated in chronic myeloid (K562) and acute B lymphoblastic (Nalm6) leukemia cells using tetrazolium assay (MTT). Two new compounds were isolated 1β-O-p-methoxy-E-cinnamoyl-5α-keto-11α-enol-albicanol (1a) and the isomer 1β-O-p-methoxy-E-cinnamoyl-5α-keto-11β-enol-albicanol (1b) and 1β-O-p-methoxy-E-cinnamoyl-isodrimeninol (2). The known compounds polygonal acid (3a) and the isomer isopolygonal acid (3b), fuegin (4a) and the isomer epifuegin (4b), the mixture drimanial (5) and 1β-O-(p-methoxy-E-cinnamoyl)-6α-hydroxypolygodial (6) were also isolated. The drimanes (1-4) and drimanial (5), 1β-(p-coumaroyloxy)-polygodial (7), 1β-(p-methoxycinnamoyl)-polygodial (8), and polygodial (9) isolated previously were assessed in tumor cells. The IC50 values were between 3.56 and 128.91 μM. 1-β-(p-cumaroiloxi)-polygodial showed the best result with IC50 8.18 and 3.56 μM by K562 and Nalm6, respectively. The chloroform extract of the stem bark of D. brasiliensis is a great source of drimane sesquiterpenes. Our experimental data suggest that drimanes are responsible for cytotoxicity activity demonstrated by this species, especially those with the aldehyde group linked to carbons C-11 and C-12.

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems.

PubMed

Grissom, C B; Cleland, W W

1987-12-18

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.

Sesquiterpenoids from roots of Taraxacum laevigatum and Taraxacum disseminatum.

PubMed

Zielińiska, K; Kisiel, W

2000-08-01

Chromatographic separation of ethanolic root extracts of Taraxacum laevigatum and Taraxacum disseminatum afforded a total of eight germacrane- and eudesmane-type sesquiterpenoids. including new compounds, 1beta,3beta,6alpha-trihydroxy-4alpha( 15)-dihydrocostic acid methyl ester and its 1-O-beta-glucopyranoside. Their structures were established by spectroscopic analyses. In addition, the structure of 4alpha(15), 11beta(13)-tetrahydroridentin B-1-O-beta-glucopyranoside was elucidated by extensive NMR studies.

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

PubMed

Takenaka, Shinji; Ozeki, Takahiro; Tanaka, Kosei; Yoshida, Ken-Ichi

2017-11-01

To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126 Leu, 132 Leu, and 135 Lys were implicated in the binding of phosphopantothenic acid, and 100 Tyr and 131 Lys in that of adenosyl biphosphate. The data supported the participation of 83 Glu and 133 Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

Effects of glucogenic and ketogenic feeding strategies on splanchnic glucose and amino acid metabolism in postpartum transition Holstein cows.

PubMed

Larsen, M; Kristensen, N B

2012-10-01

Nine periparturient Holstein cows catheterized in major splanchnic vessels were used in a complete randomized design with repeated measurements to investigate effects of glucogenic and ketogenic feeding strategies on splanchnic metabolism of glucose and amino acids. At parturition, cows were assigned to 1 of 3 feeding strategies: a glucogenic diet (GLCG) based on sodium hydroxide treated wheat grain (56.5% of diet dry matter); a ketogenic diet (KETO) based on fodder beets (40.5% of diet dry matter); or an alfalfa-glucogenic strategy (ALF-GLCG) supplying 100% alfalfa (Medicago sativa L.) haylage at the day of parturition, followed by a 6-d linear shift to the GLCG diet. Samples were obtained 14 d before expected parturition as well as at 4, 15, and 29 d in milk (DIM). The net portal release of glucose was greatest with GLCG, reflecting the higher intake of ruminal escape starch with GLCG, as compared with a lower starch intake with KETO. Postpartum, the portal recovery of feed starch was greater (28 ± 3%, mean ± SEM) with KETO as compared with GLCG (15 ± 4%). At 4 DIM, the net hepatic release of glucose was greatest with KETO and least with ALF-GLCG, whereafter it increased as lactation progressed with ALF-GLCG and GLCG, but not with KETO. The high alfalfa haylage allowance at 4 DIM with the ALF-GLCG treatment induced the lowest net release of nutrients from the splanchnic tissues at 4 DIM. The hepatic removal of lactate as percent of total influx (mean ± SEM) increased from 27 ± 3% prepartum to 56 ± 3% at 4 DIM. The hepatic removal of lactate as percent of net portal release increased from 144 ± 10% prepartum to 329 ± 17% at 4 DIM with ALF-GLCG and KETO as compared with 242 ± 20% in GLCG. No clear evidence for an amino acid sparing effect in splanchnic tissues from increasing small intestinal glucose absorption was observed. In conclusion, the glucogenic feeding strategy induced the highest glucogenic status among the tested feeding strategies due to greater release of glucose from splanchnic tissues. In contrast, the immediate postpartum high allowance of alfalfa haylage provided the lowest amount of nutrients from the splanchnic tissues, inducing low glucogenic status, pointing to the importance of allocating highly digestible diets to postpartum transition cows. Salvaging glucogenic carbon via interorgan transfer of lactate from peripheral tissues supported the immediate postpartum incremental increase in hepatic glucose release rather than hepatic catabolism of amino acids. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Therapeutic potentials of Crataegus azarolus var. eu- azarolus Maire leaves and its isolated compounds.

PubMed

Abu-Gharbieh, Eman; Shehab, Naglaa Gamil

2017-04-18

Hyperglycemia is a complicated condition accompanied with high incidence of infection and dyslipidemia. This study aimed to explore the phyto-constituents of Crataegus azarolus var. eu- azarolus Maire leaves, and to evaluate the therapeutic potentials particularly antimicrobial, antihyperglycemic and antihyperlipidemic of the extract and the isolated compound (3β-O-acetyl ursolic acid). Total phenolics and flavonoidal contents were measured by RP-HPLC analysis. Free radicals scavenging activity of different extraction solvents was tested in-vitro on DPPH free radicals. The antimicrobial activity of the ethanolic extract and its fractions as well as the isolated compounds were evaluated in-vitro on variable microorganisms. Animal models were used to evaluate the antihyperglycemic and antihyperlipidemic activities of the ethanolic extract along with the isolated compound (3β-O acetyl ursolic acid). RP- HPLC analysis of the phenolics revealed high content of rutin, salicylic and ellagic acids. Six compounds belonging to triterpenes and phenolics were isolated from chloroform and n-butanol fractions namely: ursolic acid, 3β-O-acetyl ursolic acid, ellagic acid, quercetin 3-O-β methyl ether, rutin and apigenin7-O-rutinoside. Ethanolic extract showed the highest DPPH radical scavenger activity compared to other solvents. Ethanolic extract, hexane fraction, ursolic acid, 3β-O acetyl ursolic acid and quercetin 3-O-methyl ether showed variable antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Administration of the ethanolic extract or 3β-O acetyl ursolic acid orally to the mice reduced blood glucose significantly in a time- and dose-dependent manner. Ethanolic extract significantly reduced LDL-C, VLDL-C, TC and TG and increased HDL-C in rats. Ethanolic extract and 3β-O acetyl ursolic acid reduced in-vitro activity of pancreatic lipase. This study reveals that Crataegus azarolus var. eu- azarolus Maire has the efficiency to control hyperglycemia with its associated complications. This study is the first to evaluate antihyperglycemic and antihyperlipidemic potentials of 3β-O acetyl ursolic acid.

A combined experimental and theoretical study of the supramolecular self-assembly of the natural benzopyran 2,2-dimethyl-3-hydroxy-6-acetyl-chromane and its isomeric benzofuran 10,11-dihydro-10-hydroxytremetone

NASA Astrophysics Data System (ADS)

Gil, Diego M.; Lizarraga, E.; Echeverría, G. A.; Piro, O. E.; Catalán, C. A. N.; Ben Altabef, A.

2017-10-01

Epoxidation of 4HMBA, the main metabolite of the medicinal plant Sencecionutans, produces an unstable epoxide eventually giving rise to a mixture of four derivatives, three of them previously reported as natural products. The epoxide product easily undergoes an intra-molecular attack of the phenolic hydroxyl against the epoxide group carbons to produce either a benzofuran or a chromane derivative. When dissolved in methanol-water mixture at room temperature the epoxide is completely solvolyzed to give the corresponding diol (hydrolysis) or vicinal hydroxyl-methoxy (methanolysis) derivative. All the compounds involved in the above reactions were characterized by IR, Raman, H NMR and UV-vis spectroscopies, and by mass spectrometry. Density functional theory (DFT) computations were used to optimize the structure conformations. The optimized structures were further subjected to a Natural Bond Orbital (NBO) and electrostatic potentials analysis. The crystal structures of the title compounds (for short, 3 and 4 respectively) were determined by X-ray diffraction methods. Compound 3 crystallizes in the triclinic P-1 space group with a = 6.4289 (6) Å, b = 8.7120 (6) Å, c = 10.952 (1) Å, α = 92.280 (7)°, β = 95.738 (7)°, γ = 103.973 (7)°, and Z = 2 molecules per unit cell and 4 in the monoclinic P21/c space group with a = 11.2891 (6) Å, b = 9.1902 (4) Å, c = 12.4272 (7) Å. Β = 113.689 (7)°, and Z = 4. In 3 neighboring molecules are linked to each other by OH⋯O (keto) bonds giving rise to a polymeric structure. In 4 the OH group is a bifurcate H-bond donor. It forms a weak intra-molecular OH⋯O (furan) bond and also a much stronger inter-molecular Osbnd H⋯O (keto) bond giving rise to a zig-zag polymeric structure. A detailed analysis of the solid state molecular interactions of compounds 3 and 4 has been performed using Hirshfeld surface analysis and their associated 2D fingerprint plots.

Improved training tolerance by supplementation with α-Keto acids in untrained young adults: a randomized, double blind, placebo-controlled trial

PubMed Central

2012-01-01

Background Exercise causes a variety of physiological and metabolic changes that can in turn reduce exercise tolerance. One of the potential mechanisms responsible for fatigue is “exercise-induced hyperammonemia”. Previous studies have shown that supplementation with amino acids can increase training tolerance. The α-keto acids are biochemical analogs of amino acids and can be converted to amino acids through transamination, thus reducing the cellular ammonia level. This double blind, placebo-controlled study was designed to investigate the effects of α-keto acid supplementation (KAS) on training tolerance, training effect, and stress-recovery state. Methods Thirty-three untrained young male adults underwent four weeks of training (5 sessions/week; 30 minutes running at the individual anaerobic threshold followed by 3 x 3 minute sprints/each session). Throughout the 4 weeks of training and one week of recovery, subjects took α-ketoglutarate (AKG group, 0.2 g/kg/d, n = 9), branched-chain keto acids (BCKA group, 0.2 g/kg/d, n = 12) or isocaloric placebo (control group, n = 12) daily. Results The 4th week training volume, maximum power output and muscle torque were higher in the AKG group (175 ± 42 min, 412 ± 49 Watts and 293 ± 58 Newton meters, respectively, P<0.05) and the BCKA group (158 ± 35, 390 ± 29 and 273 ± 47, P<0.05) than in the control group (92 ± 70, 381 ± 67 and 233 ± 43). The general stress and emotional exhaustion as assessed by the rest-stress-questionnaire-sport after the 3rd week of training increased significantly in the control group (P<0.05), but not in the KAS groups. Conclusions Under KAS, subjects could bear a higher training volume and reach a higher power output and peak muscle torque, accompanied by a better stress-recovery-state. Thus, KAS improves exercise tolerance and training effects along with a better stress-recovery state. Whether the improved training tolerance by KAS is associated with effects on ammonia homeostasis requires further observation. PMID:22857787

A beta-keto ester as a novel, efficient, and versatile ligand for copper(I)-catalyzed C-N, C-O, and C-S coupling reactions.

PubMed

Lv, Xin; Bao, Weiliang

2007-05-11

Employing ethyl 2-oxocyclohexanecarboxylate as a novel, efficient, and versatile ligand, the copper-catalyzed coupling reactions of various N/O/S nucleophilic reagents with aryl halides could be successfully carried out under mild conditions. A variety of products including N-arylamides, N-arylimidazoles, aryl ethers, and aryl thioethers were synthesized in good to excellent yields.

Scopadulciol, an inhibitor of gastric H+, K(+)-ATPase from Scoparia dulcis, and its structure-activity relationships.

PubMed

Hayashi, T; Asano, S; Mizutani, M; Takeguchi, N; Kojima, T; Okamura, K; Morita, N

1991-01-01

A new tetracyclic diterpenoid, scopadulciol [3], together with 6-methoxybenzoxazolinone, glutinol, and acacetin, was isolated from the 70% EtOH extract of Scoparia dulcis collected in Taiwan. Its structure was elucidated to be 6 beta-benzoyl-12-methyl-13-oxo-9(12)a,9(12)b-dihomo-18-podocarpanol on the basis of spectral data. It mildly inhibited hog gastric H+, K(+)-ATPase. Examination of the inhibitory activities of derivatives of scopadulcic acid B [2], including 3, revealed that methylation of the carboxyl group and introduction of an acetyl group or oxime at C-13 or C-18 markedly enhanced the inhibitory activity, while debenzoylation reduced the activity. Among the 30 compounds tested, compound 12, a methyl ester of scopadulcic acid B [2], showed the most potent activity.

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri).

PubMed

Islam, A; Persson, B; Zaidi, Z H; Jörnvall, H

1989-08-01

The primary structure of Rose-ringed Parakeet hemoglobin beta-chain was established, completing the analysis of this hemoglobin. Comparison with other avian beta-chains show variations smaller than those for the corresponding alpha-chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform beta-chain, and a total of 35 positions are affected by differences among all avian beta-chains analyzed (versus 61 for the alpha-chains). At three positions, the Psittacula beta-chain has residues unique to this species. Three alpha 1 beta 1 contacts are modified, by substitutions at positions beta 51, beta 116, and beta 125.

Purification and characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52.

PubMed

Masuda, N; Oda, H; Tanaka, H

1983-01-04

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.

Sesquiterpene glucosides from anti-leukotriene B4 release fraction of Taraxacum officinale.

PubMed

Kashiwada, Y; Takanaka, K; Tsukada, H; Miwa, Y; Taga, T; Tanaka, S; Ikeshiro, Y

2001-01-01

Chemical examination of the MeOH extract of the root of Taraxacum officinale, which exhibited inhibitory activity on the formation of leukotriene B4 from activated human neutrophils, has resulted in the isolation of 14-O-beta-D-glucosyl-11,13-dihydro-taraxinic acid (1) and 14-O-beta-D-glucosyl-taraxinic acid (2). The absolute stereostructure of 1 has been established by X-ray chrystallographic examination.

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

PubMed

Lee, F J; Lin, L W; Smith, J A

1988-10-15

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

Urinary biomarkers of oxidative damage in Maple syrup urine disease: the L-carnitine role.

PubMed

Guerreiro, Gilian; Mescka, Caroline Paula; Sitta, Angela; Donida, Bruna; Marchetti, Desirèe; Hammerschmidt, Tatiane; Faverzani, Jessica; Coelho, Daniella de Moura; Wajner, Moacir; Dutra-Filho, Carlos Severo; Vargas, Carmen Regla

2015-05-01

Maple syrup urine disease (MSUD) is a disorder of branched-chain amino acids (BCAA). The defect in the branched-chain α-keto acid dehydrogenase complex activity leads to an accumulation of these compounds and their corresponding α-keto-acids and α-hydroxy-acids. Studies have shown that oxidative stress may be involved in neuropathology of MSUD. L-carnitine (L-car), which has demonstrated an important role as antioxidant by reducing and scavenging free radicals formation and by enhancing the activity of antioxidant enzymes, have been used in the treatment of some metabolic rare disorders. This study evaluated the oxidative stress parameters, di-tyrosine, isoprostanes and antioxidant capacity, in urine of MSUD patients under protein-restricted diet supplemented or not with L-car capsules at a dose of 50 mg kg(-1) day(-1). It was also determined urinary α-keto isocaproic acid levels as well as blood free L-car concentrations in blood. It was found a deficiency of carnitine in patients before the L-car supplementation. Significant increases of di-tyrosine and isoprostanes, as well as reduced antioxidant capacity, were observed before the treatment with L-car. The L-car supplementation induced beneficial effects on these parameters reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary of α-ketoisocaproic acid after 2 months of L-car treatment, compared to control group. In conclusion, our results suggest that L-car may have beneficial effects in the treatment of MSUD by preventing oxidative damage to the cells and that urine can be used to monitorize oxidative damage in patients affected by this disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Acute supplementation with keto analogues and amino acids in rats during resistance exercise.

PubMed

de Almeida, Rosemeire Dantas; Prado, Eduardo Seixas; Llosa, Carlos Daniel; Magalhães-Neto, Anibal; Cameron, Luiz-Claudio

2010-11-01

During exercise, ammonia levels are related to the appearance of both central and peripheral fatigue. Therefore, controlling the increase in ammonia levels is an important strategy in ameliorating the metabolic response to exercise and in improving athletic performance. Free amino acids can be used as substrates for ATP synthesis that produces ammonia as a side product. Keto analogues act in an opposite way, being used to synthesise amino acids whilst decreasing free ammonia in the blood. Adult male rats were divided into four groups based on receiving either keto analogues associated with amino acids (KAAA) or a placebo and resistance exercise or no exercise. There was an approximately 40% increase in ammonaemia due to KAAA supplementation in resting animals. Exercise increased ammonia levels twofold with respect to the control, with a smaller increase (about 20%) in ammonia levels due to exercise. Exercise itself causes a significant increase in blood urea levels (17%). However, KAAA reduced blood urea levels to 75% of the pre-exercise values. Blood urate levels increased 28% in the KAAA group, independent of exercise. Supplementation increased glucose levels by 10% compared with control animals. Exercise did not change glucose levels in either the control or supplemented groups. Exercise promoted a 57% increase in lactate levels in the control group. Supplementation promoted a twofold exercise-induced increase in blood lactate levels. The present results suggest that an acute supplementation of KAAA can decrease hyperammonaemia induced by exercise.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

PubMed Central

Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael; Sethi, Amit

2016-09-13

Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

PubMed

Furukawa, Ayako; Tada-Oikawa, Saeko; Kawanishi, Shosuke; Oikawa, Shinji

2007-01-01

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Further sesquiterpenoids and phenolics from Taraxacum officinale.

PubMed

Kisiel, W; Barszcz, B

2000-06-01

Five germacrane- and guaiane-type sesquiterpene lactones, including two previously described taraxinic acid derivatives, were isolated from the roots of Taraxacum officinale, together with benzyl glucoside, dihydroconiferin, syringin and dihydrosyringin. The other three lactones were identified as 11beta, 13-dihydrolactucin, ixerin D and ainslioside. Moreover, the stereochemistry at C-11 in dihydrotaraxinic acid was assigned.

Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.

PubMed

Sun, Renhua; Zheng, Heng; Fang, Zhengzhi; Yao, Wenbing

2010-01-01

The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations. Copyright 2009 Elsevier Inc. All rights reserved.

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes.

PubMed

Alicigüzel, Y; Aslan, M

2004-09-01

In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species.

Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer's disease mouse model.

PubMed

Ricobaraza, Ana; Cuadrado-Tejedor, Mar; Pérez-Mediavilla, Alberto; Frechilla, Diana; Del Río, Joaquin; García-Osta, Ana

2009-06-01

Chromatin modification through histone acetylation is a molecular pathway involved in the regulation of transcription underlying memory storage. Sodium 4-phenylbutyrate (4-PBA) is a well-known histone deacetylase inhibitor, which increases gene transcription of a number of genes, and also exerts neuroprotective effects. In this study, we report that administration of 4-PBA reversed spatial learning and memory deficits in an established mouse model of Alzheimer's disease (AD) without altering beta-amyloid burden. We also observed that the phosphorylated form of tau was decreased in the AD mouse brain after 4-PBA treatment, an effect probably due to an increase in the inactive form of the glycogen synthase kinase 3beta (GSK3beta). Interestingly, we found a dramatic decrease in brain histone acetylation in the transgenic mice that may reflect an indirect transcriptional repression underlying memory impairment. The administration of 4-PBA restored brain histone acetylation levels and, as a most likely consequence, activated the transcription of synaptic plasticity markers such as the GluR1 subunit of the AMPA receptor, PSD95, and microtubule-associated protein-2. The results suggest that 4-PBA, a drug already approved for clinical use, may provide a novel approach for the treatment of AD.

METHOD FOR PREPARING NORMORPHINE

DOEpatents

Rapoport, H.; Look, M.

1959-06-01

An improved method is presented for producing normorphine from morphine. Morphine as the starting material is acetylated by treatment with acetylating agents to produce di-acetyl morphine (heroin). The acetylated compound is reacted with cyanating agents to produce di-acetyl-cyanonormorphine (cyanonorheroin). The di-acetyl-cyanonormorphine compound is then treated in accordance with the improved hydrolysis reactions of the present invention in which concentrated hydrochloric acid is employed for a limited time period to hydrolyze the acetyl group therefrom forming cyanonormorphine. Subsequently, the reaction mixture is diluted and hydrolysis of the cyano groups from the cyanonormorphine is effected with a longer contact time with dilute hydrochloric acid thereby producing normorphine. A high over-all conversion and production of a high purity product which may be radioactlvely labeled, if desired, is obtained by operation of the process.

Translocation of radiolabeled indole-3-acetic acid and indole-3-acetyl-myo-inositol from kernel to shoot of Zea mays L

NASA Technical Reports Server (NTRS)

Chisnell, J. R.; Bandurski, R. S.

1988-01-01

Either 5-[3H]indole-3-acetic acid (IAA) or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[3H]indole-3-acetic acid or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.

Two new triterpenoid glycosides isolated from Aesculus assamica GRIFF.

PubMed

Liu, Hongwei; Zhang, Xue; Gao, Hao; Wang, Nali; Jin, Sanlin; Cai, Bin; Yao, Xinsheng; Cai, Guoping

2005-10-01

Phytochemical study of the ethanol extract of the seeds of Aesculus assamica led to the isolation of two new triterpenoid saponins. The structure of the new compounds were elucidated on the basis of spectral data to be 28-O-acetyl-21-O-(4-O-angeloyl)-6-deoxy-beta-glucopyranosyl-3-O-[beta-glucopyranosyl(1-2)-O-[beta-glucopyranosyl(1-4)]-beta-glucuronopyranosyl]protoaescigenin (1), and 21-O-(4-O-angeloyl)-6-deoxy-beta-glucopyranosyl-3-O-[beta-glucopyranosyl(1-2)-O-[beta-glucopyranosyl(1-4)]-beta-glucuronopyranosyl]protoaescigenin (2). Their in vitro bioactivity against plant pathogenic fungus Pyricularia oryzae and cytotoxicity against K562 and HCT-15 cell lines were evaluated.

Peroxisomal fatty acid oxidation as detected by H2O2 production in intact perfused rat liver.

PubMed Central

Foerster, E C; Fährenkemper, T; Rabe, U; Graf, P; Sies, H

1981-01-01

1. H2O2 formation associated with the metabolism of added fatty acids was quantitatively determined in isolated haemoglobin-free perfused rat liver (non-recirculating system) by two different methods. 2. Organ spectrophotometry of catalase Compound I [Sies & Chance (1970) FEBS Lett. 11, 172-176] was used to detect H2O2 formation (a) by steady-state titration with added hydrogen donor, methanol or (b) by comparison of fatty-acid responses with those of the calibration compound, urate. 3. In the use of the peroxidatic reaction of catalase, [14C]methanol was added as hydrogen donor at an optimal concentration of 1 mM in the presence of 0.2 mM-L-methionine, and 14CO2 production rates were determined. 4. Results obtained by the different methods were similar. 5. The yield of H2O2 formation, expressed as the rate of H2O2 formation in relation to the rate of fatty-acid supply, was less than 1.0 in all cases, indicating that, regardless of chain length, less than one acetyl unit was formed per mol of added fatty acid by the peroxisomal system. In particular, the standard substrate used with isolated peroxisomal preparations (C16:0 fatty acid) gave low yield (close to zero). Long-chain monounsaturated fatty acids exhibit a relatively high yield of H2O2 formation. 6. The hypolipidaemic agent bezafibrate led to slightly increased yields for most of the acids tested, but the yield with oleate was decreased to one-half the original yield. 7. It is concluded that in the intact isolated perfused rat liver the assayable capacity for peroxisomal beta-oxidation is used to only a minor degree. However, the observed rates of H2O2 production with fatty acids can account for a considerable share of the endogenous H2O2 production found in the intact animal. PMID:7317011

A DIRECT ROUTE TO ACYLHYDROQUINONES FROM ALPHA-KETO ACIDS AND ALPHA-CARBOXAMIDO ACIDS. (R825330)

EPA Science Inventory

Abstract

The reaction of quinones with in situ generated acyl- or carboxamido radicals provides a direct route to the synthesis of acylhydroquinones not accessible by the photochemical reaction of quinones with aldehydes.

Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum.

PubMed

Chen, Zhuo; Luo, Ling; Chen, Runfa; Hu, Hanhua; Pan, Yufang; Jiang, Haibo; Wan, Xia; Jin, Hu; Gong, Yangmin

2018-03-01

N ε -lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD + -dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and Active Stie Residues of Pg1D, an N-Acetyltransferase from the Bacillosamine Synthetic Pathway Required for N-Glycan Synthesis in Campylobacter jejuni

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

Rangarajan,E.; Ruane, K.; Sulea, T.

2008-01-01

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2, 4-diacetamido-2, 4,6-trideoxy-d-Glc (QuiNAc4NAc or N, N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2, 4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Angstroms resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal {alpha}/{beta}-domain and a C-terminal left-handed {beta}-helix. Few structural differencesmore » accompany CoA binding, except in the C-terminal region following the {beta}-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the {beta}-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an 'L-shape', compared with the 'in-line' orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.« less

Aromatic amine metabolism: immunochemical relationships of N-acetyltransferase and N,O-acyltransferase

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

Land, S.; Allaben, W.T.; King, C.M.

1986-05-01

Mutagenic and carcinogenic aromatic amines are acetylated in most organisms. Acetyl CoA and arylhydroxamic acids can serve as acetyl donors for N-Acetylation of amines to yield stable amides, or by O-acetylation of hydroxylamine derivatives to produce reactive metabolites that can react covalently with nucleic acid. Polyclonal antibodies against rat arylhydroxamic acid, N,O-acyltransferase (AHAT) have been compared for their abilities to react with this enzyme and the acetyl CoA-dependent N-acetyltransferase (NAT) of the rat, rabbit, hamster, mouse and human. Liver cytosols were treated with increasing quantities of antibodies from immune or control rabbits. Immune complexes were removed by treatment with proteinmore » A-Sepharose before assay of nucleic acid adduct formation by AHAT activation of N-hydroxy-2-acetylaminofluorene and the acetylation of 2-aminofluorene by NAT. Both rat activities, the AHAT of the hamster and the NAT of the mouse and human were removed by this treatment. No decrease in NAT activity of hamster, or of either rabbit cytosol activity was observed. Neither mouse nor human liver has appreciable AHAT activity. These data support the idea that AHAT and NAT of rat, AHAT of hamster and NAT of mouse and human liver are immunochemically related, but that NAT of the hamster is an immunochemically distinct peptide.« less

Aspirin increases mitochondrial fatty acid oxidation

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

Uppala, Radha; Dudiak, Brianne; Beck, Megan E.

The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse themore » mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. - Highlights: • Aspirin increases mitochondrial—but inhibits peroxisomal—fatty acid oxidation. • Aspirin acetylates mitochondrial proteins including fatty acid oxidation enzymes. • SIRT3 does not influence the effect of aspirin on fatty acid oxidation. • Increased fatty acid oxidation is likely due to altered mitochondrial morphology and respiration.« less

{beta}-Catenin stabilization imparts crypt progenitor phenotype to hyperproliferating colonic epithelia

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

Sellin, Joseph H.; Wang Yu; Singh, Pomila

2009-01-01

Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we provide mechanistic basis of changes in {beta}-catenin/APC/CKI{epsilon} leading to progression and/or regression of hyperplasia in vivo. In response to CR-induced TMCH, crypt lengths increased significantly between days 6-27 post-infection, followed by a steep decline by day 34. {beta}-Cat{sup 45}/total {beta}-catenin were elevated on day 1 post-infection, preceding changes in crypt length, and persisted for 27 days before declining by day 34. Importantly, cellular CKI{epsilon} and {beta}-catenin co-immunoprecipitated and exhibited remarkable parallel changes in kinetics during hyperplasia/regression phases. {beta}-catenin, phosphorylated at Ser33,37 and Thr41 ({beta}-cat{sup 33,37/41}), was low tillmore » day 12, followed by gradual increase until day 27 before declining by day 34. GSK-3{beta} exhibited significant Ser{sup 9}-phosphorylation/inactivation at days 6-12 with partial recovery at days 27-34. Wild type (wt) APC (p312) levels increased at day 6 with transient proteolysis/truncation to p130 form between days 12 and 15; p312 reappeared by day 19 and returned to baseline by day 34. The kinetics of {beta}-Cat{sup 45}/{beta}-catenin nuclear accumulation and acetylation (Ac-{beta}-Cat{sup Lys49}) from days 6 to 27, followed by loss of phosphorylation/acetylation by day 34 was almost identical; Tcf-4 co-immunoprecipitated with {beta}-Cat{sup 45}/{beta}-catenin and localized immunohistochemically to {beta}-Cat{sup 41/45}-positive regions leading to elevated cyclin D1 expression, during the hyperproliferative, but not regression phases of TMCH. CKI{epsilon} mediated phosphorylation of {beta}-Cat{sup 45}, resulting in stabilization/nuclear translocation of {beta}-Cat{sup 45} may be critical for maintaining proliferation at days 6-27. Reversal of GSK-3{beta} phosphorylation and APC changes may be equally critical during the regression phase from days 27 to 34.« less

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

PubMed Central

Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

2017-01-01

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

Asymmetric synthesis of all-carbon benzylic quaternary stereocenters via Cu-catalyzed conjugate addition of dialkylzinc reagents to 5-(1-arylalkylidene) Meldrum's acids.

PubMed

Fillion, Eric; Wilsily, Ashraf

2006-03-08

The asymmetric synthesis of all-carbon benzylic quaternary stereocenters has been successfully achieved through copper-catalyzed addition of dialkylzinc reagents to 5-(1-arylalkylidene) and 5-(dihydroindenylidene) Meldrum's acids in the presence of phosphoramidite ligand. The resulting benzyl-substituted Meldrum's acids and 1,1-disubstituted indanes were obtained in good yields and up to 99% ee. The significance of substituting the position para, meta, and ortho to the electrophilic benzylic center was highlighted. A benzyl Meldrum's acid product was further transformed to a 3,3-disubstituted 1-indanone and a beta,beta-disubstituted pentanoic acid.

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

PubMed

Dennis, J A; Healy, P J

1999-08-01

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit. Copyright 1999 Harcourt Publishers Ltd.

Structural Aspects for Evolution of [beta]-Lactamases from Penicillin-Binding Proteins

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

Meroueh, Samy O.; Minasov, George; Lee, Wenlin

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and {beta}-lactamases, resistance enzymes to {beta}-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for {beta}-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7{beta}-[N-Acetyl-L-alanyl-{gamma}-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporinmore » bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC {beta}-lactamase from Escherichia coli was solved at 1.71 {angstrom} resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the {beta}-lactamase active site. Furthermore, insertion of a peptide in the {beta}-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the {beta}-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.« less

Chemical constituents and their acetyl cholinesterase inhibitory and antioxidant activities from leaves of Acanthopanax henryi: potential complementary source against Alzheimer's disease.

PubMed

Zhang, Xiao Dan; Liu, Xiang Qian; Kim, Yang Hee; Whang, Wan Kyunn

2014-05-01

The aim of this study was to investigate chemical constituents of the leaves of Acanthopanax henryi, and their antioxidant, acetyl cholinesterase inhibitory activities. Caffeoyl quinic acid derivates and flavonoids were obtained from A. henry, through column chromatography technologies, and the content of major constituents was determined by the HPLC-UV method. Anti-oxidant activity of the isolated metabolites was evaluated by free radical scavenging (DPPH, ABTS radicals) and superoxide anion scavenging. The results showed that di-caffeoyl quinic acid derivates had stronger antioxidant activity than positive controls (ascorbic acid, trolox and allopurinol). Acetyl cholinesterase inhibitory activity was estimated on the constituents, among which, quercetin, 4-caffeoyl-quinic acid and 4,5-caffeoyl quinic acid were found to have strong acetyl cholinesterase inhibitory activity with IC50 values ranging from 62.6 to 121.9 μM. The present study showed that some of the tested constituents from the leaves of A. henryi exhibit strong antioxidant and acetyl cholinesterase inhibitory effects. This suggest that the leaves of A. henryi can be used as a new natural complementary source of acetyl cholinesterase inhibitors and anti-oxidant agents, thus being a promising potential complementary source against Alzheimer's disease.

A Proteomic Approach to Analyze the Aspirin-mediated Lysine Acetylome*

PubMed Central

Tatham, Michael H.; Cole, Christian; Scullion, Paul; Wilkie, Ross; Westwood, Nicholas J.; Stark, Lesley A.; Hay, Ronald T.

2017-01-01

Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations. PMID:27913581

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

PubMed

Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

METHOD FOR PREPARING NORMORPHINE

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

Rapoport, H.; Look, M.

1959-06-01

An improved method is presented for producing normorphine from morphine. Morphine as the starting material is acetylated by treatment with acetylating agents to produce di-acetyl morphine (heroin). The acetylated compound is reacted with cyanating agents to produce di-acetyl-cyanonormorphine (cyanonorheroin). The di-acetyl-cyanonormorphine compound is then treated in accordance with the improved hydrolysis reactions of the present invention in which concentrated hydrochloric acid is employed for a limited time period to hydrolyze the acetyl group therefrom forming cyanonormorphine. Subsequently, the reaction mixture is diluted and hydrolysis of the cyano groups from the cyanonormorphine is effected with a longer contact time with dilutemore » hydrochloric acid thereby producing normorphine. A high over-all conversion and production of a high purity product which may be radioactlvely labeled, if desired, is obtained by operation of the process.« less

Synthetic approach to the AB ring system of ouabain.

PubMed

Jung, Michael E; Piizzi, Grazia

2003-04-04

Several novel hydroxylated cis-decalin derivatives, potential intermediates for the synthesis of the AB ring system of the important cardiotonic steroid ouabain, have been synthesized from commercially available starting materials. The first step in the preparation of these highly functionalized intermediates is a Robinson annulation of the beta-keto ester 6 and the 4-silyl-3-buten-2-one 5 to furnish the octalone 4 with good diastereoselectivity in fair yield (due to competition with a novel silicon-to-carbon phenyl migration). Reduction of the epoxy alcohol 3 (derived from 4 in two high-yielding steps) with LiAlH(4) gave a mixture of the desired triol 11 along with the product of an unusual reductive opening at the tertiary carbon, namely the triol 12. A plausible mechanism for this unusual reduction is presented as are possible methods for avoiding it. In particular, reduction of the corrresponding epoxy ketone 15 with aluminum amalgam proceeded in good yield to give the hydroxy ketone 16. Also reduction of the epoxide ester having the inverted stereochemistry at C3 afforded the desired tertiary alcohol 33 in good yield. Another approach using the beta,gamma-unsaturated ketal 38 permitted the formation of the tertiary alcohol 40. Fleming oxidation of the related, very functionalized silane 39 afforded the desired 1beta-alcohol 41 in fair yield. Finally a novel rearrangement was observed when the epoxy alcohol 24 was treated with DIBAL to effect loss of the angular hydroxymethyl group to produce the tetrasubstitued alkene 29 in high yield.

Differentiation- and apoptosis-inducing activities by pentacyclic triterpenes on a mouse melanoma cell line.

PubMed

Hata, Keishi; Hori, Kazuyuki; Takahashi, Saori

2002-05-01

In a study to investigate the relationship between the chemical structure and the differentiation-inducing activity of pentacyclic triterpenes, several lupane, oleanane, and ursane triterpenes were prepared and their effects on B16 2F2 melanoma cell differentiation and growth were examined. Eleven lupane triterpenes used in this study acted on the melanoma cells as a melanogen, but no induction of melanogenesis of B16 2F2 cells by oleanane and ursane was detected. The differences at C-17 of the lupane series and acetylation of the OH group at C-3 did not markedly influence their activities. However, the ED(50) value for up-regulation of melanin biosynthesis was markedly decreased by the oxidation of the OH group at C-3 of lupeol (1). Betulinic acid (11), its methyl ester (12), lup-28-al-20(29)-ene-3beta-ol (9), and lup-28-al-20(29)-en-3-one (10) inhibited B16 2F2 cell proliferation by induction of apoptosis. These findings suggested that the carbonyl group at C-17 might be essential for the apoptotic effects of these compounds on B16 2F2 cells.

Localization of cellulose synthase in Acetobacter xylinum

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

Bureau, T.E.

1987-01-01

The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme and trypsin were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxy-octulosonic acid was used to identify the outer membranes. Cellulose synthetase activity was assayed as the conversion of radioactivity from UDP-(/sup 14/C)glucose into an alkali-insoluble ..beta..-1,4-D-(/sup 14/C)glucan. The cellulosic nature of the product was demonstrated by enzymatic hydrolysis followed by thin-layer chromatography, and by methylation analysis followed by thin-layer chromatography and gas chromatography-mass spectroscopy. X-ray diffraction analysis indicated that themore » in vitro product is cellulose II which is in contrast to the in vivo product, namely cellulose I. In addition, no microfibrillar morphology could be observed from negative stained and metal shadowed preparations of the in vitro product.« less

Aspirin Increases Mitochondrial Fatty Acid Oxidation

PubMed Central

Uppala, Radha; Dudiak, Brianne; Beck, Megan E.; Bharathi, Sivakama S.; Zhang, Yuxun; Stolz, Donna B.; Goetzman, Eric S.

2016-01-01

The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 hr incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. PMID:27856258

Hb Alesha [beta67(E11)Val-->Met, GTG-->ATG] in an Argentinean girl.

PubMed

Eberle, Silvia Eandi; Noguera, Nélida I; Sciuccati, Gabriela; Bonduel, Mariana; Díaz, Lilian; Staciuk, Raquel; Targovnik, Héctor M; Feliu-Torres, Aurora

2007-01-01

Hb Alesha is caused by a GTG-->ATG mutation at codon 67 of the beta-globin gene, resulting in abnormal beta-globin chains in which the normal beta67(E11) valine is changed to methionine. This hemoglobin (Hb) is also known as Hb Bristol, the first unstable Hb described, since in a fraction of the variant the methionine is modified into an aspartic acid by a posttranslational modification. This replacement disrupts the apolar bonds between the valine and the heme group, producing an unstable Hb and severe hemolysis. We have identified this rare hemoglobinopathy in an Argentinean girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell transfusions.

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

Kita, Keiko; Fukura, Takanobu; Nakase, Kohichi

The authors cloned and sequenced the gene encoding and NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3{Beta}-hydroxysteroid dehydrogenase-plant dihydroglavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism. The ARII proteinmore » was overproduced in Escherichia coli about 2,000-fold compared to the production in the original y east cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. Salmonicolor. To examine the contribution of the dinucleotide-binding motif G{sub 19}-X-X-G{sub 22}-X-X-A{sub 25}, which is located in the N-terminal region, during ARII catalysis, they replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G{sub 19}{r{underscore}arrow}A and G{sub 22}{r{underscore}arrow}A mutant enzymes by 4-COBE die not occur. The A{sub 25}{r{underscore}arrow}G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.« less

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

PubMed

Cai, Z; Li, X; Katsumura, Y

1999-10-01

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.

Constituents of antibacterial extract of Caesalpinia paraguariensis Burk.

PubMed

Woldemichael, Girma M; Singh, Maya P; Maiese, William M; Timmermann, Barbara N

2003-01-01

The Argentinean legume Caesalpinia paraguariensis Burk. (Fabaceae) was selected for further fractionation work based on the strong antimicrobial activity of its CH2Cl2-MeOH (1:1 v/v) extract against a host of clinically significant microorganisms, including antibiotic resistant strains. 1D and 2D NMR enabled the identification of the novel benzoxecin derivative caesalpinol along with the known compounds bilobetin, stigma-5-en-3-O-beta-6'-stearoylglucopyranoside, stigma-5-en-3-beta-6'-palmitoylglucopyranoside, stigma-5-en-3-beta-glucopyranoside, oleanolic acid, 3-O-(E)-hydroxycinnamoyl oleanolic acid, betulinic acid, 3-O-(E)-hydroxycinnamoyl betulinic acid, and lupeol from the active fractions. Oleanolic acid was found active against Bacillus subtilis and both methicillin-sensitive and -resistant Staphylococcus aureus with MICs of 8 (17.5 microM), 8 and 64 (140 microM) microg/ml, respectively. The rest of the compounds, however, did not show activity.

Influence of calcium on fungal growth, hyphal morphology and citric acid production in Aspergillus niger.

PubMed

Pera, L M; Callieri, D A

1997-01-01

Addition of 0.5 g/L CaCl2 to the fermentation medium lowered the final biomass dry mass by 35% and increased the uptake of phosphate and sucrose, and the production of citric acid by 15, 35 and 50%, respectively. In a medium deprived of Ca2+ the microorganism displayed both a pelleted and a filamentous form of growth, the hyphae being scarcely branched, without bulbous cells. An addition of Ca2+ induced a pelleted form of growth, highly branched hyphae and numerous bulbous cells. Bulbous cells growing in the presence of Ca2+ exhibited cell walls composed of laminated layers, and featured vesicles associated with the wall and/or the cell membrane, containing numerous inclusions. The cytotoxic effect of high concentrations of citric acid in the medium as well as an increase of the activity of N-acetyl-beta-D-glucosaminidase, a lytic enzyme, might be involved in these morphological changes.

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

NASA Technical Reports Server (NTRS)

Komoszynski, M.; Bandurski, R. S.

1986-01-01

Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

Discovery and characterization of sialic acid O-acetylation in group B Streptococcus.

PubMed

Lewis, Amanda L; Nizet, Victor; Varki, Ajit

2004-07-27

Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal alpha 2-3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of the O-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase gene neuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.

Acetyl diacylglycerol produced by modified camelina (Camelina sativa)

USDA-ARS?s Scientific Manuscript database

Acetyl diacylglyceride (Acetyl-TAG) is a component of a commercial product, ACETEM, manufactured by transesterification reaction of triglycerides, glycerol, and triacetin or by acetylation of mono- and diglycerides with acetic acid anhydride. ACETEM is commonly used as foaming agents and coatings in...

[Effects of carbon and nitrogen sources on 5-keto-gluconic acid production].

PubMed

Tan, Zhilei; Wang, Hongcui; Wei, Yuqiao; Li, Yanyan; Zhong, Cheng; Jia, Shiru

2014-01-01

Gluconobacter oxydans is known to oxidize glucose to gluconic acid (GA), and subsequently, to 2-keto-gluconic acid (2KGA) and 5-keto-gluconic acid (5KGA), while 5KGA can be converted to L-(+)-tartaric acid. In order to increase the production of 5KGA, Gluconobacter oxydans HGI-1 that converts GA to 5KGA exclusively was chosen in this study, and effects of carbon sources (lactose, maltose, sucrose, amylum and glucose) and nitrogen sources (yeast extract, fish meal, corn steep liquor, soybean meal and cotton-seed meal) on 5KGA production were investigated. Results of experiment in 500 mL shake-flask show that the highest yield of 5KGA (98.20 g/L) was obtained using 100 g/L glucose as carbon source. 5KGA reached 100.20 g/L, 109.10 g/L, 99.83 g/L with yeast extract, fish meal and corn steep liquor as nitrogen source respectively, among which the optimal nitrogen source was fish meal. The yield of 5KGA by corn steep liquor is slightly lower than that by yeast extract. For the economic reason, corn steep liquor was selected as nitrogen source and scaled up to 5 L stirred-tank fermentor, and the final concentration of 5KGA reached 93.80 g/L, with its maximum volumetric productivity of 3.48 g/(L x h) and average volumetric productivity of 1.56 g/(L x h). The result obtained in this study showed that carbon and nitrogen sourses for large-scale production of 5KGA by Gluconobacter oxydans HGI-1 were glucose and corn steep liquor, respectively, and the available glucose almost completely (85.93%) into 5KGA.

FeS/S/FeS2 Redox System and Its Oxidoreductase-like Chemistry in the Iron-Sulfur World

NASA Astrophysics Data System (ADS)

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS2 redox system on the interconversion between several pairs of ±-hydroxy acids and ±-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized ±-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS2 redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world.

Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus.

PubMed

Zarzycki, Jan; Brecht, Volker; Müller, Michael; Fuchs, Georg

2009-12-15

The phototrophic bacterium Chloroflexus aurantiacus uses a yet unsolved 3-hydroxypropionate cycle for autotrophic CO(2) fixation. It starts from acetyl-CoA, with acetyl-CoA and propionyl-CoA carboxylases acting as carboxylating enzymes. In a first cycle, (S)-malyl-CoA is formed from acetyl-CoA and 2 molecules of bicarbonate. (S)-Malyl-CoA cleavage releases the CO(2) fixation product glyoxylate and regenerates the starting molecule acetyl-CoA. Here we complete the missing steps devoted to glyoxylate assimilation. In a second cycle, glyoxylate is combined with propionyl-CoA, an intermediate of the first cycle, to form beta-methylmalyl-CoA. This condensation is followed by dehydration to mesaconyl-C1-CoA. An unprecedented CoA transferase catalyzes the intramolecular transfer of the CoA moiety to the C4 carboxyl group of mesaconate. Mesaconyl-C4-CoA then is hydrated by an enoyl-CoA hydratase to (S)-citramalyl-CoA. (S)-Citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by a tri-functional lyase, which previously cleaved (S)-malyl-CoA and formed beta-methylmalyl-CoA. Thus, the enigmatic disproportionation of glyoxylate and propionyl-CoA into acetyl-CoA and pyruvate is solved in an elegant and economic way requiring only 3 additional enzymes. The whole bicyclic pathway results in pyruvate formation from 3 molecules of bicarbonate and involves 19 steps but only 13 enzymes. Elements of the 3-hydroxypropionate cycle may be used for the assimilation of small organic molecules. The 3-hydroxypropionate cycle is compared with the Calvin-Benson-Bassham cycle and other autotrophic pathways.

Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein.

PubMed

Ohta, M; Hamako, J; Yamamoto, S; Hatta, H; Kim, M; Yamamoto, T; Oka, S; Mizuochi, T; Matsuura, F

1991-10-01

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. After N-acetylation, the oligosaccharides were labelled with a UV-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz 1H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc alpha 1-3Man7-9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man5-9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc. The glucosylated oligosaccharides, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, have not previously been reported in mature glycoproteins from any source.

A Chemical Biology Solution to Problems with Studying Biologically Important but Unstable 9-O-Acetyl Sialic Acids.

PubMed

Khedri, Zahra; Xiao, An; Yu, Hai; Landig, Corinna Susanne; Li, Wanqing; Diaz, Sandra; Wasik, Brian R; Parrish, Colin R; Wang, Lee-Ping; Varki, Ajit; Chen, Xi

2017-01-20

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac 2 ). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac 2 , with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac 2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

PubMed

González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

2010-05-01

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

The Influence of pH and Sodium Hydroxide Exposure Time on Glucosamine and Acrylamide Levels in California-Style Black Ripe Olives.

PubMed

Charoenprasert, Suthawan; Zweigenbaum, Jerry A; Zhang, Gong; Mitchell, Alyson E

2017-07-01

Acrylic acid, N-acetyl-glucosamine and glucosamine were investigated for their role in the formation of acrylamide in California-style black ripe olives [CBROs]. Levels of acrylic acid and glucosamine are reported for the first time in fresh (333.50 ± 21.88 and 243.59 ± 10.06 nmol/g, respectively) and in brine-stored olives (184.50 ± 6.02 and 165.88 ± 11.51 nmol/g, respectively). Acrylamide levels significantly increased when acrylic acid (35.2%), N-acetyl-glucosamine (29.9%), and glucosamine (124.0%) were added to olives prior to sterilization. However, isotope studies indicate these compounds do not contribute carbon and/or nitrogen atoms to acrylamide. The base-catalyzed degradation of glucosamine is demonstrated in olive pulp and a strong correlation (r 2 = 0.9513) between glucosamine in olives before sterilization and acrylamide formed in processed CBROs is observed. Treatment with sodium hydroxide (pH > 12) significantly reduces acrylamide levels over 1 to 5 d without impacting olive fruit texture. © 2017 Institute of Food Technologists®.

Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.

PubMed

Lemieux, M Joanne; Mark, Brian L; Cherney, Maia M; Withers, Stephen G; Mahuran, Don J; James, Michael N G

2006-06-16

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.

Synthesis of specifically deuterated S-benzylcysteines and of oxytocin and related diastereomers deuterated in the half-cystine positions

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

Upson, D.A.; Hruby, V.J.

1976-04-16

S-Benzylcysteine derivatives specifically deuterated at the ..cap alpha.. carbon only, the ..beta.. carbon only, and at both the ..cap alpha.. and ..beta.. carbons have been synthesized. These labeled compounds have been enzymatically resolved and the enantiomers and reacemates have been converted to the N-tert-butyloxycarbonyl derivatives. The deuterium labels were not exchanged under the conditions of the syntheses. Condensation of the sodium salt of diethyl ..cap alpha..-acetami-domalonate with benzyl chloromethyl sulfide followed by hydrolysis with DCl afforded S-benzyl-DL-(..cap alpha..-/sup 2/H/sub 1/) cysteine. Acetylation followed by treatment with hog renal acylase separated the stereoisomers. A Mannich reaction with (/sup 2/H/sub 2/) methylenemore » diacetate, diethyl ..cap alpha..-acetamido-..cap alpha..-dimethylamino(/sup 2/H/sub 2/)methylmalonate methiodide (15). Treatment of 15 with sodium benzylmercaptide gave diethyl ..cap alpha..-acetamido-..cap alpha..-benzylthio(/sup 2/H/sub 2/)methylmalonate, which was hydrolyzed with HCl to yield S-benzyl-DL-(..beta..,..beta..-/sup 2/H/sub 2/)cysteine or with DCl to afford S-benzyl-DL-(..cap alpha..,..beta..,..beta..,-/sup 2/H/sub 3/)cysteine. These compounds were resolved as before. The preparation of S-benzyl-DL-(..cap alpha..,..beta..,..beta..-/sup 2/H/sub 3/)cysteine required an efficient source of ethanol-d. This deuterated solvent was prepared in quantitative yield in 2 h from tetraethoxysilane, D/sub 2/O, and a catalytic amount of thionyl chloride. The protected deuterated amino acids were used in the preparation of several oxytocin analogues in which the specific deuteration appears in either the 1-hemicystine or the 6-hemicystine residues.« less

Identification of an active acidic residue in the catalytic site of beta-hexosaminidase.

PubMed

Tse, R; Vavougios, G; Hou, Y; Mahuran, D J

1996-06-11

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression. Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m). Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively. The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

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

Shi, Rong; Pineda, Marco; Ajamian, Eunice

2009-01-15

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+},more » which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.« less

Diabetes and branched-chain amino acids: What is the link?

PubMed

Bloomgarden, Zachary

2018-05-01

Branched-chain amino acids (BCAA) have increasingly been studied as playing a role in diabetes, with the PubMed search string "diabetes" AND "branched chain amino acids" showing particular growth in studies of the topic over the past decade (Fig. ). In the Young Finn's Study, BCAA and, to a lesser extent, the aromatic amino acids phenylalanine and tyrosine were associated with insulin resistance (IR) in men but not in women, whereas the gluconeogenic amino acids alanine, glutamine, or glycine, and several other amino acids (i.e. histidine, arginine, and tryptophan) did not show an association with IR. Obesity may track more strongly than metabolic syndrome and diabetes with elevated BCAA. In a study of 1302 people aged 40-79; higher levels of BCAA tracked with older age, male sex, and metabolic syndrome, as well as with obesity, cardiovascular risk, dyslipidemia, hypertension, and uric acid. Medium- and long-chain acylcarnitines, by-products of mitochondrial catabolism of BCAAs, as well as branched-chain keto acids and the BCAA themselves distinguished obese people having versus not having features of IR, and in a study of 898 patients with essential hypertension, the BCAA and tyrosine and phenylalanine were associated with metabolic syndrome and impaired fasting glucose. In a meta-analysis of three genome-wide association studies, elevations in BCAA and, to a lesser extent, in alanine tracked with IR, whereas higher levels of glutamine and glycine were associated with lesser likelihood of IR. Given these associations with IR, it is not surprising that a number of studies have shown higher BCAA levels in people with and prior to development of type 2 diabetes (T2D), although this has particularly been shown in Caucasian and Asian ethnic groups while not appearing to occur in African Americans. Similarly, higher BCAA levels track with cardiovascular disease. [Figure: see text] The metabolism of BCAA involves two processes: (i) a reversible process catalysed by a branched-chain aminotransferase (BCAT), either cytosolic or mitochondrial, requiring pyridoxal to function as an amino group carrier, by which the BCAA with 2-ketoglutarate produce a branched-chain keto acid plus glutamate; and (ii) the irreversible mitochondrial process catalysed by branched-chain keto acid dehydrogenase (BCKDH) leading to formation of acetyl-coenzyme A (CoA), propionyl-CoA, and 2-methylbutyryl-CoA from leucine, valine, and isoleucine, respectively, which enter the tricarboxylic acid (Krebs) cycle as acetyl-CoA, propionyl-CoA, and 2-methylbutyryl-CoA, respectively, leading to ATP formation. The BCAA stimulate secretion of both insulin and glucagon and, when given orally, of both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), with oral administration leading to greater and more prolonged insulin and glucagon secretion. Insulin may particularly reduce BCAA turnover to a greater extent than that of other amino acids, and decreases the appearance and increases the uptake of amino acids. However, older studies of the effect of glucose or insulin on BCAA concentrations and rates of leucine appearance and oxidation showed no reduction in T2D, although the higher baseline levels of BCAA in obesity have long been recognized. Impaired function of BCAT and BCKDH has been posited, either as a primary genetic abnormality or due to effects of elevated fatty acids, proinflammatory cytokines, or insulin levels with consequent accumulation of branched-chain keto acids and metabolites such as diacylglycerol and ceramide, potentially contributing to the development of further insulin resistance, and decreased skeletal muscle BCAT and BCKDH expression has been shown in people with diabetes, supporting this concept. A Mendelian randomization study used measured variation in genes involved in BCAA metabolism to test the hypothesis of a causal effect of modifiable exposure on IR, showing that variants in protein phosphatase, Mg 2+ /Mn 2+ dependent 1K (PPM1K), a gene encoding the mitochondrial phosphatase activating the BCKDH complex, are associated with T2D, but another such study suggested that genetic variations associated with IR are causally related to higher BCAA levels. Another hypothesis involves the mammalian target of rapamycin complex 1 (mTORC1), which is activated by BCAA, as well as by insulin and glucose via cellular ATP availability. If this is the relevant pathway, BCAA overload may cause insulin resistance by activation of mammalian target of rapamycin (mTOR), as well as by leading to increases in acylcarnitines, with mTOR seen in this scenario as a central signal of cross-talk between the BCAA and insulin. At this point, whether whole-body or tissue-specific BCAA metabolism is increased or decreased in states of insulin-resistant obesity and T2D is uncertain. Insulin action in the hypothalamus induces but overfeeding decreases hepatic BCKDH, leading to the concept that hypothalamic insulin resistance impairs BCAA metabolism in obesity and diabetes, so that plasma BCAAs may be markers of hypothalamic insulin action rather than direct mediators of changes in IR. A way to address this may be to understand the effects of changes in diet and other interventions on BCAA, as well as on IR and T2D. In an animal model, lowering dietary BCAA increased energy expenditure and improved insulin sensitivity. Two large human population studies showed an association of estimated dietary BCAA intake with T2D risk, although another population study showed higher dietary BCAA to be associated with lower T2D risk. Ethnic differences, reflecting underlying differences in genetic variants, may be responsible for such differences. In the study of Asghari et al. in the current issue of the Journal of Diabetes, BCAA intake was associated with the development of subsequent IR. Studies of bariatric surgery suggest lower basal and post-insulin infusion BCAA levels are associated with greater insulin sensitivity, with reductions in BCAA not seen with weight loss per se with gastric band procedures, but occurring after Roux-en-Y gastric bypass, an intervention that may have metabolic benefits over and above those from reduction in body weight. The gut microbiota may be important for the supply of the BCAA to mammalian hosts, either by de novo biosynthesis or by modifying nutrient absorption. A final fascinating preliminary set of observations is that of the effects of empagliflozin on metabolomics; evidence of increased Krebs cycle activation and of higher levels of BCAA metabolites, such as acylcarnitines, suggests that sodium-glucose cotransporter 2 (SGLT2) inhibition may, to some extent, involve BCAA metabolism. Certainly, we do not yet have a full understanding of these complex associations. However, the suggestion of multiple roles of BCAA in the development of IR promises to be important and to lead to the development of novel effective T2D therapies. © 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Acetylation Suppresses the Proapoptotic Activity of GD3 Ganglioside

PubMed Central

Malisan, Florence; Franchi, Luigi; Tomassini, Barbara; Ventura, Natascia; Condò, Ivano; Rippo, Maria Rita; Rufini, Alessandra; Liberati, Laura; Nachtigall, Claudia; Kniep, Bernhard; Testi, Roberto

2002-01-01

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3. PMID:12486096

[Study on the chemical constituents in Pouzolzia zeylanica].

PubMed

Fu, Ming; Niu, You-Ya; Yu, Juan; Kong, Qing-Tong

2012-11-01

To study the chemical constituents of Pouzolzia zeylanica. Many chromatography means were used in separation and purification, and the structures of all compounds were identified by the means of spectroscopic analysis and physicochemical properties. 14 compounds were elucidated as: beta-sitosterol (1), daucosterol (2), oleanolic acid (3), epicatechin (4), alpha-amyrin (5), eugenyl-beta-rutinoside (6), 2alpha, 3alpha, 19alpha-trihydroxyurs-12-en-28-oic (7), scopolin (8), scutellarein-7-O-alpha-L-rhamnoside (9), scopoletin (10), quercetin (11), quercetin-3-O-beta-D-glucoside (12), apigenin (13), 2alpha-hydroxyursolic acid (14). All compounds are obtained from this plant for the first time.

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

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

Ghosh, Amit; Ando, David; Gin, Jennifer

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

DOE PAGES

Ghosh, Amit; Ando, David; Gin, Jennifer; ...

2016-10-05

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

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

PubMed

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

2008-05-23

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

4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.

PubMed

Urashima, Tadasu; Inamori, Hiroaki; Fukuda, Kenji; Saito, Tadao; Messer, Michael; Oftedal, Olav T

2015-06-01

Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(α2-3)Gal(β1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (≥ 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Metabolism and elimination of benzocaine by rainbow-trout, Oncorhynchus mykiss

USGS Publications Warehouse

Meinertz, J.R.; Gingerich, W.H.; Allen, J.L.

1991-01-01

1. Branchial and urinary elimination of benzocaine residues was evaluated in adult rainbow trout, oncorhynchus mykiss, given a single dorsal aortic dose of c-14-benzocaine hydrochloride.^2. Branchial elimination of benzocaine residues was rapid and accounted for 59.2% Of the dose during the first 3 h after dosing. Renal elimination of radioactivity was considerably slower; the kidney excreted 2.7% Dose within 3 h and 9.0% Within 24 h. Gallbladder bile contained 2.0% Dose 24 h after injection.^3. Of the radioactivity in radiochromatograms from water taken 3 min after injection, 87.3% Was benzocaine and 12.7% Was n-acetylated benzocaine. After 60 min, 32.7% Was benzocaine and 67.3% Was n-acetylated benzocaine.^4. Of the radioactivity in radiochromatograms from urine taken 1 h after dosing, 7.6% Was para-aminobenzoic acid, 59.7% Was n-acetylated para-aminobenzoic acid, 19.5% Was benzocaine, and 8.0% Was n-acetylated benzocaine. The proportion of the radioactivity in urine changed with time so that by 20 h, 1.0% Was para-aminobenzoic acid and 96.6% Was n-acetylated para-aminobenzoic acid.^5. Benzocaine and a more hydrophobic metabolite, n-acetylated benzocaine, were eliminated primarily through the gills; renal and biliary pathways were less significant elimination routes for benzocaine residues.

Small-Molecule Inhibitor Leads of Ribosome-Inactivating Proteins Developed Using the Doorstop Approach

DTIC Science & Technology

2011-03-01

stereochemistry of R22 used in our virtual screen. R20 or R20b was prepared by coupling 4-formylbenzoic acid with a substituted pyrrole in the keto form for R20 or...a mixture of keto and enol forms for R20b according to a reported process [42] (Figure 4). The substituted pyrrole was obtained via cyclization of 2...1H- pyrrole -3-carboxylate—a close ana- log of R20b—has been reported to have the Z stereochemistry [45]. Therefore, identification of R20 as an active

Mycobacterium tuberculosis Arylamine N-Acetyltransferase Acetylates and Thus Inactivates para-Aminosalicylic Acid.

PubMed

Wang, Xude; Yang, Shanshan; Gu, Jing; Deng, Jiaoyu

2016-12-01

Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) is able to acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) techniques. The antituberculosis activity of the acetylated PAS is significantly reduced. As a result, overexpression of TBNAT in M. tuberculosis results in PAS resistance, as determined by MIC tests and drug exposure experiments. Taken together, our results suggest that TBNAT from M. tuberculosis is able to inactivate PAS by acetylating the compound. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

PubMed Central

Boger, Dale L.; Sato, Haruhiko; Lerner, Aaron E.; Hedrick, Michael P.; Fecik, Robert A.; Miyauchi, Hiroshi; Wilkie, Gordon D.; Austin, Bryce J.; Patricelli, Matthew P.; Cravatt, Benjamin F.

2000-01-01

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones. PMID:10805767

Ultrasound-assisted synthesis of santalbic acid and a study of triacylglycerol species in Santalum album (Linn.) seed oil.

PubMed

Lie Ken Jie, M S; Pasha, M K; Ahmad, F

1996-10-01

Methyl ricinoleate (1) was treated with bromine and the dibromo derivative (2) was reacted with ethanolic KOH under ultrasonic irradiation to give 12-hydroxy-octadec-9-ynoic acid upon acidification with dil. HCI. The latter compound was methylated with BF3/methanol to give methyl 12-hydroxy-octadec-9-ynoate (3). Compound 3 was treated with methanesulfonyl chloride in the presence of triethylamine in CH2Cl2 to give methyl 12-mesyloxy-octadec-9-ynoate (4). Reaction of methyl 12-mesyloxy-octadec-9-ynoate with aqueous KOH under ultrasonic irradiation (20 kHz) gave (11E)-octadecen-9-ynoic acid (5, santalbic acid, 40%) and (11Z)-octadecen-9-ynoic acid (6, 60%) on acidification with dil. HCI. These isomers were separated by urea fractionation. The 13C nuclear magnetic resonance (NMR) spectroscopic properties of the methyl ester and the triacylglycerol (TAG) esters of these enynoic fatty acid isomers were studied. The carbon shifts of the unsaturated carbon nuclei of the methyl ester of the E-isomer were unambiguously assigned as 88.547 (C-9), 79.287 (C-10), 109.760 (C-11), and 143.450 (C-12) ppm, while the unsaturated carbon shifts of the (Z)-enynoate isomer appeared at 94.277 (C-9), 77.561 (C-10), 109.297 (C-11), and 142.668 (C-12) ppm. In the 13C NMR spectral analysis of the TAG molecules of type AAA containing either the (Z)- or (E)-enyne fatty acid, the C-1 to C-6 carbon atoms on the alpha- and beta-acyl positions were differentiated. The unsaturated carbon atoms in the alpha- and beta-acyl chains were also resolved into two signals except that of the C-11 olefinic carbon. Sandal (Santalum album) wood seed oil (a source of santalbic acid) was separated by silica chromatography into three fractions. The least polar fraction (7.2 wt%) contained TAG which had a random distribution of saturated and unsaturated fatty acids, of which oleic acid (69%) was the predominant component. The second fraction (3.8 wt%) contained santalbic acid (58%) and oleic acid (28%) together with some other normal fatty acids. Santalbic acid in this fraction was found in both the alpha- and beta-acyl positions of the glycerol "backbone." The most polar fraction (89 wt%) consisted of TAG containing santalbic acid only. The distribution of the various fatty acids on the glycerol "backbone" was supported by the results from the 13C NMR spectroscopic analysis.

Chemoenzymatic synthesis of differentially protected 3-deoxysugars

NASA Astrophysics Data System (ADS)

Gillingham, Dennis G.; Stallforth, Pierre; Adibekian, Alexander; Seeberger, Peter H.; Hilvert, Donald

2010-02-01

3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great promise as a source of synthetically useful catalytic transformations.

Paviosides A-H, eight new oleane type saponins from Aesculus pavia with cytotoxic activity.

PubMed

Lanzotti, Virginia; Termolino, Pasquale; Dolci, Marcello; Curir, Paolo

2012-05-15

A phytochemical analysis of Aesculus pavia has led to the isolation of eight novel triterpenoid saponins, based on oleane type skeleton and named paviosides A-H (1a, 1b-4a, 4b). On the basis of chemical, and 2D NMR and mass spectrometry data, the structures of the new compounds were elucidated as 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-d-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (1a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (1b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (2a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (2b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (3a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-d-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (3b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl protoaescigenin (4a), and 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl protoaescigenin (4b). The compounds showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines. Among them, paviosides E-H (3a, 3b and 4a, 4b) showed higher activity with values ranging from 2.1 to 3.6 μg/mL. Structure-activity relationship studies indicated the positive effect on the activity of xylose unit in the place of glucose, while a little detrimental effect is observed when glucose is substituted by galactose. The aglycone structure and the presence of a tigloyl or an angeloyl group at C-21 do not affect significantly the inhibitory activity on both tested cell lines. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-03-01

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

Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria

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

McInerney, M.J.; Amos, D.A.; Kealy, K.S.

1992-12-31

Anaerobic syntrophic bacteria degrade fatty acids and some aromatic compounds which are important intermediates in the degradation of organic matter to CO{sub 2} and CH{sub 4} in methanogenic environments. Several of the described syntrophic species produce poly-{beta}-hydroxyalkanoate (PHA) suggesting that the synthesis and use of PHA is important in their physiology. In the fatty acid-degrading, syntrophic bacterium, Syntrophomonas wolfei, PHA is made during exponential phase of growth and used after growth has stopped and substrate levels are low. Altering the carbon to nitrogen ratio of the medium does not affect the amount of PHA made or its monomeric composition. Itmore » is hypothesized that PHA serves as an endogenous energy source for syntrophic bacteria when the concentrations of hydrogen or acetate are too high for the degradation of the growth substrate to be thermodynamically favorable. In S. wolfei, PHA is synthesized by two routes, the direct incorporation of 3-ketoacyl-coenzyme A (CoA) generated in {beta}-oxidation without cleavage of a C-C bond, and by the condensation and subsequent reduction of two acetyl-CoA molecules. Genes that encode for the synthesis of PHA in S. wolfei have been cloned into Escherichia coli in order to understand the molecular mechanisms that regulate PHA synthesis. 61 refs., 1 fig., 4 tabs.« less

Conformational studies of bacterial peptidoglycan: structure and stereochemistry of N-acetyl-β- D-glucosamine and N-acetyl-β- D-muramic acid

NASA Astrophysics Data System (ADS)

Yadav, P. N. S.; Rai, D. K.; Yadav, J. S.

1989-03-01

The energies of various conformations of N-acetyl-β- D-glucosamine (NAG) and its 3-O- D-lactic acid derivative N-acetyl-β- D-muramic acid (NAM) have been calculated by geometry optimization using the molecular mechanics program MM2. The geometries of these systems have been analyzed in the light of ring torsion, bond lengths, bond angles and conformational states of side groups of the pyranosyl ring and compared with available experimental data of similar pyranose derivatives. The present study indicates the presence of hydrogen bonds to stabilize the side group conformations. Discrepancies with experimental data that are seen in a few cases are ascribed to the nature of the side groups and their geometry.

In Vitro Enzymatic Synthesis of New Penicillins Containing Keto Acids as Side Chains

PubMed Central

Ferrero, Miguel A.; Reglero, Angel; Martínez-Blanco, Honorina; Fernández-Valverde, Martiniano; Luengo, Jose M.

1991-01-01

Seven different penicillins containing α-ketobutyric, β-ketobutyric, γ-ketovaleric, α-ketohexanoic, δ-ketohexanoic, ε-ketoheptanoic, and α-ketooctanoic acids as side chains have been synthesized in vitro by incubating the enzymes phenylacetyl coenzyme A (CoA) ligase from Pseudomonas putida and acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum with CoA, ATP, Mg2+, dithiothreitol, 6-aminopenicillanic acid, and the corresponding side chain precursor. PMID:1952871

A novel small RNA S042 increases acid tolerance in Lactococcus lactis F44.

PubMed

Wu, Hao; Song, Shunyi; Tian, Kairen; Zhou, Dandan; Wang, Binbin; Liu, Jiaheng; Zhu, Hongji; Qiao, Jianjun

2018-06-07

Lactococcus lactis, a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. Small RNAs (sRNAs) that serve as regulators at post-transcriptional level play important roles in acid stress response. Here, a novel sRNA S042 was identified by RNA-Seq, RT-PCR and Northern blot. The transcription level of s042 was upregulated 2.29-fold under acid stress by Quantitative RT-PCR (qRT-PCR) analysis. Acid tolerance assay showed that overexpressing s042 increased the survival rate of L. lactis F44 and deleting s042 significantly inhibited the viability under acidic conditions. Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR (arginine regulator) and accD (acetyl-CoA carboxylase carboxyl transferase subunit beta) were validated to be the direct targets activated by S042 through reporter fusion assay. The regulatory mechanism between S042 and its targets was further investigated through Bioinformatics and qRT-PCR. This study served to highlight the role of the novel sRNA S042 in acid resistance of L. lactis and provided new insights into the response mechanism of acid stress. Copyright © 2018 Elsevier Inc. All rights reserved.

A new look at acid catalyzed deacetylation of carbohydrates: A regioselective synthesis and reactivity of 2-O-acetyl aryl glycopyranosides.

PubMed

Stepanova, Elena V; Nagornaya, Marina O; Filimonov, Victor D; Valiev, Rashid R; Belyanin, Maxim L; Drozdova, Anna K; Cherepanov, Victor N

2018-03-22

In the present work we report that acetyl groups of per - acetylated aryl glycosides have different reactivity during the acidic deacetylation using HCl/EtOH in CHCl 3, which leads to preferential deacetylation at O-3, O-4 and O-6. Thereby, the one-step preparation of 2-O-acetyl aryl glycosides with simple aglycon was accomplished for the first time. It was proved that the found reagent is to be general and unique for the preparation of series of 2-О-acetyl aryl glycosides. We have determined the influence of both carbohydrate moiety and the aglycon on the selectivity of deacetylation reaction by kinetic experiments. Using DFT/B3LYP/6-31G(d,p) and semi-empirical АМ1 methods we have found that the highest activation barrier is for 2-О-acetyl group. This completely explains the least reactivity of 2-О-acetyl group. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mastitis detection: current trends and future perspectives.

PubMed

Viguier, Caroline; Arora, Sushrut; Gilmartin, Niamh; Welbeck, Katherine; O'Kennedy, Richard

2009-08-01

Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.

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

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

He, H.; Ding, Y.; Bartlam, M.

2003-01-31

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

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

PubMed

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

2003-01-31

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

Determination of the structure of lecithins via the formation of acetylated 1,2-diglycerides.

PubMed

Privett, O S; Nutter, L J

1967-03-01

A detailed procedure for quantitative determinations of molecular species of lecithins is described and applied to several lecithins isolated from natural sources. The method is based on the conversion of lecithin to acetylated 1,2-diglycerides and analysis of these compounds by methodology used for the determination of triglyceride structure.The preparation of the acetylated 1,2-diglycerides was carried out via hydrolysis with phospholipase C and acetylation of the resultant, 1,2-diglycerides with pyridine-acetic anhydride. Preparation of acetylated 1,2-diglycerides from lecithin by acetolysis with acetic acid-acetic anhydride was shown to be accompanied by intermolecular as well as intramolecular rearrangement of the fatty acids.The structure of the acetylated 1,2-diglycerides was determined by a combination of argentation-TLC and pancreatic lipase hydrolysis using internal standards for quantification. The method was applied to lecithins isolated from milk serum, egg, soybean, safflower seed and wheat germ lipids.

(13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

PubMed

Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2015-12-01

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

PubMed

Lewis, Amanda L; Cao, Hongzhi; Patel, Silpa K; Diaz, Sandra; Ryan, Wesley; Carlin, Aaron F; Thon, Vireak; Lewis, Warren G; Varki, Ajit; Chen, Xi; Nizet, Victor

2007-09-21

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.

PubMed

Sawado, Tomoyuki; Halow, Jessica; Bender, M A; Groudine, Mark

2003-04-15

To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult beta-globin promoters of the DeltaLCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.

Interactions of Enolizable Barbiturate Dyes.

PubMed

Schade, Alexander; Schreiter, Katja; Rüffer, Tobias; Lang, Heinrich; Spange, Stefan

2016-04-11

The specific barbituric acid dyes 1-n-butyl-5-(2,4-dinitro-phenyl) barbituric acid and 1-n-butyl-5-{4-[(1,3-dioxo-1H-inden-(3 H)-ylidene)methyl]phenyl}barbituric acid were used to study complex formation with nucleobase derivatives and related model compounds. The enol form of both compounds shows a strong bathochromic shift of the UV/Vis absorption band compared to the rarely coloured keto form. The keto-enol equilibria of the five studied dyes are strongly dependent on the properties of the environment as shown by solvatochromic studies in ionic liquids and a set of organic solvents. Enol form development of the barbituric acid dyes is also associated with alteration of the hydrogen bonding pattern from the ADA to the DDA type (A=hydrogen bond acceptor site, D=donor site). Receptor-induced altering of ADA towards DDA hydrogen bonding patterns of the chromophores are utilised to study supramolecular complex formation. As complementary receptors 9-ethyladenine, 1-n-butylcytosine, 1-n-butylthymine, 9-ethylguanidine and 2,6-diacetamidopiridine were used. The UV/Vis spectroscopic response of acid-base reaction compared to supramolecular complex formation is evaluated by (1)H NMR titration experiments and X-ray crystal structure analyses. An increased acidity of the barbituric acid derivative promotes genuine salt formation. In contrast, supramolecular complex formation is preferred for the weaker acidic barbituric acid. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

PubMed

Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

2016-05-10

The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

Identification and semi-quantification of biogenic organic nitrates in ambient particulate matters by UHPLC/ESI-MS

NASA Astrophysics Data System (ADS)

Li, Rui; Wang, Xinfeng; Gu, Rongrong; Lu, Chunying; Zhu, Fanping; Xue, Likun; Xie, Huijun; Du, Lin; Chen, Jianmin; Wang, Wenxing

2018-03-01

Particulate biogenic organic nitrates (PBONs) are important components of secondary organic aerosols and play an important role in the tropospheric atmosphere chemistry. However, the concentrations and the chemistry of PBONs remain poorly understood due to the lack of accurate measurement techniques on specific organic nitrates. In this study, ultra high performance liquid chromatography/electrospray mass spectrometry was applied in detection of individual PBONs in ambient atmosphere. Total five kinds of PBONs were identified in PM2.5 samples collected in urban Ji'nan in spring according to characteristic fragments of NO2, NO3, HNO3, CO2, and H2O, including monoterpene hydroxyl nitrate (MW = 215, MHN215), pinene keto nitrate (MW = 229, PKN229), limonene di-keto nitrate (MW = 247, LDKN247), oleic acid keto nitrate (MW = 359, OAKN359), and oleic acid hydroxyl nitrate (MW = 361, OAHN361). Among them, three kinds of PBONs originated from biogenic volatile organic compounds of pinene and limonene and two kinds of PBONs came from chemical conversion of oleic acid. The concentrations of these PBONs were roughly quantified with surrogate standards of (1R,2R,5R)-(+)-2-hydroxy-3-pinanone and ricinoleic acid. The average concentrations of MHN215, PKN229, LDKN247, OAKN359, and OAHN361 were 111.6 ± 23.0, 93.1 ± 49.6, 55.3 ± 7.4, 23.4 ± 14.5, 36.8 ± 18.3 ng m-3, respectively. The total concentration of these PBONs was 325.4 ± 116.7 ng m-3, contributing to 1.64 ± 0.34‰ of PM2.5.

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

PubMed Central

Uemura, K; Roelcke, D; Nagai, Y; Feizi, T

1984-01-01

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6204642

New indole, aminoindole and pyranoindole derivatives with anti-inflammatory activity.

PubMed

Nakkady, S S; Fathy, M M; Hishmat, O H; Mahmond, S S; Ebeid, M Y

2000-01-01

6-Methoxy-1-methyl-2,3-diphenyl indol-5-carboxaldehyde (2) was demethylated to give the 6-hydroxy derivative (3) which was cyclized to the pyrano[3,2-f]indole derivatives (4a-d) by the action of ethyl acetoacetate, diethyl malonate, malononitrile, ethyl cyanoacetate. When 4c was boiled in acetic acid, it gave 4d. Reduction of 4c by sodium borohydride yielded the orthoaminonitrile (5). Friedel Craft's acetylation of 1b yielded the 5-acetyl derivative (6), which reacted with hydrazine hydrate, o-toluidine and o-aminophenol to afford (7a-c). Demethylation of (1b) yielded the hydroxyl derivative (8), which differs from compound (9) obtained by demethylation of 6-methoxy-2,3-diphenyl-indole (1a). Friedel Craft's acetylation of 9 gave the 7-acetyl compound (10) which yielded the hydrazone (11). The reaction of primary aromatic amines, (i.e. p-nitroaniline, p-anisidine and p-bromo aniline) with 6-methoxy-1-methyl-2,3-diphenyl-indol-5-carboxaldehyde (2) gave the Schiff bases (12a-c). The latter compounds were reduced by sodium borohydride to yield the corresponding Mannich bases (13a-c). Treatment of 12a-c with thioglycolic acid led to the thiazolidin-4-one-derivatives (14a-c). When (12a-c) reacted with cyanoacetamide, the amino group was replaced by the active methylene to form the cyano compound (15). The structure was confirmed by reacting the carboxaldehyde (2) with cyanoacetamide to yield (15). Pharmacological screening was has been carried out to test the anti-inflammatory activity, ulcerogenecity, effect on the isolated rabbit intestine and the antispasmodic activity.

A new lignan glycoside from the rhizomes of Imperata cylindrica.

PubMed

Lee, Dae-Young; Han, Kyung-Min; Song, Myoung-Chong; Lee, Do-Gyeong; Rho, Yeong-Deok; Baek, Nam-In

2008-01-01

A new lignan glycoside, 6-acetyl-1-[1,3-(4,4'-dihydroxy-3,3'-dimethoxy-beta-truxinyl)-beta-d-fructofuranosyl]-alpha-d-glucopyranoside (1), named impecyloside, was isolated from the rhizomes of Imperata cylindrica. The structure of the compound was determined by spectroscopic data including FABMS, UV, IR, 1H NMR and 13C NMR (DEPT) and 2D NMR (COSY, HSQC, HMBC).

Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids

PubMed Central

Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V.; Zhang, Xiangli; Fristensky, Brian; Cicek, Nazim; Sparling, Richard; Levin, David. B.

2015-01-01

Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. ‘Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the ‘Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams. PMID:26544181

Compositional, Atomic and Molecular Analysis in Support of Materials Needs of the U.S. Air Force.

DTIC Science & Technology

1982-09-01

internally hydrogen-bonded monomer in .which the keto group is involved in the hydrogen bond but the acid carbonyl is not. 3 ) 3 - Bromopyruvic Acid...The spectra and structure of 3 - bromopyruvic acid were investigated and compared to those of pyruvic acid. It has been found that the spectra of 3 ...phase, cyclic monomer in dilute solution). The solid state spectra are quite different, however. The solid states spectra of 3 - bromopyruvic acid show a

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

Beta-cell metabolic alterations under chronic nutrient overload in rat and human islets

USDA-ARS?s Scientific Manuscript database

The aim of this study was to assess multifactorial Beta-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of ...

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

DTIC Science & Technology

2011-09-28

presented according to [51]. Kdo, 2- keto -3-deoxy-octulosonic acid; Ko, D-glycero-D-talo-oct-2-ulosonic acid; Hep, heptulose (ketoheptose); Glc, glucose...pestis. J Bacteriol 98: 1404–1406. 54. Jawetz E, Meyer KF (1943) Avirulent strains of Pasteurella pestis. J Infect Dis 73: 124– 143 . 55. Burrows TW

Optical isomer separation of potential analgesic drug candidates by using capillary electrophoresis.

PubMed

Ferrara, G; Santagati, N A; Aturki, Z; Fanali, S

1999-09-01

Using cyclodextrin capillary zone electrophoresis (CD-CZE), baseline separation of synthetic potential analgesic drug diastereoisomer candidates 6,11-dimethyl-1,2,3,4,5,6-hexahydro-3-[(2'-methoxycarbonyl-2'-phenylc yclopropyl)methyl]-2,6-methano-3-benzazocin-8-ol (MPCB) and 6,11-dimethyl-1,2,3,4,5,6-hexahydro-3-[[2'-methoxycarbonyl-2'(4-chloroph enyl)cyclopropyl]methyl]-2,6-methano-3-benzazocin-8-ol (CCB) was achieved. Among the cyclodextrins tested (hydroxypropyl-, carboxymethyl- and sulfobutyl-beta-cyclodextrin (HP-beta-CD, CM-beta-CD and SBE-beta-CD)) SBE-beta-CD was found to be the most effective complexing agent, allowing good optical isomer separation. Resolution was also influenced by the CD concentration, pH of the buffer and presence of organic modifier in the background electrolyte. The optimum experimental conditions for the separation of studied analgesic drugs were found using 25 mM borate buffer at pH 9 containing 40 mM of SBE-beta-CD and 20% v/v of methanol. Using the above-mentioned background electrolyte, it was also possible to separate, in the same run, the enantiomers of normetazocine (NMZ) as well as the optical isomers of (+/-)-cis-2-chloromethyl-1-phenyl cyclopropancarboxylic acid methyl ester (PCE) or (+/-)-cis-2-chloromethyl-1-(4-chlorophenyl)cyclopropancarboxylic acid methyl ester (CPCE) reagents used in the synthesis of the studied analgesic drugs).

Interaction of metal ions and amino acids - Possible mechanisms for the adsorption of amino acids on homoionic smectite clays

NASA Technical Reports Server (NTRS)

Gupta, A.; Loew, G. H.; Lawless, J.

1983-01-01

A semiempirical molecular orbital method is used to characterize the binding of amino acids to hexahydrated Cu(2+) and Ni(2+), a process presumed to occur when they are adsorbed in the interlamellar space of homoionic smectite clays. Five alpha-amino acids, beta-alanine, and gamma-aminobutyric acid were used to investigate the metal ion and amino acid specificity in binding. It was assumed that the alpha, beta, and gamma-amino acids would bind as bidentate anionic ligands, forming either 1:1 or 1:2 six-coordinated five, six, and seven-membered-ring chelate complexes, respectively. Energies of complex formation, optimized geometries, and electron and spin distribution were determined; and steric constraints of binding of the amino acids to the ion-exchanged cations in the interlamellar spacing of a clay were examined. Results indicate that hexahydrated Cu(2+) forms more stable complexes than hexahydrated Ni(2+) with all the amino acids studied. However, among these amino acids, complex formation does not favor the adsorption of the biological subset. Calculated energetics of complex formation and steric constraints are shown to predict that 1:1 rather than 1:2 metal-amino acid complexes are generally favored in the clay.

Identification and analysis of o-acetylated sialoglycoproteins.

PubMed

Mandal, Chandan; Mandal, Chitra

2013-01-01

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell-cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7-C8-C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES

DTIC Science & Technology

bound lipoic acid and 17 moles of bound FAD. Alpha -ketoglutarate dehydrogenase complex contains approximately 10 moles of protein-bound lipoic acid , 9...typical metal activators of oxidative decarboxylation reaction of alpha -keto acid . These activating effects were in good agreement with the results of...A coenzyme A- and NAD-linked pyruvate and alpha -ketoglutarate dehydrogenase complexes have been isolated from pig heart muscle as multienzyme units

Biting deterrence and insecticidal activity of hydrazide-hydrazones and their corresponding 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles against Aedes aegypti.

PubMed

Tabanca, Nurhayat; Ali, Abbas; Bernier, Ulrich R; Khan, Ikhlas A; Kocyigit-Kaymakcioglu, Bedia; Oruç-Emre, Emine E; Unsalan, Seda; Rollas, Sevim

2013-06-01

Taking into account the improvement in insecticidal activity by the inclusion of fluorine in the hydrazone moiety, the authors synthesized new 4-fluorobenzoic acid hydrazides and 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles, substituting a phenyl group or a heteroaryl ring carrying one or two atoms of F, Cl and Br, and investigated their biting deterrent and larvicidal activities against Aedes aegypti for the first time. The compound 3-acetyl-5-(4-fluorophenyl)-2-[4-(dimethylamino)phenyl]-2,3-dihydro-1,3,4-oxadiazole (17) produced the highest biting deterrent activity (BDI = 1.025) against Ae. Aegypti, followed by 4-fluorobenzoic acid [(phenyl)methylene] hydrazide (1). These activity results were similar to those of N,N-diethyl-meta-toluamide (DEET), which showed a proportion not biting of 0.8-0.92. When compounds 1 and 17 were tested on cloth worn on human volunteers, compound 1 was not repellent for some volunteers until present in excess of 500 nmol cm(-2) , while compound 17 was not repellent at the highest concentration tested (1685 nmol cm(-2) ). In the larvicidal screening bioassays, only compounds 10, 11, 12 and 17 showed 100% mortality at the highest screening dose of 100 ppm against Ae. aegypti larvae. Compounds 11 and 12 with LD50 values of 24.1 and 30.9 ppm showed significantly higher mortality than 10 (80.3 ppm) and 17 (58.7 ppm) at 24-h post-treatment. The insecticidal and biting deterrent activities were correlated with the presence of a halogen atom on the phenyl or heteroaryl substituent of the hydrazone moiety. © 2012 Society of Chemical Industry.

Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis.

PubMed

Choi, Kwon-Young; Wernick, David G; Tat, Christine A; Liao, James C

2014-05-01

The non-recyclable use of nitrogen fertilizers in microbial production of fuels and chemicals remains environmentally detrimental. Conversion of protein wastes into biofuels and ammonia by engineering nitrogen flux in Escherichia coli has been demonstrated as a method to reclaim reduced-nitrogen and curb its environmental deposition. However, protein biomass requires a proteolysis process before it can be taken up and converted by any microbe. Here, we metabolically engineered Bacillus subtilis to hydrolyze polypeptides through its secreted proteases and to convert amino acids into advanced biofuels and ammonia fertilizer. Redirection of B. subtilis metabolism for amino-acid conversion required inactivation of the branched-chain amino-acid (BCAA) global regulator CodY. Additionally, the lipoamide acyltransferase (bkdB) was deleted to prevent conversion of branched-chain 2-keto acids into their acyl-CoA derivatives. With these deletions and heterologous expression of a keto-acid decarboxylase and an alcohol dehydrogenase, the final strain produced biofuels and ammonia from an amino-acid media with 18.9% and 46.6% of the maximum theoretical yield. The process was also demonstrated on several waste proteins. The results demonstrate the feasibility of direct microbial conversion of polypeptides into sustainable products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Determination of Clara cell protein urinary elimination as a marker of tubular dysfunction.

PubMed

Martín-Granado, Ascensión; Vázquez-Moncholí, Carmen; Luis-Yanes, María Isabel; López-Méndez, Marisela; García-Nieto, Víctor

2009-04-01

Clara cell 16-kDa protein (CC16) is a protein expressed primarily by the bronchial cells. It is rapidly eliminated by glomerular filtration, reabsorbed almost entirely, and catabolized in proximal tubule cells. To date, normal values for urinary CC16 in healthy children have not been determined. We have studied 63 pediatric patients (mean age 8.17 +/- 3.91 years) and 31 healthy children (control group; mean age 8.83 +/- 3.65 years). In the control group, the CC16/creatinine ratio was 1.22 +/- 1.52 microg/g. In 16 out of 31 control children, the value of the ratio was zero. Fourteen patients (22.2%) showed a high CC16/creatinine ratio; in contrast, among these same patients, the ratio N-acetyl-beta-D: -glucosaminidase (NAG)/creatinine was elevated in seven cases (11.1%) and the ratio beta2-microglobulin/creatinine was elevated in seven cases (11.1%). The three parameters were in agreement in 51 patients (80.9%). Among the patients, the CC16/creatinine ratio was correlated with both the beta2-microglobulin/creatinina ratio (r = 0.76, P < 0.001) and the NAG/creatinine ratio (r = 0.6, P < 0.001). Our findings indicate that CC16 is a good marker of proximal tubular function in childhood. The highest observed values were in children with proximal tubulopathies, in children with chronic renal failure, and in those treated with cyclosporine.

Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum.

PubMed

Schräder, T; Andreesen, J R

1992-05-15

Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl-Sepharose and Sepharose S. The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an alpha 4 beta 4 composition. The smaller subunit was identified as an acetyl-group-transferring protein, the 57-kDa protein was hydrophobic. N-terminal amino acid sequences were determined for both subunits. Antibodies raised against the 48-kDa subunit showed cross-reactions with extracts of E. acidaminophilum grown on different substrates and with extracts from other glycine-utilizing anaerobic bacteria such as Clostridium purinolyticum, C. sticklandii, and C. sporogenes. The respective protein from the former two organisms corresponded in molecular mass. When protein PA was chemically carboxymethylated by iodo[2-14C]acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E. acidaminophilum.

Phytochemical Study of the Ecuadorian Species Lepechinia mutica (Benth.) Epling and High Antifungal Activity of Carnosol against Pyricularia oryzae.

PubMed

Ramírez, Jorge; Gilardoni, Gianluca; Ramón, Erika; Tosi, Solveig; Picco, Anna Maria; Bicchi, Carlo; Vidari, Giovanni

2018-04-19

The plant Lepechinia mutica (Benth.) Epling (family Lamiaceae) is endemic to Ecuador. In the present study, we report some major non-volatile secondary metabolites from the leaves and the chemistry of the essential oil distilled from the flowers. The main identified compounds were carnosol, viridiflorol, ursolic acid, oleanolic acid, chrysothol, and 5-hydroxy-4′,7-dimethoxy flavone. Their structures were determined by X-ray diffraction and NMR and MS techniques. The essential oil showed a chemical composition similar to that distilled from the leaves, but with some qualitative and quantitative differences regarding several minor compounds. The main constituents (>4%) were: δ-3-carene (24.23%), eudesm-7(11)-en-4-ol (13.02%), thujopsan-2-α-ol (11.90%), β-pinene (7.96%), valerianol (5.19%), and co-eluting limonene and β-phellandrene (4.47%). The volatile fraction was also submitted to enantioselective analysis on a β-cyclodextrin column, obtaining the separation and identification of the enantiomers for α-thujene, β-pinene, sabinene, α-phellandrene, limonene and β-phellandrene. Furthermore, the anti-fungal activity of non-volatile secondary metabolites was tested in vitro, with carnosol resulting in being very active against the “blast disease” caused by the fungus Pyricularia oryzae .

Highly Functionalized 1,2–Diamino Compounds through Reductive Amination of Amino Acid-Derived β–Keto Esters

PubMed Central

Pérez-Faginas, Paula; Aranda, M. Teresa; García-López, M. Teresa; Infantes, Lourdes; Fernández-Carvajal, Asia; González-Ros, José Manuel; Ferrer-Montiel, Antonio; González-Muñiz, Rosario

2013-01-01

1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived β–keto esters are a suitable starting point for the synthesis of β,γ–diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α–amino esters. AcOH and NaBH3CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the β,γ–diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity. PMID:23308167

Effects of water temperature and diets containing palm oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes of rainbow trout (Oncorhynchus mykiss).

PubMed

Tocher, Douglas R; Fonseca-Madrigal, Jorge; Dick, James R; Ng, Wing-Keong; Bell, J Gordon; Campbell, Patrick J

2004-01-01

Food grade fisheries have reached their sustainable limits while aquaculture production has increased to meet consumer demands. However, for growth in aquaculture to continue and utilise sustainable, feeding ingredients, alternatives to fish oil (FO), the predominant lipid component of fish diets, must be developed. Therefore, there is currently considerable interest in the regulation of fatty acid metabolism in fish in order to determine strategies for the best use of plant oils in diets for commercially important cultured fish species. Plant oils are characteristically rich in C18 polyunsaturated fatty acids (PUFA) but devoid of C20 and C22 highly unsaturated fatty acids (HUFA) found in FO. The fatty acyl desaturase enzyme activities involved in the biosynthesis of HUFA from PUFA are known to be under nutritional regulation and can be increased in fish fed diets rich in plant oils. However, fatty acid desaturase activity is also known to be modulated by water temperature in fish. The present study aimed to investigate the interaction between water temperature and diet in the regulation of fatty acid metabolism in rainbow trout. Trout, acclimatized to 7, 11 or 15 degrees C, were fed for 4 weeks on diets in which the FO was replaced in a graded manner by palm oil. At the end of the trial, fatty acyl desaturation/elongation and beta-oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1-14C]18:3n-3 as substrate, and samples of liver were collected for analysis of lipid and fatty acid composition. The most obvious effect of temperature was that fatty acid desaturation/elongation and beta-oxidation were reduced in both hepatocytes and intestinal enterocytes from fish maintained at the highest water temperature (15 degrees C). There were differences between the two tissues with the highest desaturation/elongation and beta-oxidation activities tending to be in fish held at 11 degrees C in the case of hepatocytes, but 7 degrees C in enterocytes. Correlations between fatty acid metabolism and dietary palm oil were most clearly observed in desaturation/elongation activities in both hepatocytes and enterocytes at 11 degrees C. The highest beta-oxidation activities were generally observed in fish fed FO alone in both hepatocytes and enterocytes with palm oil having differential effects in the two cell types.

Holy smoke in medieval funerary rites: chemical fingerprints of frankincense in southern Belgian incense burners.

PubMed

Baeten, Jan; Deforce, Koen; Challe, Sophie; De Vos, Dirk; Degryse, Patrick

2014-01-01

Frankincense, the oleogum resin from Boswellia sp., has been an early luxury good in both Western and Eastern societies and is particularly used in Christian funerary and liturgical rites. The scant grave goods in late medieval burials comprise laterally perforated pottery vessels which are usually filled with charcoal. They occur in most regions of western Europe and are interpreted as incense burners but have never been investigated with advanced analytical techniques. We herein present chemical and anthracological results on perforated funerary pots from 4 Wallonian sites dating to the 12-14th century AD. Chromatographic and mass spectrometric analysis of lipid extracts of the ancient residues and comparison with extracts from four Boswellia species clearly evidence the presence of degraded frankincense in the former, based on characteristic triterpenoids, viz. boswellic and tirucallic acids, and their myriad dehydrated and oxygenated derivatives. Cembrane-type diterpenoids indicate B. sacra (southern Arabia) and B. serrata (India) as possible botanical origins. Furthermore, traces of juniper and possibly pine tar demonstrate that small amounts of locally available fragrances were mixed with frankincense, most likely to reduce its cost. Additionally, markers of ruminant fats in one sample from a domestic context indicate that this vessel was used for food preparation. Anthracological analysis demonstrates that the charcoal was used as fuel only and that no fragrant wood species were burned. The chars derived from local woody plants and were most likely recovered from domestic fires. Furthermore, vessel recycling is indicated by both contextual and biomarker evidence. The results shed a new light on funerary practices in the Middle Ages and at the same time reveal useful insights into the chemistry of burned frankincense. The discovery of novel biomarkers, namely Δ2-boswellic acids and a series of polyunsaturated and aromatic hydrocarbons, demonstrates the high potential for organic chemical analyses of incense residues.

Holy Smoke in Medieval Funerary Rites: Chemical Fingerprints of Frankincense in Southern Belgian Incense Burners

PubMed Central

Baeten, Jan; Deforce, Koen; Challe, Sophie; De Vos, Dirk; Degryse, Patrick

2014-01-01

Frankincense, the oleogum resin from Boswellia sp., has been an early luxury good in both Western and Eastern societies and is particularly used in Christian funerary and liturgical rites. The scant grave goods in late medieval burials comprise laterally perforated pottery vessels which are usually filled with charcoal. They occur in most regions of western Europe and are interpreted as incense burners but have never been investigated with advanced analytical techniques. We herein present chemical and anthracological results on perforated funerary pots from 4 Wallonian sites dating to the 12–14th century AD. Chromatographic and mass spectrometric analysis of lipid extracts of the ancient residues and comparison with extracts from four Boswellia species clearly evidence the presence of degraded frankincense in the former, based on characteristic triterpenoids, viz. boswellic and tirucallic acids, and their myriad dehydrated and oxygenated derivatives. Cembrane-type diterpenoids indicate B. sacra (southern Arabia) and B. serrata (India) as possible botanical origins. Furthermore, traces of juniper and possibly pine tar demonstrate that small amounts of locally available fragrances were mixed with frankincense, most likely to reduce its cost. Additionally, markers of ruminant fats in one sample from a domestic context indicate that this vessel was used for food preparation. Anthracological analysis demonstrates that the charcoal was used as fuel only and that no fragrant wood species were burned. The chars derived from local woody plants and were most likely recovered from domestic fires. Furthermore, vessel recycling is indicated by both contextual and biomarker evidence. The results shed a new light on funerary practices in the Middle Ages and at the same time reveal useful insights into the chemistry of burned frankincense. The discovery of novel biomarkers, namely Δ2-boswellic acids and a series of polyunsaturated and aromatic hydrocarbons, demonstrates the high potential for organic chemical analyses of incense residues. PMID:25391130

Interleukin-1beta-induced hyperresponsiveness to [Sar9,Met(O2)11]substance P in isolated human bronchi.

PubMed

Barchasz, E; Naline, E; Molimard, M; Moreau, J; Georges, O; Emonds-Alt, X; Advenier, C

1999-08-20

Interleukin-1beta has been reported to induce airway hyperresponsiveness in several animal models. In this study, we have investigated whether interleukin-1beta was able to potentiate the contractions of human isolated small bronchi (internal diameter < or = 1 mm) provoked by a specific tachykinin NK1 receptor agonist, [Sar9,Met(O2)11]substance P. Pre-incubation of human isolated small bronchi with interleukin-1beta (10 ng/ml, in Krebs-Henseleit solution, at 21 degrees C for 15 h) potentiated the contractile response to [Sar9,Met(O2)11]substance P. It also increased the [Sar9,Met(O2)11]substance P-induced release of thromboxane B2, the stable metabolite of thromboxane A2. Indomethacin (10(-6) M), a non-specific cyclooxygenase inhibitor, or GR 32191 ((1R-(1alpha(Z)),2beta,3beta,5alpha))-(+)-7-(5-(((1,1' -biphenyl)-4-yl)-methoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-hept enoic acid, hydrochloride) (10(-6) M), a prostanoid TP-receptor antagonist, blocked the contractions induced by [Sar9,Met(O2)11]substance P both in control experiments and after interleukin-1beta pre-treatment, indicating that prostanoids and thromboxane receptors are directly implicated in the [Sar9,Met(O2)11]substance P-induced contractile response. The thromboxane mimetic U-46619 (10(-8)-10(-6) M) (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F2alpha)-induced contractions of human isolated small bronchi were not enhanced by interleukin-1beta pre-treatment, suggesting that no up-regulation of thromboxane receptors occurred. Furthermore, the cyclooxygenase-2 inhibitor CGP 28238 (6-(2,4-difluorophenoxy)-5-methyl-sulfonylamino-1-indanon e) (10(-6) M) had no direct effect on [Sar9,Met(O2)11]substance P-provoked contractions, but inhibited the interleukin-1beta-induced potentiation of [Sar9,Met(O2)11]substance P response. In conclusion, our results show that interleukin-1beta pre-treatment is able to potentiate the contractions of isolated human small bronchi provoked by [Sar9,Met(O2)11]substance P both by increasing prostanoid synthesis and by inducing a cyclooxygenase-2 pathway.

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes.

PubMed

Harnik, M; Kashman, Y; Cojocaru, M; Bauer, H; Laux, M; Lewicka, S; Vecsei, P

1990-10-01

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%

Effect of acetyl salicylic acid on increased production of thromboxane after aortic graft surgery.

PubMed

Lewin, J; Swedenborg, J; Egberg, N; Vesterqvist, O; Green, K

1989-06-01

Contact between blood and foreign surfaces, e.g. vascular grafts, causes activation and release of platelets. One consequence of platelet activation is production of thromboxane A2 (TxA2). The physiological effects of TxA2, i.e. platelet aggregation and vaso-constriction are counteracted by another prostanoid, prostacyclin (PGI2). Acetylsalicylic acid (ASA) causes a longlasting inhibition of platelet TxA2 production and a more shortlasting inhibition of PGI2 production. The present study examines TxA2 and PGI2 synthesis in patients receiving synthetic arterial grafts, some of which were treated with ASA. The prostanoid synthesis was evaluated by measurement of their main urinary metabolites with gas chromatography-mass spectrometry. Platelet release was evaluated by measurements of beta-thromboglobulin (beta-TG) and the plasma coagulation by measurements of fibrinopeptide A (FPA). These compounds were also measured in urine in order to avoid artifacts caused by activation of platelets and plasma coagulation during blood sampling. Following replacement of the abdominal aorta with a synthetic vascular graft there was a marked increase in the synthesis of TxA2 and PGI2. Increased levels of beta-TG and FPA were also demonstrated. Administration of ASA on the first and second postoperative days significantly reduced the synthesis of TxA2 but caused no significant effects on the other parameters measured. It is concluded that ASA may be beneficial in the postoperative period since it counteracts TxA2 with vasoconstricting and platelet aggregating properties but leaves PGI2 with vasodilating and antiaggregating properties relatively uneffected.

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.

PubMed

Kajiwara, S; Kakizono, T; Saito, T; Kondo, K; Ohtani, T; Nishio, N; Nagai, S; Misawa, N

1995-10-01

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells*

PubMed Central

Galdieri, Luciano; Gatla, Himavanth; Vancurova, Ivana; Vancura, Ales

2016-01-01

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. PMID:27733682

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

PubMed

Choi, Mun Hwan; Xu, Ju; Rho, Jong Kook; Zhao, Xu Ping; Yoon, Sung Chul

2010-06-01

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07. (c) 2010 Elsevier Ltd. All rights reserved.

[C-11]{beta}CNT: A new monoamine uptake ligand for studying serotonin and dopamine transporter sites in the living brain with PET

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

Mulholland, G.K.; Zheng, Q.H.; Zhou, F.C.

1996-05-01

There is considerable interest in measuring serotonin (5HT) and dopamine (DA) function in the human brain. Altered levels of 5HT and DA are recognized in drug abuse, neurotoxicities, psychiatric disorders, and neurodegenerative conditions including Alzheimer`s and Parkinson`s disease. Several phenyltropane analogs of cocaine bind tightly to both DA and 5HT uptake proteins. We have made a new agent from this class called {beta}CNT, 2{beta}-carboxymethyl-3{beta}-(2-naphthyl)-tropane, the isosteric O-for-CH{sub 2} analog of a compound reported to have among the highest measured affinities for DA and 5HT transporters and studied its in vivo brain distributions in animals for the first time. Optically puremore » {beta}CNT was made from cocaine, and labeled at the O-methyl position by esterification of {beta}CNT-acid with [C-11]CH{sub 3}OTfl under conditions similar to Wilson`s. HPLC-purified (99+%) final products (15-50% eob yield from CO{sub 2}, 40 min synth) had specific activities 0.1-1.2 Ci/{mu}mol at the time of injection. Preliminary [C-11]{beta}{beta}CNT rodent distribution showed very high brain uptake (3% ID at 60 min) and localization (striat: fr cort: hypo: cer: blood, 11: 5: 4: 1: 06). {beta}CNT-PET studies in juvenile pigs (5-20 mCi, 20-35 kg) found rapid brain uptake, and prominent retention (85 min) in midbrain, anterior brainstem and striatum, followed by cortex and olfactory bulb. Paroxetine pretreatment (5HT uptake blocker, 2mg/kg), diminished retention in most brain areas; nomifensine (DA/NE uptake blocker, 6 mg/kg) reduced striatum selectively. Direct comparisons of [C-11]{beta}CNT with other PET transporter radioligands {beta}CFT, {beta}CIT, and {beta}CTT (RTI-32) in the same pig found {beta}CNT had highest overall brain uptake among the agents. These initial results suggest {beta}CNT has favorable properties for imaging both 5HT and DA transporters in vivo, and further evaluation of its potential as a human PET agent is warranted.« less

Insights into the development of fungal biomarkers for metal ecotoxicity assessment: case of Trametes versicolor exposed to copper.

PubMed

Lebrun, Jérémie D; Trinsoutrot-Gattin, Isabelle; Laval, Karine; Mougin, Christian

2010-04-01

The relationship between the physiological state of fungi and the response of their functional system to metals is not known, limiting the use of fungal enzymes as tools for assessing metal ecotoxicity in terrestrial ecosystems. The present study attempts to establish how the development phases modulate the secretion of enzymes in the filamentous fungus Trametes versicolor after exposure to Cu. For that purpose, extracellular hydrolases (acid and alkaline phosphatases, aryl-sulfatase, beta-glucosidase, beta-galactosidase, and N-acetyl-beta-glucosaminidase) and oxidoreductases (laccase, manganese and lignin peroxidases) were monitored in liquid cultures for 2 weeks. Copper was added during either the growth or the stationary phases at 20 or 200 ppm. Results of the present study showed that Cu at the highest concentration modifies the secretion of enzymes, regardless of the development phase to which the fungus was exposed. However, the sensitivity of enzyme responses to Cu depended on the phase development and the type of secreted enzyme. In a general way, the production of hydrolases was decreased by Cu, whereas that of oxidoreductases was highly increased. Furthermore, lignin peroxidase was not detected in control cultures and was specifically produced in the presence of Cu. In conclusion, fungal oxidoreductases may be enzymatic biomarkers of copper exposure for ecotoxicity assessment. (c) 2009 SETAC.

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

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

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

Characterization of alpha-ketobutyrate metabolism in rat tissues: effects of dietary protein and fasting.

PubMed

Steele, R D; Weber, H; Patterson, J I

1984-04-01

The oxidative decarboxylation of alpha-ketobutyrate was studied in rat tissue preparations. Decarboxylation was confined to the mitochondrial fraction and required coenzyme A, NAD, TPP and FAD for optimal activity in solubilized preparations. The pH optimum for this reaction in liver was 7.8, somewhat higher than that reported for other alpha-keto acid dehydrogenases. An apparent Km of 0.63 mM for alpha-ketobutyrate was determined for the rat liver system. Competition by other alpha-keto acids at 10 mM concentrations inhibited enzyme activity up to 75%. Tissue distribution of alpha-ketobutyrate dehydrogenase activity relative to liver activity was (in percent): liver, 100; heart, 127; brain, 63; kidney, 57; skeletal muscle, 38; and small intestine, 7. Total liver alpha-ketobutyrate dehydrogenase was decreased by 40% after a 24-hour fast. Similar results were found for kidney and heart activity. alpha-Aminobutyrate-pyruvate aminotransferase activity in liver or kidney was not affected by fasting; however, it was induced in liver by 50% after feeding a 40% casein diet for 10 days compared to rats fed a 20% casein diet. Increasing the dietary casein content from 6 through 40% of the diet resulted in about a fivefold increase in liver alpha-ketobutyrate dehydrogenase activity. The substantial extrahepatic capacity for alpha-ketobutyrate metabolism makes it unlikely that a loss of liver function results in an inability to metabolize alpha-ketobutyrate. Whether alpha-ketobutyrate is decarboxylated by a specific enzyme or by an already characterized complex such as pyruvate dehydrogenase or the branched-chain keto acid dehydrogenase remains to be established.

Specific tritium labeling of gangliosides at the 3-position of sphingosines.

PubMed

Ghidoni, R; Sonnino, S; Masserini, M; Orlando, P; Tettamanti, G

1981-11-01

GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively.

Unique occurrence of unusual fatty acids and their industrial utilization

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

Hosamani, K.M.

1996-01-01

Ebenaceae plant family consists of 7 genera and more than 320 species. The present investigation describes the unique occurrence of hitherto unknown 9-keto-cis-13-octadecenoic acid (29.0%) and the cyclopropenoid fatty acids (malvalic acid 12.7% and sterculic acid 8.7%) in the seed oil of Diospyros melanoxylon as well as in the Ebenaceae plant family. Other normal fatty acids are also detected. The identification and characterization were based on FTIR, {sup 1}H NMR, {sup 13}C NMR, MS, and GLC techniques and chemical degradations.

Metabolomic Analysis of the Secretome of Human Embryonic Stem Cells Following Methyl Parathion and Methyl Paraoxon Exposure Phase 2: Metabolite Downselection for Structural Confirmation

DTIC Science & Technology

2013-12-01

degradation 2 Pipecolic acid II 2-keto-6- aminocaproate II Pyruvate metabolism 1 Malic acid I Purine metabolism 1 Guanine I Propanoate metabolism 1...acetamidobutanoic acid II cis-4-hydroxy-D-proline II D-arginine and D-ornithine metabolism 4 Ornithine II 5-amino-2-oxopentanoic acid II 2-amino-4-oxo...pentanoic acid II (2R,4S)-2,4-diaminopentanoate II Gly, Ser, and Thr metabolism 3 L-cystathionine II Choline II 5-aminolevulinic acid II Val

Three-residue turns in alpha/beta-peptides and their application in the design of tertiary structures.

PubMed

Sharma, Gangavaram V M; Nagendar, Pendem; Ramakrishna, Kallaganti V S; Chandramouli, Nagula; Choudhary, Madavi; Kunwar, Ajit C

2008-06-02

A new three-residue turn was serendipitously discovered in alpha/beta hybrid peptides derived from alternating C-linked carbo-beta-amino acids (beta-Caa) and L-Ala residues. The three-residue beta-alpha-beta turn at the C termini, nucleated by a helix at the N termini, resulted in helix-turn (HT) supersecondary structures in these peptides. The turn in the HT motif is stabilized by two H bonds-CO(i-2)-NH(i), with a seven-membered pseudoring (gamma turn) in the backward direction, and NH(i-2)-CO(i), with a 13-membered pseudoring in the forward direction (i being the last residue)--at the C termini. The study was extended to generalize the new three-residue turn (beta-alpha-beta) by using different alpha- and beta-amino acids. Furthermore, the HT motifs were efficiently converted, by an extension with helical oligomers at the C termini, into peptides with novel helix-turn-helix (HTH) tertiary structures. However, this resulted in the destabilization of the beta-alpha-beta turn with the concomitant nucleation of another three-residue turn, alpha-beta-beta, which is stabilized by 11- and 15-membered bifurcated H bonds. Extensive NMR spectroscopic studies were carried out to delineate the secondary and tertiary structures in these peptides, which are further supported by molecular dynamics (MD) investigations.

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11.

PubMed

Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2015-01-01

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.