Non-critically phase-matched second harmonic generation and third order nonlinearity in organic crystal glucuronic acid γ-lactone

NASA Astrophysics Data System (ADS)

Saripalli, Ravi Kiran; Katturi, Naga Krishnakanth; Soma, Venugopal Rao; Bhat, H. L.; Elizabeth, Suja

2017-12-01

The linear, second order, and third order nonlinear optical properties of glucuronic acid γ-lactone single crystals were investigated. The optic axes and principal dielectric axes were identified through optical conoscopy and the principal refractive indices were obtained using the Brewster's angle method. Conic sections were observed which is perceived to be due to spontaneous non-collinear phase matching. The direction of collinear phase matching was determined and the deff evaluated in this direction was 0.71 pm/V. Open and closed aperture Z-scan measurements with femtosecond pulses revealed high third order nonlinearity in the form of self-defocusing, two-photon absorption, as well as saturable absorption.

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).

Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme.

PubMed

Mochizuki, Hideo; Yamagishi, Kiwamu; Suzuki, Kiyoshi; Kim, Yeong Shik; Kimata, Koji

2015-07-01

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides

PubMed Central

Hsieh, Po-Hung; Xu, Yongmei; Keire, David A; Liu, Jian

2014-01-01

Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate. PMID:24770491

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation.

PubMed

Griffeth, L K; Rosen, G M; Rauckman, E J

1985-01-01

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.

The value of grip test, lysophosphatidlycholines, glycerophosphocholine, ornithine, glucuronic acid decrement in assessment of nutritional and metabolic characteristics in hepatitis B cirrhosis

PubMed Central

Zhang, Lei; Xiao, Huijuan; Qi, Yumei; Liu, Shuye; Qian, Baoxin; Wang, Fengmei; Han, Tao

2017-01-01

The liver is essential for the regulation of energy, protein and amino acids, as well as in other aspects of metabolism. To identify efficient indexes for evaluation of nutritional status and metabolic characteristics during different Child-Pugh stages of hepatitis B cirrhosis, 83 patients and 35 healthy individuals were enrolled in our study. We found that grip strength, triceps skinfold thickness (TSF), body fat and skeletal muscle of the patients were reduced compared to the control group (P<0.05). Ultra-high-performance liquid chromatography data combined with mass spectrometry (UPLC-MS) showed that levels of a variety of metabolites, including lysophosphatidylcholines (LysoPCs), glycerophosphocholine, ornithine and glucuronic acid were reduced in the serum of patients with hepatitis B cirrhosis (P<0.001). However, glycerophosphoserine and taurocholic acid levels were higher than in the control group (P<0.001). Moreover, grip strength was correlated with the Child-Pugh score (P<0.05). Serum albumin, total cholesterol, LDL, LysoPCs, glycerophosphocholine, ornithine, glucuronic acid, glycerophosphoserine and taurocholic acid were correlated with the Child-Pugh score (P<0.01). These findings suggested that grip strength and the above small molecular substances might be considered as sensitive and important indexes for evaluating nutritional status and metabolic characteristics of patients with hepatitis B cirrhosis, which may help assess prognosis and adjust nutritional treatment. PMID:28384211

Characterization of a uronate dehydrogenase from Thermobispora bispora for production of glucaric acid from hemicellulose substrate.

PubMed

Li, Yaxian; Xue, Yemin; Cao, Zhigang; Zhou, Tao; Alnadari, Fawze

2018-06-23

A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60 °C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1 h at 60 °C. The K m and V max values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165 mM and 117.7 U mg -1 , respectively, those for galacturonic acid (GalUA) were 0.115 mM and 104.2 U mg -1 , respectively, and those for NAD + were 0.120 mM and 133.3 U mg -1 , respectively; the turnover number (k cat ) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (K m ) for GalUA was lower than that for GluUA. After 60 min of incubation at 50 °C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.

Different temperatures select distinctive acetic acid bacteria species and promotes organic acids production during Kombucha tea fermentation.

PubMed

De Filippis, Francesca; Troise, Antonio Dario; Vitaglione, Paola; Ercolini, Danilo

2018-08-01

Kombucha is a traditional beverage produced by tea fermentation, carried out by a symbiotic consortium of bacteria and yeasts. Acetic Acid Bacteria (AAB) usually dominate the bacterial community of Kombucha, driving the fermentative process. The consumption of this beverage was often associated to beneficial effects for the health, due to its antioxidant and detoxifying properties. We characterized bacterial populations of Kombucha tea fermented at 20 or 30 °C by using culture-dependent and -independent methods and monitored the concentration of gluconic and glucuronic acids, as well as of total polyphenols. We found significant differences in the microbiota at the two temperatures. Moreover, different species of Gluconacetobacter were selected, leading to a differential abundance of gluconic and glucuronic acids. Copyright © 2018 Elsevier Ltd. All rights reserved.

Lactic acid bacteria: promising supplements for enhancing the biological activities of kombucha.

PubMed

Nguyen, Nguyen Khoi; Dong, Ngan Thi Ngoc; Nguyen, Huong Thuy; Le, Phu Hong

2015-01-01

Kombucha is sweetened black tea that is fermented by a symbiosis of bacteria and yeast embedded within a cellulose membrane. It is considered a health drink in many countries because it is a rich source of vitamins and may have other health benefits. It has previously been reported that adding lactic acid bacteria (Lactobacillus) strains to kombucha can enhance its biological functions, but in that study only lactic acid bacteria isolated from kefir grains were tested. There are many other natural sources of lactic acid bacteria. In this study, we examined the effects of lactic acid bacteria from various fermented Vietnamese food sources (pickled cabbage, kefir and kombucha) on kombucha's three main biological functions: glucuronic acid production, antibacterial activity and antioxidant ability. Glucuronic acid production was determined by high-performance liquid chromatography-mass spectrometry, antibacterial activity was assessed by the agar-well diffusion method and antioxidant ability was evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity. Four strains of food-borne pathogenic bacteria were used in our antibacterial experiments: Listeria monocytogenes ATCC 19111, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028 and Bacillus cereus ATCC 11778. Our findings showed that lactic acid bacteria strains isolated from kefir are superior to those from other sources for improving glucuronic acid production and enhancing the antibacterial and antioxidant activities of kombucha. This study illustrates the potential of Lactobacillus casei and Lactobacillus plantarum isolated from kefir as biosupplements for enhancing the bioactivities of kombucha.

Intake of Hydrolyzed Casein is Associated with Reduced Body Fat Accretion and Enhanced Phase II Metabolism in Obesity Prone C57BL/6J Mice

PubMed Central

Clausen, Morten Rahr; Zhang, Xumin; Yde, Christian C.; Ditlev, Ditte B.; Lillefosse, Haldis H.; Madsen, Lise; Kristiansen, Karsten; Liaset, Bjørn; Bertram, Hanne C.

2015-01-01

The amount and form of dietary casein have been shown to affect energy metabolism and lipid accumulation in mice, but the underlying mechanisms are not fully understood. We investigated 48 hrs urinary metabolome, hepatic lipid composition and gene expression in male C57BL/6J mice fed Western diets with 16 or 32 energy% protein in the form of extensively hydrolyzed or intact casein. LC-MS based metabolomics revealed a very strong impact of casein form on the urinary metabolome. Evaluation of the discriminatory metabolites using tandem mass spectrometry indicated that intake of extensively hydrolyzed casein modulated Phase II metabolism associated with an elevated urinary excretion of glucuronic acid- and sulphate conjugated molecules, whereas glycine conjugated molecules were more abundant in urine from mice fed the intact casein diets. Despite the differences in the urinary metabolome, we observed no differences in hepatic expression of genes involved in Phase II metabolism, but it was observed that expression of Abcc3 encoding ATP binding cassette c3 (transporter of glucuronic acid conjugates) was increased in livers of mice fed hydrolyzed casein. As glucuronic acid is derived from glucose and sulphate is derived from cysteine, our metabolomic data provided evidence for changes in carbohydrate and amino acid metabolism and we propose that this modulation of metabolism was associated with the reduced glucose and lipid levels observed in mice fed the extensively hydrolyzed casein diets. PMID:25738501

Bioactive and metal uptake studies of carboxymethyl chitosan-graft-D-glucuronic acid membranes for tissue engineering and environmental applications.

PubMed

Jayakumar, R; Rajkumar, M; Freitas, H; Sudheesh Kumar, P T; Nair, S V; Furuike, T; Tamura, H

2009-08-01

Carboxymethyl chitosan-graft-D-glucuronic acid (CMCS-g-D-GA) was prepared by grafting D-GA onto CMCS in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and then the membranes were made from it. In this work, the bioactivity studies of CMCS-g-D-GA membranes were carried out and then characterized by SEM, CLSM, XRD and FT-IR. The CMCS-g-D-GA membranes were found to be bioactive. The adsorption of Ni2+, Zn2+ and Cu2+ ions onto CMCS-g-D-GA membranes has also been investigated. The maximum adsorption capacity of CMCS-g-D-GA for Ni2+, Zn2+ and Cu2+ was found to be 57, 56.4 and 70.2 mg/g, respectively. Hence, these membranes were useful for tissue engineering, environmental and water purification applications.

Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov., Antarctic basidioblastomycetes

NASA Technical Reports Server (NTRS)

Vishniac, H. S.

1985-01-01

New yeasts from the Ross Desert (dry valley area) of Antarctica include Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov. Cryptococcus socialis MYSW A801-3aY1 (= ATCC 56685) requires no vitamins, assimilates L-arabinose, cellobiose, D-glucuronate, maltose, melezitose, raffinose, soluble starch, sucrose, and trehalose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production, cellobiose assimilation, and failure to utilize nitrate, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%. Cryptococcus consortionis MYSW A801-3aY92 (= ATCC 56686) requires thiamine, assimilates L-arabinose, D-glucuronate, 2-ketogluconate, salicin, succinate, sucrose, trehalose, and D-xylose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production and failure to utilize nitrate, cellobiose, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%.

FT-IR study of the polysaccharides isolated from the skin juice, gel juice, and flower of Aloe vera tissues affected by fertilizer treatment

PubMed Central

2012-01-01

Background This experiment was conducted to evaluate the effect of different amounts of fertilizers on the polysaccharides of Aloe vera plant. There were four different treatments, viz. T1 = 150% N, T2 = 150% P, T3 = 150% K, and T4 = 150% NPK (50% N + 50% P + 50% K) soil. Crude water-soluble polysaccharides were isolated from the gel juice, skin juice, and flowers of A. vera planted in these soils. Results Result indicates that skin juice contained 2.4 times the level of polysaccharides in gel juice from one plant, suggesting the potential industrial application of A. vera skin rather than discarding it. After anion-exchange chromatography, neutral polysaccharides accounted for 58.1% and 78.5% of the total recovered neutral and acidic polysaccharide preparations from the gel juice and skin juice, respectively, whereas the crude flower polysaccharides were largely composed of weakly acidic polysaccharides (84.2%). Sugar analysis of the polysaccharides after gel permeation chromatography revealed that glucose and galactose were the most abundant monosaccharide in the neutral polysaccharides from the gel juice and skin juice, respectively. The acidic polysaccharides from the two juices consisted of glucuronic acid, galactose, glucose, mannose, and xylose with variable proportions. Conclusions Except glucuronic acid (15.4%) in flower acidic polysaccharide, the flower neutral and acidic polysaccharides contained galactose, glucose, and mannose as the main sugar components. Glucuronic acid was the major uronic acid in all acidic polysaccharides from different tissues. PMID:23095284

Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy.

PubMed

Lyczakowski, Jan J; Wicher, Krzysztof B; Terrett, Oliver M; Faria-Blanc, Nuno; Yu, Xiaolan; Brown, David; Krogh, Kristian B R M; Dupree, Paul; Busse-Wicher, Marta

2017-01-01

Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce ( Picea glauca ) gene, PgGUX , encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.

Glucuronoyl esterases are active on polymeric substrate, methyl esterified glucuronoxylan

USDA-ARS?s Scientific Manuscript database

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic d...

Structural and Functional Characterization of a Novel Family GH115 4-O-Methyl-α-Glucuronidase with Specificity for Decorated Arabinogalactans.

PubMed

Aalbers, Friso; Turkenburg, Johan P; Davies, Gideon J; Dijkhuizen, Lubbert; Lammerts van Bueren, Alicia

2015-12-04

Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-β-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemical characteristics and anticoagulant activities of two sulfated polysaccharides from Enteromorpha linza (Chlorophyta)

NASA Astrophysics Data System (ADS)

Qi, Xiaohui; Mao, Wenjun; Chen, Yin; Chen, Yanli; Zhao, Chunqi; Li, Na; Wang, Chunyan

2013-03-01

Two sulfated polysaccharides, designated MP and SP, were extracted from the marine green alga Enteromorpha linza using hot water and then purified using ion-exchange and size-exclusion chromatography. The anticoagulant activities of MP and SP were examined by determination of their activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. Results showed that MP and SP were composed of abundant rhamnose with small amounts of xylose and glucuronic acid, whereas SP also contained a small amount of galactose. Approximate molecular weights of MP and SP were 535 and 502 kDa, respectively. As compared with SP, MP had higher contents of sulfate ester (19.0%) and uronic acid (14.9%). The MP mainly consisted of (1→4)-linked rhamnose residues with partially sulfated groups at the C-3 position, and small amounts of (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid and (1→4)-linked xylose residues. The SP contained abundant (1→4)-linked rhamnose with minor amounts of (1→3)-linked rhamnose, (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid, (1→4)-linked xylose, and (1→3)-linked galactose residues. The sulfate groups were mainly located at C-3 of (1→4)-linked rhamnose residues. Both MP and SP, in particular the former, effectively prolonged APTT and TT. This work demonstrates that MP and SP have unique structural characteristics distinct from those of other sulfated polysaccharides from Enteromorpha. The MP is a potential source of anticoagulant, and the difference in anticoagulant activities of the two sulfated polysaccharides is directly linked to the discrepancy of their chemical features.

Molecular weight determination and correlation analysis of Dalbergia sissoo polysaccharide with constituent oligosaccharides.

PubMed

Kumar, Vineet; Rana, Vikas; Soni, P L

2013-01-01

Mucilaginous polysaccharide extracted from Dalbergia sissoo Roxb. leaves has a number of medicinal applications. Molecular weight studies and correlation analysis of the structure of polysaccharide with oligosaccharides can be helpful for further utilisation, modification and structure-activity relationship for biological applications. To determine molecular weight of medicinally important polysaccharide. To establish an unequivocal correlation of the polysaccharide monosugars with constituting oligosaccharides and glucuronic acid content based on gas-liquid chromatography (GLC) with the spectrophotometric method. Complete and partial hydrolytic studies of pure polysaccharide yielded constituting monosugars and oligosaccharides. The ratio of sugars in polysaccharide and oligosaccharides was studied by preparation of alditol acetates and analysed using GLC. The uronic acid content was studied by GLC analysis and spectrophotometry. Molecular weight of the polysaccharide was determined using the viscometric method. Dalbergia sissoo leaves yielded 14.0% pure polysaccharide, containing 15.7% of glucuronic acid. Complete hydrolysis and GLC analysis of alditol acetate derivatives of reduced and unreduced monosugars indicated the presence of L-rhamnose, D-glucuronic acid, D-galactose and D-glucose in 1.00:1.00:2.00:2.33 molar ratios. Partial hydrolysis followed by monosugar analysis of oligosaccharides established the monosugar ratio in complete agreement with polysaccharide, thereby corroborating the sugar ratio. Similar uronic acid content was obtained by GLC and spectrophotometry. The polysaccharide had an average molecular weight of 1.5 × 10⁵  Da. The study has established an obvious correlation of the structure of polysaccharide with oligosaccharides, leading to unambiguous identification of monosaccharides, which normally is not studied conclusively while reporting the polysaccharide structure. The molecular weight of the polysaccharide was determined. Copyright © 2012 John Wiley & Sons, Ltd.

Dual responsive dysprosium-doped hydroxyapatite particles and toxicity reduction after functionalization with folic and glucuronic acids.

PubMed

Sánchez Lafarga, Ana Karen; Pacheco Moisés, Fermín P; Gurinov, Andrey; Ortiz, Genaro Gabriel; Carbajal Arízaga, Gregorio Guadalupe

2015-03-01

The development of probes for biomedical applications demands materials with low toxicity levels besides fluorescence or magnetic properties to be detected by confocal microscopes or MRI resonators. Several drug delivery systems or other biomedical materials prepared with hydroxyapatite have been proposed, however, toxicity effects might arise when the size of particles is nanometric. In this study, hydroxyapatite functionalized with glucuronic or folic acids presented lower oxidative stress, measured from lipoperoxides and nitric oxide indicators in rats than pure hydroxyapatite. In separated experiments, hydroxyapatite was doped with dysprosium cations by coprecipitation producing a single crystal phase with fluorescent properties easily visualized by confocal microscopy when excited at 488nm. These particles also presented the ability to modify the proton relaxation time in T1 maps collected by magnetic resonance imaging. These modified hydroxyapatite nanoparticles could be candidates to design bimodal probes with low toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

PubMed

d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

2016-02-10

Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production. Copyright © 2015 Elsevier B.V. All rights reserved.

Desorption electrospray ionization mass spectrometry for the analysis of pharmaceuticals and metabolites.

PubMed

Kauppila, Tiina J; Wiseman, Justin M; Ketola, Raimo A; Kotiaho, Tapio; Cooks, R Graham; Kostiainen, Risto

2006-01-01

The performance of desorption electrospray ionization (DESI) in the analysis of a group of pharmaceuticals and their glucuronic acid conjugates is reported. The suitability of different sprayer solvents and different surfaces was examined. In the positive ion mode, water/methanol/trifluoroacetic acid performed best, whereas, in the negative ion mode, water/methanol/ammonium hydroxide was found to be the most suitable spray solvent. Of the surfaces investigated, polymethylmethacrylate (PMMA) was found to give the best performance in terms of sensitivity. Spray solution flow rate and the distance of the sprayer tip from the surface were also found to have significant effects on the signal intensity. Analytes with basic groups efficiently formed the corresponding protonated molecules in the positive ion mode, whereas acidic analytes, such as the glucuronic acid conjugates, formed intense signals due to the deprotonated molecules in the negative ion mode. Ionization of neutral compounds was less efficient and in many cases it was achieved through adduct formation with simple anions or cations. Copyright (c) 2005 John Wiley & Sons, Ltd.

Conformational free energies of methyl-α-L-iduronic and methyl-β-D-glucuronic acids in water

NASA Astrophysics Data System (ADS)

Babin, Volodymyr; Sagui, Celeste

2010-03-01

We present a simulation protocol that allows for efficient sampling of the degrees of freedom of a solute in explicit solvent. The protocol involves using a nonequilibrium umbrella sampling method, in this case, the recently developed adaptively biased molecular dynamics method, to compute an approximate free energy for the slow modes of the solute in explicit solvent. This approximate free energy is then used to set up a Hamiltonian replica exchange scheme that samples both from biased and unbiased distributions. The final accurate free energy is recovered via the weighted histogram analysis technique applied to all the replicas, and equilibrium properties of the solute are computed from the unbiased trajectory. We illustrate the approach by applying it to the study of the puckering landscapes of the methyl glycosides of α-L-iduronic acid and its C5 epimer β-D-glucuronic acid in water. Big savings in computational resources are gained in comparison to the standard parallel tempering method.

Conformational free energies of methyl-alpha-L-iduronic and methyl-beta-D-glucuronic acids in water.

PubMed

Babin, Volodymyr; Sagui, Celeste

2010-03-14

We present a simulation protocol that allows for efficient sampling of the degrees of freedom of a solute in explicit solvent. The protocol involves using a nonequilibrium umbrella sampling method, in this case, the recently developed adaptively biased molecular dynamics method, to compute an approximate free energy for the slow modes of the solute in explicit solvent. This approximate free energy is then used to set up a Hamiltonian replica exchange scheme that samples both from biased and unbiased distributions. The final accurate free energy is recovered via the weighted histogram analysis technique applied to all the replicas, and equilibrium properties of the solute are computed from the unbiased trajectory. We illustrate the approach by applying it to the study of the puckering landscapes of the methyl glycosides of alpha-L-iduronic acid and its C5 epimer beta-D-glucuronic acid in water. Big savings in computational resources are gained in comparison to the standard parallel tempering method.

In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

PubMed

Chimphango, Annie F A; Görgens, J F; van Zyl, W H

2016-06-05

The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Extraction and determination of chondroitin sulfate from fish processing byproducts

USDA-ARS?s Scientific Manuscript database

Chondroitin sulfate (CS) refers to a group of sulfated glycosaminoglycan containing a chain of alternating N-acetylgalactosamine and glucuronic acid sugars. It is a major component of the extracellular matrix of cartilage and attached to proteins. CS is usually an over the counter dietary supplement...

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques.

PubMed

Cescutti, P; Ravenscroft, N; Ng, S; Lam, Z; Dutton, G G

1993-06-21

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and 1H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage phi SK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the parent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide. [Formula: see text] The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.

Apple peel bioactive rich extracts effectively inhibit in vitro human LDL cholesterol oxidation.

PubMed

Thilakarathna, Surangi H; Rupasinghe, H P Vasantha; Needs, Paul W

2013-05-01

Apple peels are rich in antioxidant bioactives and hence can possess the ability to inhibit human low density lipoprotein cholesterol (LDL-C) oxidation. LDL-C oxidation is known to initiate atherosclerotic plaque formation. Unique quercetin-rich (QAE) and triterpene-rich (TAE) apple peel extracts, their constituent compounds and three in vivo quercetin metabolites were investigated for in vitro LDL-C oxidation inhibition. Both extracts effectively inhibited Cu(2+)-induced LDL-C oxidation. IC(50) of QAE and TAE for LDL-C oxidation products were 0.06-8.29 mg/L and 29.58-95.49 mg/L, respectively. Quercetin compounds, chlorogenic acid and phloridzin could contribute more to the effectiveness of QAE at physiological concentrations. The three in vivo quercetin metabolites; quercetin-3'-sulfate, quercetin-3-glucuronic acid and isorhamnetin-3-glucuronic acid were effective at physiological concentrations and therefore, QAE can be effective in LDL-C oxidation inhibition under physiological conditions. Constituent TAE compounds did not perform well under Cu(2+)-induction. Overall, both extracts effectively inhibited LDL-C oxidation in vitro. Copyright © 2012 Elsevier Ltd. All rights reserved.

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

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

Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu

2010-10-15

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellularmore » spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.« less

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

Synthesis, characterization and properties of uridine 5'-( -D-apio-D-furanosyl pyrophosphate).

PubMed

Kindel, P K; Watson, R R

1973-06-01

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

PubMed Central

Kindel, Paul K.; Watson, Ronald R.

1973-01-01

1. A method was developed for synthesizing UDP-apiose [uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5′-(α-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5′-(α-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [3H]UDP-[U-14C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the 3H/14C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100°C for 15min; (b) degraded at pH8.0 and 100°C for 3min; (c) used as a substrate in the enzymic synthesis of [14C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [3H]UDP-[U-14C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-14C]apiose and phosphate formed on alkaline degradation of UDP-[U-14C]apiose was α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-14C]apiose and phosphate formed on acid hydrolysis of α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-14C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-14C]apiose to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80°C, at pH8.0 and 25°C and at pH8.0 and 4°C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-14C]-apiose to d-[U-14C]apiose and UDP at pH3.0 and 40°C was 4.67min. After 20 days at pH6.2–6.6 and 4°C, 17% of the starting UDP-[U-14C]apiose was degraded to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-14C]apiose and UDP. After 120 days at pH6.4 and −20°C 2% of the starting UDP-[U-14C]apiose was degraded and 4% was hydrolysed. PMID:4723773

Identification of glyceollin metabolites derived from conjugation with glutathione and glucuronic acid in rats by on-line liquid chromatography-electrospray ionization tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Glyceollin-related metabolites produced in rats following oral glyceollin administration were screened and identified by precursor and product ion scanning using liquid chromatography, coupled on-line with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), to identify all glyceollin me...

Xylanase 30 A from Clostridium thermocellum functions as a glucuronoxylan xylanohydrolase

Treesearch

Franz J. St John; Casey Crooks; Diane Dietrich; Jason Hurlbert

2017-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) have been shown to hydrolyze glucuronoxylan with dependence upon the glucuronic acid (GlcA) appendage. In a recent report, the GH30-8 xylanase from Clostridium thermocellum (CtXyn30A) was shown to hydrolyze arabinoxylan which contains no GlcA. Protein structure...

Ascorbate synthesis pathway, dual role of ascorbate in bone homeostasis

USDA-ARS?s Scientific Manuscript database

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of D-glucuronate to L-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) m...

Variations of pH as an additional tool in the analysis of crowded NMR spectra of fucosylated chondroitin sulfates.

PubMed

Ustyuzhanina, Nadezhda E; Dmitrenok, Andrey S; Bilan, Maria I; Shashkov, Alexander S; Gerbst, Alexey G; Usov, Anatolii I; Nifantiev, Nikolay E

2016-03-24

The influence of pH variation on chemical shift values in NMR spectra of fucosylated chondroitin sulfates was studied using polysaccharides isolated from three sea cucumber species Apostichopus japonicus, Actinopyga mauritiana and Cucumaria japonica. The signals of glucuronic acid residues were found to be the most sensitive to pH changes in comparison to the chemical shifts of the sulfated galactosamine and fucosyl units, most of which were altered insignificantly. It was shown that in the presence of imidazole-HCl buffer (pH 7.2) NMR spectra of the polysaccharides from A. japonicus and A. mauritiana were sufficiently resolved, whereas under acidic conditions their (1)H NMR spectra were complicated by overlapping of H-1 signals of GlcA and GalNAc. In the case of polysaccharide from C. japonica bearing 3-O-fucosylated and 3-O-sulfated glucuronic acid residues in the backbone, acidification of the medium led to separation of H-1 signals of GlcA3S and GalNAc. Therefore, the combination of data obtained at different pH values may be useful for interpretation of overcrowded spectra of fucosylated chondroitin sulfates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of Xylan Side-Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass.

PubMed

Pereira, Caroline S; Silveira, Rodrigo L; Dupree, Paul; Skaf, Munir S

2017-04-10

Lignocellulosic biomass is mainly constituted by cellulose, hemicellulose, and lignin and represents an important resource for the sustainable production of biofuels and green chemistry materials. Xylans, a common hemicellulose, interact with cellulose and often exhibit various side chain substitutions including acetate, (4-O-methyl) glucuronic acid, and arabinose. Recent studies have shown that the distribution of xylan substitutions is not random, but follows patterns that are dependent on the plant taxonomic family and cell wall type. Here, we use molecular dynamics simulations to investigate the role of substitutions on xylan interactions with the hydrophilic cellulose face, using the recently discovered xylan decoration pattern of the conifer gymnosperms as a model. The results show that α-1,2-linked substitutions stabilize the binding of single xylan chains independently of the nature of the substitution and that Ca 2+ ions can mediate cross-links between glucuronic acid substitutions of two neighboring xylan chains, thus stabilizing binding. At high temperature, xylans move from the hydrophilic to the hydrophobic cellulose surface and are also stabilized by Ca 2+ cross-links. Our results help to explain the role of substitutions on xylan-cellulose interactions, and improve our understanding of the plant cell wall architecture and the fundamentals of biomass pretreatments.

Trichothecenes Mycotoxin Studies

DTIC Science & Technology

1986-02-01

15-monoacetoxyscirpenol with C-UDP-glucuronic acid (data not shown). We were unable to hydrolyze this metabolite by incu- bation with either bovine ...then, this metabolite appears to be non- toxic. This coupled with our findings that this glucuronide is not hydrolyzed by E. coli or bovine liver a...leakage or violation of the skin patches by two of the animals toward the end of these early experiments. Because of their small size and higher level of

Design and synthesis of unnatural heparosan and chondroitin building blocks

PubMed Central

Bera, Smritilekha; Linhardt, Robert J.

2011-01-01

Triazole linked heparosan and chondroitin disaccharide and tetrasaccharide building blocks were synthesized in a stereoselective manner by applying a very efficient Copper Catalyzed Azide-Alkyne Cycloadditions (CuAAC) reaction of appropriately substituted azido-glucuronic acid and propargyluted N-acetyl glucosamine and N-acetyl galactosamine derivative respectively. The resulting suitably substituted tetrasaccharide analogs can be easily converted into azide and alkyne unit for further synthesis of higher oligosaccharide analogs. PMID:21438620

Molecular MR Imaging of CD44 in Breast Cancer with Hyaluronan-Based Contrast Agents

DTIC Science & Technology

2009-09-01

linear polysaccharide composed of alternating (β-1,4)-linked d- glucuronic acid and (β-1,3) N-acetyl-d-glucosamine residues with molecular weights as...enzymatic reactions in-vivo that generate polysaccharides of decreasing sizes, which in principle may facilitate the timely excretion of HA based...14CO2) or in urine (as low molecular weight HA or monosaccharide fragments). The same authors also reported that the total amount of excretion into

The UDP-glucuronosyltransferases of the blood-brain barrier: their role in drug metabolism and detoxication

PubMed Central

Ouzzine, Mohamed; Gulberti, Sandrine; Ramalanjaona, Nick; Magdalou, Jacques; Fournel-Gigleux, Sylvie

2014-01-01

UDP-glucuronosyltransferases (UGTs) form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-D-glucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds) by the linkage of glucuronic acid from the high energy donor, UDP-α-D-glucuronic acid. In brain, UGTs actively participate to the overall protection of the tissue against the intrusion of potentially harmful lipophilic substances that are metabolized as hydrophilic glucuronides. These metabolites are generally inactive, except for important pharmacologically glucuronides such as morphine-6-glucuronide. UGTs are mainly expressed in endothelial cells and astrocytes of the blood brain barrier (BBB). They are also associated to brain interfaces devoid of BBB, such as circumventricular organ, pineal gland, pituitary gland and neuro-olfactory tissues. Beside their key-role as a detoxication barrier, UGTs play a role in the steady-state of endogenous compounds, like steroids or dopamine (DA) that participate to the function of the brain. UGT isoforms of family 1A, 2A, 2B and 3A are expressed in brain tissues to various levels and are known to present distinct but overlapping substrate specificity. The importance of these enzyme species with regard to the formation of toxic, pharmacologically or physiologically relevant glucuronides in the brain will be discussed. PMID:25389387

Disposition in male volunteers of a subanaesthetic intravenous dose of an oil in water emulsion of 14C-propofol.

PubMed

Simons, P J; Cockshott, I D; Douglas, E J; Gordon, E A; Hopkins, K; Rowland, M

1988-04-01

1. An intravenous dose of 14C-propofol (0.47 mg/kg) administered to six male volunteers was rapidly eliminated with 88% recovered in the urine in 5 days and less than 2% in faeces. 2. The dose was cleared by metabolism with less than 0.3% excreted unchanged. The major metabolites were the glucuronic acid conjugate of propofol and the glucuronic acid and sulphate conjugates of its hydroxylated derivative, 2,6-diisopropyl-1,4-quinol. Propofol glucuronide accounted for about 53% of the urinary radioactivity and was the major metabolite in plasma from 30 min post dose. 3. The blood concentration of propofol declined in a biphasic manner from a maximum mean value of 0.44 microgram/ml, 2 min after injection. The half-lives of the first and second exponential phases, mean values 5 min and 97 min respectively, varied widely among subjects. A proportion of the dose was cleared slowly, probably due to slow release from less well perfused tissues. Propofol accounted for 94% of the total blood radioactivity at 2 min but only about 6% from 3 to 8 h post dose. 4. Propofol has a volume of distribution equivalent to about 3 to 4 times body weight, and a mean total body clearance of 2.2 1/min.

Modifications of a calcium phosphate cement with biomolecules--influence on nanostructure, material, and biological properties.

PubMed

Vater, Corina; Lode, Anja; Bernhardt, Anne; Reinstorf, Antje; Nies, Berthold; Gelinsky, Michael

2010-12-01

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

The UDP-glucose dehydrogenase of Escherichia coli K-12 displays substrate inhibition by NAD that is relieved by nucleotide triphosphates.

PubMed

Mainprize, Iain L; Bean, Jordan D; Bouwman, Catrien; Kimber, Matthew S; Whitfield, Chris

2013-08-09

UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes*

PubMed Central

Courville, Pascal; Quick, Matthias; Reimer, Richard J.

2010-01-01

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H+-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members. PMID:20424173

Bluephage: A rapid method for the detection of somatic coliphages used as indicators of fecal pollution in water.

PubMed

Muniesa, M; Ballesté, E; Imamovic, L; Pascual-Benito, M; Toribio-Avedillo, D; Lucena, F; Blanch, A R; Jofre, J

2018-01-01

The use of somatic coliphages as indicators of fecal and viral pollution in water and food has great potential due to the reliability, reproducibility, speed and cost effectiveness of methods for their detection. Indeed, several countries already use this approach in their water management policies. Although standardized protocols for somatic coliphage detection are available, user-friendly commercial kits would facilitate their routine implementation in laboratories. The new method presented here allows detection of up to 1 somatic coliphage in under 3.5 h, well within one working day. The method is based on a modified Escherichia coli strain with knocked-out uidB and uidC genes, which encode the transport of glucuronic acid inside cells, and overexpressing uidA, which encodes the enzyme β-glucuronidase. The enzyme accumulated in the bacterial cells only has contact with its substrate after cell lysis, such as that caused by phages, since the strain cannot internalize the substrate. When the enzyme is released into the medium, which contains a chromogen analogous to glucuronic acid, it produces a change of color from yellow to dark blue. This microbiological method for the determination of fecal pollution via the detection of culturable microorganisms can be applied to diverse sample types and volumes for qualitative (presence/absence) and quantitative analysis and is the fastest reported to date. Copyright © 2017 Elsevier Ltd. All rights reserved.

Importance of Dichloroacetate and Trichloroacetate to the Hepatocarcinogenic Response to Trichloroeylene in B6C3F1 Mice

DTIC Science & Technology

1989-10-15

Baker Co., polysorbate (Tween 80), beta- glucuronidase (Type VII), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), clofibrate , phenobllbital, DCA, TCA...TCOH conjugated wit’a glucuronic acid was determined in an aliquot of sample treated with beta-glucuronidase. A Varian Model 3700 gas chromatQgraph...diaminobenzoic acid fluorimetric assay. The fraction of DNA unwound during the two hour incubation at OC was calculated as: (Total DNA - DS DNA)t

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

PubMed Central

Bethke, Gerit; Thao, Amanda; Xiong, Guangyan; Hatsugai, Noriyuki; Katagiri, Fumiaki; Pauly, Markus

2016-01-01

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions. PMID:26813622

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.

PubMed

Dawson, J; Knowles, R G; Pogson, C I

1992-03-03

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

PubMed

d'Errico, Clotilde; Jørgensen, Jonas O; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

2015-05-01

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass. © 2014 Wiley Periodicals, Inc.

Glucuronic Acid Epimerase Is Associated with Plasma Triglyceride and High Density Lipoprotein Cholesterol Levels in Turks

PubMed Central

Hodoğlugil, Uğur; Williamson, David W.; Yu, Yi; Farrer, Lindsay A.; Mahley, Robert W.

2011-01-01

Summary We narrowed chromosome 15q21-23 linkage to plasma high density lipoprotein cholesterol (HDL-C) levels in atherogenic dyslipidemic Turkish families by fine mapping, then focused on glucuronic acid epimerase (GLCE), a heparan sulfate proteoglycan (HSPG) biosynthesis enzyme. HSPGs participate in lipid metabolism along with apolipoprotein (apo) E. Of 31 SNPs in the GLCE locus, nine analyzed by haplotype were associated with plasma HDL-C and triglyceride levels (permuted p = 0.006 and 0.013, respectively) in families. Of five tagging GLCE SNPs in two cohorts of unrelated subjects, three (rs16952868, rs11631403, rs3865014) were associated with triglyceride and HDL-C levels in males (non-permuted p < 0.05). The association was stronger in APOE 2/3 subjects (apoE2 has reduced binding to HSPGs) and reached multiple-testing significance (p < 0.05) in both males and females (n = 2612). Similar results were obtained in the second cohort (n = 1164). Interestingly, at the GLCE locus, bounded by recombination hotspots, Turks had a minor allele frequency of SNPs resembling Chinese more than European ancestry; adjoining regions on chromosome 15 resembled the European pattern. Studies of glce+/–apoe–/– mice fed a chow or high-fat diet supported a role for GLCE in lipid metabolism. Thus, SNPs in GLCE are associated with triglyceride and HDL-C levels in Turks, and mouse studies support a role for glce in lipid metabolism. PMID:21488854

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

PubMed Central

Hughes, R. C.; Thurman, P. F.

1970-01-01

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[3H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods. PMID:5419741

Baicalin ameliorates isoproterenol-induced acute myocardial infarction through iNOS, inflammation and oxidative stress in rat

PubMed Central

Chen, Huaguo; Xu, Yongfu; Wang, Jianzhong; Zhao, Wei; Ruan, Huihui

2015-01-01

Baicalin belongs to glucuronic acid glycosides and after hydrolysisbaicalein and glucuronic acid come into being. It has such effects as clearing heat and removing toxicity, anti-inflammation, choleresis, bringing high blood pressure down, diuresis, anti-allergic reaction and so on. In this study, we investigated whether baicalin ameliorates isoproterenol-induced acute myocardial infarction and its mechanism. Rat model of acute myocardial infarction was induced by isoproterenol. Casein kinase (CK), the MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), cardiac troponin T (cTnT) and infarct size measurement were used to measure the protective effect of baicalin on isoproterenol-induced acute myocardial infarction. iNOS protein expression in rat was analyzed using western blot analysis. Tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), malondialdehyde (MDA) and superoxide dismutase (SOD) and caspase-3 activation levels were explored using commercial ELISA kits. In the acute myocardial infarction experiment, baicalin effectively ameliorates the level of CK, CK-MB, LDH and cTnT, reduced infarct size in acute myocardial infarction rat model. Meanwhile, treatment with baicalin effectively decreased the iNOS protein expression, inflammatory factors and oxidative stresses in a rat model of acute myocardial infarction. However, baicalin emerged that anti-apoptosis activity and suppressed the activation of caspase-3 in a rat model of acute myocardial infarction. The data suggest that the protective effect of baicalin ameliorates isoproterenol-induced acute myocardial infarction through iNOS, inflammation and oxidative stress in rat. PMID:26617721

Identification of three new phase II metabolites of a designer drug methylone formed in rats by N-demethylation followed by conjugation with dicarboxylic acids.

PubMed

Židková, Monika; Linhart, Igor; Balíková, Marie; Himl, Michal; Dvořáčková, Veronika; Lhotková, Eva; Páleníček, Tomáš

2018-06-01

1. Methylone (3,4-methylenedioxy-N-methylcathinone, MDMC), which appeared on the illicit drug market in 2004, is a frequently abused synthetic cathinone derivative. Known metabolic pathways of MDMC include N-demethylation to normethylone (3,4-methylenedioxycathinone, MDC), aliphatic chain hydroxylation and oxidative demethylenation followed by monomethylation and conjugation with glucuronic acid and/or sulphate. 2. Three new phase II metabolites, amidic conjugates of MDC with succinic, glutaric and adipic acid, were identified in the urine of rats dosed subcutaneously with MDMC.HCl (20 mg/kg body weight) by LC-ESI-HRMS using synthetic reference standards to support identification. 3. The main portion of administered MDMC was excreted unchanged. Normethylone, was a major urinary metabolite, of which a minor part was conjugated with dicarboxylic acids. 4. Previously identified ring-opened metabolites 4-hydroxy-3-methoxymethcathinone (4-OH-3-MeO-MC), 3-hydroxy-4-methoxymeth-cathinone (3-OH-4-MeO-MC) and 3,4-dihydroxymethcathinone (3,4-di-OH-MC) mostly in conjugated form with glucuronic and/or sulphuric acids were also detected. 5. Also, ring-opened metabolites derived from MDC, namely, 4-hydroxy-3-methoxycathinone (4-OH-3-MeO-C), 3-hydroxy-4-methoxycathinone (3-OH-4-MeO-C) and 3,4-dihydroxycathinone (3,4-di-OH-C) were identified for the first time in vivo.

Biochemistry of metalocenes. The organ distribution of hydroxyacetyl (/sup 103/Ru)ruthenocene and its glucuronide in mice

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

Taylor, A.J.; Macha, J.; Wenzel, M.

1980-01-01

Hydroxyacetyl(/sup 103/Ru)ruthenocene and its o-glucuronide were prepared in vitro by incubation of acetyl(/sup 103/Ru)ruthenocene with rat-liver homogenate, NADPH, and UDP-glucuronate. The factors affecting hydroxylation and glucuronidation in vitro were optimized for acetylruthenocene. Hydroxyacetyl(/sup 103/Ru)ruthenocene glucuronide showed no affinity for the adrenal glands, but after iv administration of hydroxyacetyl(/sup 103/Ru)ruthenocene there was a distinct accumulation of Ru-103 in adrenals, similar to that found after administration of acetyl(/sup 103/Ru)ruthenocene.

Inherited Disorders of Bilirubin Clearance

PubMed Central

Memon, Naureen; Weinberger, Barry I; Hegyi, Thomas; Aleksunes, Lauren M

2016-01-01

Inherited disorders of hyperbilirubinemia may be caused by increased bilirubin production or decreased bilirubin clearance. Reduced hepatic bilirubin clearance can be due to defective 1) unconjugated bilirubin uptake and intrahepatic storage, 2) conjugation of glucuronic acid to bilirubin (e.g. Gilbert syndrome, Crigler-Najjar syndrome, Lucey-Driscoll syndrome, breast milk jaundice), 3) bilirubin excretion into bile (Dubin-Johnson syndrome), or 4) conjugated bilirubin re-uptake (Rotor syndrome). In this review, the molecular mechanisms and clinical manifestations of these conditions are described, as well as current approaches to diagnosis and therapy. PMID:26595536

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

PubMed

Feyerabend, Thorsten B; Li, Jin-Ping; Lindahl, Ulf; Rodewald, Hans-Reimer

2006-04-01

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

[Separation, purification and primary reverse cholesterol transport study of Cordyceps militaris polysaccharide].

PubMed

Guo, Shou-Dong; Cui, Ying-Jie; Wang, Ren-Zhong; Wang, Ren-Yuan; Wu, Wen-Xue; Ma, Teng

2014-09-01

The authors designed to separate, purify and determine the monosaccharide composition of the polysaccharide from Cordyceps militaris, and study its effect on reverse cholesterol transport in vivo by isotope tracing assay. Polysaccharides were separate and purify by ion exchange column Q-sepharose Fast Flow and size exclusion column Sephacryl S200HR; the molecular weight and monosaccharide composition of the polysaccharides were determined by high performance gel permeation chromatography and high performance liquid chromatography coming with pre-column derivation, respectively. Finally, three purified polysaccharides CMBW1, CMBW2 and CMYW1 were obtained, their total carbohydrate contents were 87%, 89%, 95%, respectively; their protein contents were 6.5%, 1.3%, 2.8%, respectively; their molecular weights were 772.1, 20.9, 13.2 kDa, respectively; CMBW1 was composed of mannose, glucosamine, rhamnose, glucuronic acid, glucose, galactose and arabinose with a molar ratio of 7.25: 0.17: 1.29: 0.23: 6.30: 11.08: 0.79; CMBW2 was composed of mannose, glucosamine, galactose and arabinose with a molar ratio of 2.40: 0.16: 2.92: 0.24; CMYW1 was composed of mannose, glucosamine, glucuronic acid and glucose with a molar ratio of 0.59: 0.57: 0.45: 25.61. Polysaccharide at 50 mg x kg(-1) could significantly improve the transport of 3H- cholesterol to blood and excretion from feces. All of the three purified polysaccharides CMBW1, CMBW2 and CMYW1 were heteropolysaccharide; and they could improve reverse cholesterol transport in vivo, the underlying mechanisms are being studied.

Internal Hydrolysis Indicator for Sample Specific Monitoring of β-Glucuronidase Activity.

PubMed

Taylor, Lacy L; Flint, Noah A; Ma, Vinh; Hill, Brandy M; Clark, Chantry J; Strathmann, Frederick G

2017-06-01

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO.

PubMed

Rydevik, Axel; Bondesson, Ulf; Thevis, Mario; Hedeland, Mikael

2013-10-01

A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost. Copyright © 2013 Elsevier B.V. All rights reserved.

Action of a GH115 α-glucuronidase from Amphibacillus xylanus at alkaline condition promotes release of 4-O-methylglucopyranosyluronic acid from glucuronoxylan and arabinoglucuronoxylan.

PubMed

Yan, Ruoyu; Vuong, Thu V; Wang, Weijun; Master, Emma R

2017-09-01

Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing. Copyright © 2017 Elsevier Inc. All rights reserved.

Energetic costs and implications of the intake of plant secondary metabolites on digestive and renal morphology in two austral passerines.

PubMed

Barceló, Gonzalo; Ríos, Juan Manuel; Maldonado, Karin; Sabat, Pablo

2016-07-01

Seed-eating birds have a diet of high nutritional value; however, they must cope with plant secondary metabolites (PSM). We postulated that the detoxification capacity of birds is associated with a metabolic cost, given that the organs responsible for detoxification significantly contribute to energetic metabolism. We used an experimental approach to assess the effects of phenol-enriched diets on two passerines with different feeding habits: the omnivorous rufous-collared sparrow (Zonotrichia capensis) and the granivorous common diuca-finch (Diuca diuca). The birds were fed with one of three diets: control diet, supplemented with tannic acid, or supplemented with Opuntia ficus-indica phenolic extract (a common food of the sparrow but not the finch). After 5 weeks of exposure to the diets, we measured basal metabolic rates (BMR), energy intake, glucuronic acid output and digestive and kidney structure. In both species, detoxification capacity expressed as glucuronic acid output was higher in individuals consuming phenol-enriched diets compared to the control diet. However, whereas sparrows increase energy intake and intestinal mass when feeding on phenol-enriched diets, finches had lower intestinal mass and energy intake remains stable. Furthermore, sparrows had higher BMR on phenol-enriched diets compared to the control group, whereas in the finches BMR remains unchanged. Interspecific differences in response to phenols intake may be determined by the dietary habits of these species. While both species can feed on moderate phenolic diets for 5 weeks, energy costs may differ due to different responses in food intake and organ structure to counteract the effects of PSM intake.

Cryptococcus friedmannii, a new species of yeast from the Antarctic

NASA Technical Reports Server (NTRS)

Vishniac, H. S.

1985-01-01

Cryptococcus friedmannii Vishniac sp. nov. from an Antarctic cryptoendolithic community is a psychrophilic basidioblastomycete characterized by cream-colored colonies of cells with smooth, layered walls, budding monopolarly, producing amylose and extracellular proteinase, utilizing nitrate and D-alanine (inter alia) as nitrogen sources and L-arabinose, arbutin, cellobiose, D-glucuronate, maltose, melezitose, salicin, soluble starch, trehalose, and D-xylose as carbon sources. This species differs from all other basidiomycetous yeasts in possessing the following combination of characters: amylose production (positive), assimilation of cellobiose (positive), D-galactose (negative), myo-inositol (negative), D-mannitol (negative), and sucrose (negative).

Mapping the UDP-Glucuronic Acid Binding Site in UDP-Glucuronosyltransferase-1 A10 by Homology-based Modeling: Confirmation with Biochemical Evidence†

PubMed Central

Banerjee, Rajat; Pennington, Matthew W.; Garza, Amanda; Owens, Ida S.

2008-01-01

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3- and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater- and 50% less-label in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321--equivalent to K314-- in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10. PMID:18570380

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution.

PubMed

Smith, James; Yang, Yiwen; Levy, Shahar; Adelusi, Oluwatoyin Oluwayemi; Hahn, Michael G; O'Neill, Malcolm A; Bar-Peled, Maor

2016-10-07

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan

PubMed Central

Higman, Victoria A.; Briggs, David C.; Mahoney, David J.; Blundell, Charles D.; Sattelle, Benedict M.; Dyer, Douglas P.; Green, Dixy E.; DeAngelis, Paul L.; Almond, Andrew; Milner, Caroline M.; Day, Anthony J.

2014-01-01

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation. PMID:24403066

Direct detection of glucuronide metabolites of lidocaine in sheep urine.

PubMed

Doran, Gregory S; Smith, Alistair K; Rothwell, Jim T; Edwards, Scott H

2018-02-15

The anaesthetic lidocaine is metabolised quickly to produce a series of metabolites, including several hydroxylated metabolites, which are further metabolised by addition of a glucuronic acid moiety. Analysis of these glucuronide metabolites in urine is performed indirectly by cleaving the glucuronic acid group using β-glucuronidase. However, direct analysis of intact glucuronide conjugates is a more straightforward approach as it negates the need for long hydrolysis incubations, and minimises the oxidation of sensitive hydrolysis products, while also distinguishing between the two forms of hydroxylated metabolites. A method was developed to identify three intact glucuronides of lidocaine in sheep urine using LC-MS/MS, which was further confirmed by the synthesis of glucuronide derivatives of 3OH-MEGX and 4OH-LIDO. Direct analysis of urine allowed the detection of the glucuronide metabolites of hydroxylidocaine (OH-LIDO), hydroxyl-monoethylglycinexylidide (OH-MEGX), and hydroxy-2,6-xylidine (OH-XYL). Analysis of urine before and after β-glucuronidase digestion showed that the efficiency of hydrolysis of these glucuronide metabolites may be underestimated in some studies. Analysis of urine in the current study from three different sheep with similar glucuronide metabolite concentrations resulted in different hydrolysis efficiencies, which may have been a result of different levels of substrate binding by matrix components, preventing enzyme cleavage. The use of direct analysis of intact glucuronides has the benefit of being less influenced by these matrix effects, while also allowing analysis of unstable metabolites like 4OH-XYL, which rapidly oxidises after hydrolysis. Additionally, direct analysis is less expensive and less time consuming, while providing more information about the status of hydroxylated metabolites in urine. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution*

PubMed Central

Smith, James; Yang, Yiwen; Levy, Shahar; Adelusi, Oluwatoyin Oluwayemi; Hahn, Michael G.; O'Neill, Malcolm A.; Bar-Peled, Maor

2016-01-01

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans. PMID:27551039

Unique Extracellular Matrix Heparan Sulfate from the Bivalve Nodipecten nodosus (Linnaeus, 1758) Safely Inhibits Arterial Thrombosis after Photochemically Induced Endothelial Lesion*

PubMed Central

Gomes, Angélica M.; Kozlowski, Eliene O.; Pomin, Vitor H.; de Barros, Cintia Monteiro; Zaganeli, José L.; Pavão, Mauro S. G.

2010-01-01

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing ∼4.6 mg g−1 of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional 1H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg−1, 5-fold lower than porcine heparin (180 IU mg−1), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC50 = 0.835 μg ml−1) and thrombin (IC50 = 9.3 μg ml−1) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg−1, the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose. PMID:20053999

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

PubMed Central

Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

2015-01-01

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis.

PubMed

Kärkönen, Anna; Fry, Stephen C

2006-03-01

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.

PubMed

Oikari, Sanna; Venäläinen, Tuula; Tammi, Markku

2014-01-03

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues.

PubMed

Vieira, R P; Mulloy, B; Mourão, P A

1991-07-25

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.

On the calculation of puckering free energy surfaces

NASA Astrophysics Data System (ADS)

Sega, M.; Autieri, E.; Pederiva, F.

2009-06-01

Cremer-Pople puckering coordinates appear to be the natural candidate variables to explore the conformational space of cyclic compounds and in literature different parametrizations have been used to this end. However, while every parametrization is equivalent in identifying conformations, it is not obvious that they can also act as proper collective variables for the exploration of the puckered conformations free energy surface. It is shown that only the polar parametrization is fit to produce an unbiased estimate of the free energy landscape. As an example, the case of a six-membered ring, glucuronic acid, is presented, showing the artifacts that are generated when a wrong parametrization is used.

Advanced Processing for Biomedical Informatics (APBI)

DTIC Science & Technology

2009-10-01

phosphatidic acid phosphatase type 2 domain containing 1A PPAPDC1A 1 96051 2.71E-06 4.39E-05 4.55 8.14 12.1 205030_at fatty acid binding protein 7...W81XWH‐06‐2‐0072    Principal Investigator: Craig D. Shriver, COL MC    54    209355_s_at phosphatidic acid phosphatase type 2B PPAP2B 8613 1.62E-06...Investigator: Craig D. Shriver, COL MC    24    209711_at solute carrier family 35 (UDP- glucuronic acid /UDP-N- acetylgalactosamine dual transporter), member

On the calculation of puckering free energy surfaces.

PubMed

Sega, M; Autieri, E; Pederiva, F

2009-06-14

Cremer-Pople puckering coordinates appear to be the natural candidate variables to explore the conformational space of cyclic compounds and in literature different parametrizations have been used to this end. However, while every parametrization is equivalent in identifying conformations, it is not obvious that they can also act as proper collective variables for the exploration of the puckered conformations free energy surface. It is shown that only the polar parametrization is fit to produce an unbiased estimate of the free energy landscape. As an example, the case of a six-membered ring, glucuronic acid, is presented, showing the artifacts that are generated when a wrong parametrization is used.

Tetranuclear copper(II) complexes bridged by alpha-D-glucose-1-phosphate and incorporation of sugar acids through the Cu4 core structural changes.

PubMed

Kato, Merii; Sah, Ajay Kumar; Tanase, Tomoaki; Mikuriya, Masahiro

2006-08-21

Tetranuclear copper(II) complexes containing alpha-D-glucose-1-phosphate (alpha-D-Glc-1P), [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(bpy)4(H2O)2]X3 [X = NO3 (1a), Cl (1b), Br (1c)], and [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(phen)4(H2O)2](NO3)3 (2) were prepared by reacting the copper(II) salt with Na2[alpha-D-Glc-1P] in the presence of diimine ancillary ligands, and the structure of 2 was characterized by X-ray crystallography to comprise four {Cu(phen)}2+ fragments connected by the two sugar phosphate dianions in 1,3-O,O' and 1,1-O mu4-bridging fashion as well as a mu-hydroxo anion. The crystal structure of 2 involves two chemically independent complex cations in which the C2 enantiomeric structure for the trapezoidal tetracopper(II) framework is switched according to the orientation of the alpha-D-glucopyranosyl moieties. Temperature-dependent magnetic susceptibility data of 1a indicated that antiferromagnetic spin coupling is operative between the two metal ions joined by the hydroxo bridge (J = -52 cm(-1)) while antiferromagnetic interaction through the Cu-O-Cu sugar phosphate bridges is weak (J = -13 cm(-1)). Complex 1a readily reacted with carboxylic acids to afford the tetranuclear copper(II) complexes, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-CA)2(bpy)4](NO3)2 [CA = CH3COO (3), o-C6H4(COO)(COOH) (4)]. Reactions with m-phenylenediacetic acid [m-C6H4(CH2COOH)2] also gave the discrete tetracopper(II) cationic complex [Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)(CH2COOH))2(bpy)4](NO3)2 (5a) as well as the cluster polymer formulated as {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)2)(bpy)4](NO3)2}n (5b). The tetracopper structure of 1a is converted into a symmetrical rectangular core in complexes 3, 4, and 5b, where the hydroxo bridge is dissociated and, instead, two carboxylate anions bridge another pair of Cu(II) ions in a 1,1-O monodentate fashion. The similar reactions were applied to incorporate sugar acids onto the tetranuclear copper(II) centers. Reactions of 1a with delta-D-gluconolactone, D-glucuronic acid, or D-glucaric acid in dimethylformamide resulted in the formation of discrete tetracopper complexes with sugar acids, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-SA)2(bpy)4](NO3)2 [SA = D-gluconate (6), D-glucuronate (7), D-glucarateH (8a)]. The structures of 6 and 7 were determined by X-ray crystallography to be almost identical with that of 3 with additional chelating coordination of the C-2 hydroxyl group of D-gluconate moieties (6) or the C-5 cyclic O atom of D-glucuronate units (7). Those with D-glucaric acid and D-lactobionic acid afforded chiral one-dimensional polymers, {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-glucarate)(bpy)4](NO3)2}n (8b) and {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-lactobionate)(bpy)4(H2O)2](NO3)3}n (9), respectively, in which the D-Glc-1P-bridged tetracopper(II) units are connected by sugar acid moieties through the C-1 and C-6 carboxylate O atoms in 8b and the C-1 carboxylate and C-6 alkoxy O atoms of the gluconate chain in 9. When complex 7 containing d-glucuronate moieties was heated in water, the mononuclear copper(II) complex with 2-dihydroxy malonate, [Cu(mu-O2CC(OH)2CO2)(bpy)] (10), and the dicopper(II) complex with oxalate, [Cu2(mu-C2O4)(bpy)2(H2O)2](NO3)2 (11), were obtained as a result of oxidative degradation of the carbohydrates through C-C bond cleavage reactions.

Vibrational Studies of Saccharide-Induced Lipid Film Reorganization at Aqueous/Air Interfaces

DOE PAGES

Link, Katie A.; Hsieh, Chia -Yun; Tuladhar, Aashish; ...

2018-02-09

Vibrational sum frequency generation (VSFG) and surface tension experiments were used to examine the effects of aqueous phase soluble saccharides on the structure and organization of insoluble lipid monolayers adsorbed to aqueous-air interfaces. Changes in dipalmitoylphosphocholine (DPPC) chain structure as a function of aqueous phase saccharide concentration and pH are reported. Complementary differential scanning calorimetry (DSC) measurements performed on solutions containing soluble saccharides and DPPC vesicles measured the effects of the saccharides on the lipid membrane phase behavior. Here, data show that the saccharides glucosamine and glucuronic acid induce a higher degree of organization in compressed DPPC monolayers regardless ofmore » the saccharide’s charge.« less

An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

PubMed

Fratz-Berilla, Erica J; Ketcham, Stephanie A; Parhiz, Hamideh; Ashraf, Muhammad; Madhavarao, Chikkathur N

2017-12-01

Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme. Published by Elsevier Inc.

Rhodotorula rosulata sp. nov., Rhodotorula silvestris sp. nov. and Rhodotorula straminea sp. nov., novel myo-inositol-assimilating yeast species in the Microbotryomycetes.

PubMed

Golubev, Wladyslav I; Scorzetti, Gloria

2010-10-01

Three novel species are described as Rhodotorula rosulata sp. nov. (type strain VKM Y-2962(T) =CBS 10977(T)), Rhodotorula silvestris sp. nov. (type strain VKM Y-2971(T) =CBS 11420(T)) and Rhodotorula straminea sp. nov. (type strain VKM Y-2964(T) =CBS 10976(T)) based on the study of eight isolates from needle litter. The new species, phylogenetically located within the Microbotryomycetes, are related to glucuronate-assimilating species of the genus Rhodotorula. Sequencing of the D1/D2 domains of the LSU rDNA gene and the internal transcribed spacer (ITS) region, as well as physiological characterization, revealed their distinct taxonomic positions.

Vibrational Studies of Saccharide-Induced Lipid Film Reorganization at Aqueous/Air Interfaces

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

Link, Katie A.; Hsieh, Chia -Yun; Tuladhar, Aashish

Vibrational sum frequency generation (VSFG) and surface tension experiments were used to examine the effects of aqueous phase soluble saccharides on the structure and organization of insoluble lipid monolayers adsorbed to aqueous-air interfaces. Changes in dipalmitoylphosphocholine (DPPC) chain structure as a function of aqueous phase saccharide concentration and pH are reported. Complementary differential scanning calorimetry (DSC) measurements performed on solutions containing soluble saccharides and DPPC vesicles measured the effects of the saccharides on the lipid membrane phase behavior. Here, data show that the saccharides glucosamine and glucuronic acid induce a higher degree of organization in compressed DPPC monolayers regardless ofmore » the saccharide’s charge.« less

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

Extraction and characterization of polysaccharides from Semen Cassiae by microwave-assisted aqueous two-phase extraction coupled with spectroscopy and HPLC.

PubMed

Chen, Zhi; Zhang, Wei; Tang, Xunyou; Fan, Huajun; Xie, Xiujuan; Wan, Qiang; Wu, Xuehao; Tang, James Z

2016-06-25

A novel and rapid method for simultaneous extraction and separation of the different polysaccharides from Semen Cassiae (SC) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/ammonium sulfate system as a multiphase solvent, the effects of MAATPE on the extraction of polysaccharides from SC such as the composition of the ATPS, extraction time, temperature and solvent-to-material ratio were investigated by UV-vis analysis. Under the optimum conditions, the yields of polysaccharides were 4.49% for the top phase, 8.80% for the bottom phase and 13.29% for total polysaccharides, respectively. Compared with heating solvent extraction and ultrasonic assisted extraction, MAATPE exhibited the higher extraction yields in shorter time. Fourier-transform infrared spectra showed that two polysaccharides extracted from SC to the top and bottom phases by MAATPE were different from each other in their chemical structures. Through acid hydrolysis and PMP derivatization prior to HPLC, analytical results by indicated that a polysaccharide of the top phases was a relatively homogeneous homepolysaccharide composed of dominant gucose glucose while that of the bottom phase was a water-soluble heteropolysaccharide with multiple components of glucose, xylose, arabinose, galactose, mannose and glucuronic acid. Molar ratios of monosaccharides were 95.13:4.27:0.60 of glucose: arabinose: galactose for the polysaccharide from the top phase and 62.96:14.07:6.67: 6.67:5.19:4.44 of glucose: xylose: arabinose: galactose: mannose: glucuronic acid for that from the bottom phase, respectively. The mechanism for MAATPE process was also discussed in detail. MAATPE with the aid of microwave and the selectivity of the ATPS not only improved yields of the extraction, but also obtained a variety of polysaccharides. Hence, it was proved as a green, efficient and promising alternative to simultaneous extraction of polysaccharides from SC. Copyright © 2016 Elsevier Ltd. All rights reserved.

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

PubMed Central

de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap

1998-01-01

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

PubMed

de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

1998-01-01

An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

PubMed Central

Videira, P; Fialho, A; Geremia, R A; Breton, C; Sá-Correia, I

2001-01-01

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily. PMID:11513745

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune.

PubMed

Spániková, Silvia; Biely, Peter

2006-08-21

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.

Human β-glucuronidase: structure, function, and application in enzyme replacement therapy.

PubMed

Naz, Huma; Islam, Asimul; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Imtaiyaz

2013-10-01

Lysosomal storage diseases occur due to incomplete metabolic degradation of macromolecules by various hydrolytic enzymes in the lysosome. Despite structural differences, most of the lysosomal enzymes share many common features including a lysosomal targeting motif and phosphotransferase recognition sites. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glucuronate-containing glycosaminoglycan. The deficiency of GUSB causes mucopolysaccharidosis type VII (MPSVII), leading to lysosomal storage in the brain. GUSB is a well-studied protein for its expression, sequence, structure, and function. The purpose of this review is to summarize our current understanding of sequence, structure, function, and evolution of GUSB and its lysosomal enzyme targeting. Enzyme replacement therapy reported for this protein is also discussed.

Solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae K40.

PubMed

Flaibani, A; Leonhartsberger, S; Navarini, L; Cescutti, P; Paoletti, S

1994-04-01

This paper reports some physicochemical properties of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40 (K40-CPS) in aqueous solution. The polymer has a linear hexasaccharide repeating unit containing one glucuronic acid residue as the only ionizable group. Potentiometric, viscometric, chiro-optical and rheological measurements have been carried out over a range of ionic strength, pH and temperature, with the aim of characterizing the conformational state of the polysaccharide in aqueous solution. All the data reported indicate that the K40-CPS does not undergo a cooperative conformational transition under the investigated experimental conditions. Furthermore, the viscosity data and the viscoelastic spectra suggest that the K40-CPS is rather flexible and adopts a random coil conformation in solution.

Exopolysaccharides produced by Burkholderia cenocepacia recA lineages IIIA and IIIB.

PubMed

Chiarini, Luigi; Cescutti, Paola; Drigo, Laura; Impallomeni, Giuseppe; Herasimenka, Yury; Bevivino, Annamaria; Dalmastri, Claudia; Tabacchioni, Silvia; Manno, Graziana; Zanetti, Flavio; Rizzo, Roberto

2004-08-01

Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production. The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy. All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues. This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex. One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-D-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B. cepacia genomovar I and B. cenocepacia IIIA.

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

PubMed

Mengele, R; Sumper, M

1992-04-25

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

PubMed

da Silva, F R; Vettore, A L; Kemper, E L; Leite, A; Arruda, P

2001-09-25

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Metabolism and disposition of a novel antineoplastic JS-38 (Benzamide, N-[4-(2,4-dimethoxyphenyl)-4,5-dihydro-5-oxo-1,2-dithiolo[4,3-b]pyrrol-6-yl]-3,5-bis (trifluoromethyl)-(9Cl)) in rats.

PubMed

Zhang, Hong; Liu, Quanhai; Fan, Tingting; Fang, Yu; Li, Ying; Wang, Guoping

2012-03-01

The metabolism and catabolism of a novel antineoplastic (ID code JS-38),Benzamide, N-[4-(2,4-dimethoxyphenyl)-4,5-dihydro-5-oxo-1,2-dithiolo[4,3-b]pyrrol-6-yl]-3,5-bis (trifluoromethyl)-(9Cl), were investigated in Wistar rats (3 female, 3 male). LC/UV, LC/MS, LC/MS/MS, NMR and acid hydrolysis methods showed that the metabolic process of JS-38 consists of a series of acetylation and glucoronation that form a metabolic product with a unique pharmacologic property of accelerating bone-marrow cell formation, and also showed a novel metabolic pathway of being acetylated and glucuronated in series.

Investigation of miR-136-5p key target genes and pathways in lung squamous cell cancer based on TCGA database and bioinformatics analysis.

PubMed

Xie, Zu-Cheng; Li, Tian-Tian; Gan, Bin-Liang; Gao, Xiang; Gao, Li; Chen, Gang; Hu, Xiao-Hua

2018-05-01

Lung squamous cell cancer (LUSC) is a common but challenging malignancy. It is important to illuminate the molecular mechanism of LUSC. Thus, we aim to explore the molecular mechanism of miR-136-5p in relation to LUSC. We used the Cancer Genome Atlas (TCGA) database to investigate the expression of miR-136-5p in relation to LUSC. Then, we identified the possible miR-136-5p target genes through intersection of the predicted miR-136-5p target genes and LUSC upregulated genes from TCGA. Bioinformatics analysis was performed to determine the key miR-136-5p targets and pathways associated with LUSC. Finally, the expression of hub genes, correlation between miR-136-5p and hub genes, and expected significance of hub genes were evaluated via the TCGA and Genotype-Tissue Expression (GTEx) project. MiR-136-5p was significantly downregulated in LUSC patients. Glucuronidation, glucuronosyltransferase, and the retinoic acid metabolic process were the most enriched metabolic interactions in LUSC patients. Ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism were identified as crucial pathways. Seven hub genes (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A10, SRD5A1, and ADH7) were found to be upregulated, and UGT1A1, UGT1A3, UGT1A6, UGT1A7, and ADH7 were negatively correlated with miR-136-5p. UGT1A7 and ADH7 were the most significantly involved miR-136-5p target genes, and high expression of these genes was correlated with better overall survival and disease-free survival of LUSC patients. Downregulated miR-136-5p may target UGT1A7 and ADH7 and participate in ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and retinol metabolism. High expression of UGT1A7 and ADH7 may indicate better prognosis of LUSC patients. Copyright © 2018. Published by Elsevier GmbH.

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.

β-Glucuronidase-coupled assays of glucuronoyl esterases.

PubMed

Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

2016-10-01

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. Copyright © 2016 Elsevier Inc. All rights reserved.

Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis.

PubMed

Bjørnsdottir, I; Kepp, D R; Tjørnelund, J; Hansen, S H

1998-03-01

A capillary electrophoresis method for determination of the enantiomers of ibuprofen and its major phase I metabolites: 2'-hydroxyibuprofen and 2'-carboxyibuprofen in urine samples have been developed. Cyclodextrins and linear dextrins have been investigated as chiral selectors. Simultaneous chiral separation of the enantiomers of ibuprofen, 2'-hydroxyibuprofen and 2'-carboxyibuprofen was obtained using a mixture of dextrin 10 and heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin in a 2-[N-morpholino]ethanesulphonic acid buffer, pH 5.26. The electroosmotic flow was reversed using hexadimethrine bromide as a buffer additive. The method can be used for the determination of the free enantiomers of ibuprofen, 2'-hydroxyibuprofen and 2'-carboxyibuprofen as well as for the indirect determination of their glucuronic acid conjugates in urine samples.

PAPAIN-INDUCED CHANGES IN RABBIT CARTILAGE

PubMed Central

Tsaltas, Theodore T.

1958-01-01

Some biochemical aspects of the collapse of the rabbit ears produced by the intravenous injection of papain have been studied. A marked depletion of chondromucoprotein (M.C.S.) and a reduction of the S35 content of cartilage matrix were found to coincide with the gross and histologic changes in the cartilage. At the same time there was a marked increase in the amount of S35 in the serum and an increase of S35 and glucuronic acid excreted in the urine. Alteration in the composition of the M.C.S. remaining in the cartilage of the papain-injected animals was detected. The findings indicate that the collapse of the rabbit ears is due to loss of chondromucoprotein from cartilage and reduction of chondroitin sulfate in the chondromucoprotein that remains. All these changes were reversed in recovery. PMID:13575681

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

PubMed

Laezza, Antonio; Casillo, Angela; Cosconati, Sandro; Biggs, Caroline I; Fabozzi, Antonio; Paduano, Luigi; Iadonisi, Alfonso; Novellino, Ettore; Gibson, Matthew I; Randazzo, Antonio; Corsaro, Maria M; Bedini, Emiliano

2017-08-14

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum.

PubMed

Boyko, Alevtina S; Konnova, Svetlana A; Fedonenko, Yulia P; Zdorovenko, Evelina L; Smol'kina, Olga N; Kachala, Vadim V; Ignatov, Vladimir V

2011-10-20

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments. Copyright © 2011 Elsevier GmbH. All rights reserved.

Optimised deconjugation of androgenic steroid conjugates in bovine urine.

PubMed

Pedersen, Mikael; Frandsen, Henrik L; Andersen, Jens H

2017-04-01

After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase and glucuronidase resulting in free steroids in the extract. It is well known that some sulphates are not deconjugated using aryl sulphatase; instead, for example, solvolysis can be used for deconjugation of these aliphatic sulphates. The effectiveness of solvolysis on androgenic steroid sulphates was tested with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid in ethyl acetate were used for deconjugation and the extract was purified by solid-phase extraction. The final extract was evaporated to dryness, re-dissolved and analysed by LC-MS/MS.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

PubMed

Souffriau, Ben; den Abt, Tom; Thevelein, Johan M

2012-07-30

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Separation of carbohydrates using hydrophilic interaction liquid chromatography.

PubMed

Fu, Qing; Liang, Tu; Li, Zhenyu; Xu, Xiaoyong; Ke, Yanxiong; Jin, Yu; Liang, Xinmiao

2013-09-20

A strategy was developed to rapidly evaluate chromatographic properties of hydrophilic interaction chromatography (HILIC) columns for separating carbohydrates. Seven HILIC columns (Silica, Diol, TSK Amide-80, XAmide, Click Maltose, Click β-CD, and Click TE-Cys columns) were evaluated by using three monosaccharide and seven disaccharides as probes. The influence of column temperature on the peak shape and tautomerization of carbohydrates, as well as column selectivity were investigated. The influence of surface charge property on the retention was also studied by using glucose, glucuronic acid, and glucosamine, which indicated that buffer salt concentration and pH value in mobile phase was necessary to control the ionic interactions between ionic carbohydrates and HILIC columns. According to evaluation results, the XAmide column was selected as an example to establish experimental schemes for separation of complex mixtures of oligosaccharide. Copyright © 2013 Elsevier Ltd. All rights reserved.

Resolution and partial characterization of two aldehyde reductases of mammalian liver.

PubMed

Tulsiani, D R; Touster

1977-04-25

Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.

Papain-induced changes in rabbit cartilage; alterations in the chemical structure of the cartilage matrix.

PubMed

TSALTAS, T T

1958-10-01

Some biochemical aspects of the collapse of the rabbit ears produced by the intravenous injection of papain have been studied. A marked depletion of chondromucoprotein (M.C.S.) and a reduction of the S(35) content of cartilage matrix were found to coincide with the gross and histologic changes in the cartilage. At the same time there was a marked increase in the amount of S(35) in the serum and an increase of S(35) and glucuronic acid excreted in the urine. Alteration in the composition of the M.C.S. remaining in the cartilage of the papain-injected animals was detected. The findings indicate that the collapse of the rabbit ears is due to loss of chondromucoprotein from cartilage and reduction of chondroitin sulfate in the chondromucoprotein that remains. All these changes were reversed in recovery.

Biocompatible blood pool MRI contrast agents based on hyaluronan

PubMed Central

Zhu, Wenlian; Artemov, Dmitri

2010-01-01

Biocompatible gadolinium blood pool contrast agents based on a biopolymer, hyaluronan, were investigated for magnetic resonance angiography application. Hyaluronan, a non-sulfated linear glucosaminoglycan composed of 2000–25,000 repeating disaccharide subunits of D-glucuronic acid and N-acetylglucosamine with molecular weight up to 20 MDa, is a major component of the extracellular matrix. Two gadolinium contrast agents based on 16 and 74 kDa hyaluronan were synthesized, both with R1 relaxivity around 5 mM−1 s−1 per gadolinium at 9.4 T at 25°C. These two hyaluronan based agents show significant enhancement of the vasculature for an extended period of time. Initial excretion was primarily through the renal system. Later uptake was observed in the stomach and lower gastrointestinal tract. Macromolecular hyaluronan-based gadolinium agents have a high clinical translation potential as hyaluronan is already approved by FDA for a variety of medical applications. PMID:21504061

Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments.

PubMed

Roca, Christophe; Lehmann, Mareen; Torres, Cristiana A V; Baptista, Sílvia; Gaudêncio, Susana P; Freitas, Filomena; Reis, Maria A M

2016-06-25

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

PubMed

Qinghua, He; Dairong, Qiao; Qinglian, Zhang; Shunji, He; Yin, Li; Linhan, Bai; Zhirong, Yang; Yi, Cao

2005-06-01

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.

Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish.

PubMed

Li, Qiang-Ming; Wang, Jing-Fei; Zha, Xue-Qiang; Pan, Li-Hua; Zhang, Hai-Lin; Luo, Jian-Ping

2017-03-01

A new polysaccharide (JSP-11) with a molecular weight of 1.25×10 6 Da was extracted and purified from jellyfish. Monosaccharide analysis showed that JSP-11 was composed of mannose, galactose and glucuronic acid with a molar ratio of 2.18:1.00:1.94. According to the analysis of fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, JSP-11 was determined to contain a linear backbone which consisted of (1→3,6)-linked β-d-Manp and (1→6)-linked β-d-Galp. The branch of (1→)-linked α-d-GlcpA was attached to the C-3 position of (1→3,6)-linked β-d-Manp in the backbone. The immunomodulatory assay exhibited that JSP-11 could significantly enhance the viability of RAW 264.7 macrophage cells, and promote the release of NO, TNF-α, and IL-1β via activating NF-κB, MAPKs and PI3K/Akt signal pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pathways of metabolism of [1'-14C]-trans-anethole in the rat and mouse.

PubMed

Bounds, S V; Caldwell, J

1996-07-01

This study describes the metabolic fate of trans-4'-methoxyprop-[1-14C]enylbenzene, the natural flavor compound trans-anethole, in rats and mice given single doses of 250 mg/kg body weight. In both rats and mice, an essentially quantitative (> 95% of dose) recovery of 14C was obtained with the majority in the 0-24 hr urine. Separation and identification of 18 urinary anethole metabolites were achieved by radio-HPLC, chemical derivatization, and GC/ MS. Anethole undergoes three primary oxidation pathways-O-demethylation, omega-side chain oxidation, and side chain epoxidation-followed by a variety of secondary pathways of oxidation and hydration, the products of which are extensively conjugated with sulfate, glucuronic acid, glycine, and glutathione. A novel major metabolite has been characterized in the rat, apparently originating from conjugation of the epoxide with glutathione, namely S-[1-(4'-methoxyphenyl)-2-hydroxypropane]-N-acetylcysteine. These metabolites are discussed in terms of the pathways responsible for and the toxicological consequences of their formation.

Metabolism of Skin-Absorbed Resveratrol into Its Glucuronized Form in Mouse Skin

PubMed Central

Pluskal, Tomáš; Ito, Ken; Hori, Kousuke; Ebe, Masahiro; Yanagida, Mitsuhiro; Kondoh, Hiroshi

2014-01-01

Resveratrol (RESV) is a plant polyphenol, which is thought to have beneficial metabolic effects in laboratory animals as well as in humans. Following oral administration, RESV is immediately catabolized, resulting in low bioavailability. This study compared RESV metabolites and their tissue distribution after oral uptake and skin absorption. Metabolomic analysis of various mouse tissues revealed that RESV can be absorbed and metabolized through skin. We detected sulfated and glucuronidated RESV metabolites, as well as dihydroresveratrol. These metabolites are thought to have lower pharmacological activity than RESV. Similar quantities of most RESV metabolites were observed 4 h after oral or skin administration, except that glucuronidated RESV metabolites were more abundant in skin after topical RESV application than after oral administration. This result is consistent with our finding of glucuronidated RESV metabolites in cultured skin cells. RESV applied to mouse ears significantly suppressed inflammation in the TPA inflammation model. The skin absorption route could be a complementary, potent way to achieve therapeutic effects with RESV. PMID:25506824

Purification, structural characterization and anticoagulant properties of fucosylated chondroitin sulfate isolated from Holothuria mexicana.

PubMed

Mou, Jiaojiao; Wang, Cong; Li, Wenjing; Yang, Jie

2017-05-01

A novel fucosylated chondroitin sulfate (HmG) was isolated from sea cucumber Holothuria mexicana, the structure of which was characterized by monosaccharide composition, disaccharide composition, IR, 1 H and 13 C NMR spectrum, additionally with two dimensional NMR spectrum of degraded HmG (DHmG). The backbone of HmG was identified as chondroitin 6-O sulfate, while the major O-4 sulfated fucose branches linked to O-3 position of glucuronic acid in almost every disaccharide unit. The anticoagulant activities of HmG and DHmG were assessed and compared with heparin and low molecular weight heparin. The results indicated that HmG and DHmG both could significantly prolong the activated partial thrombo-plastin time, and the properties were well related to its molecular weight. DHmG showed similar anticoagulant properties to low molecular weight heparin with less bleeding risks, making it a safer anticoagulant drug. Copyright © 2017 Elsevier B.V. All rights reserved.

Inter vs. intraglycosidic acetal linkages control sulfation pattern in semi-synthetic chondroitin sulfate.

PubMed

Laezza, Antonio; De Castro, Cristina; Parrilli, Michelangelo; Bedini, Emiliano

2014-11-04

Microbial-sourced unsulfated chondroitin could be converted into chondroitin sulfate (CS) polysaccharide by a multi-step strategy relying upon benzylidenation and acetylation reactions as key-steps for its regioselective protection. By conducting the two reactions one- or two-pots, CSs with different sulfation patterns could be obtained at the end of the semi-synthesis. In particular, a CS polysaccharide possessing sulfate groups randomly distributed between positions 4 and 6 of N-acetyl-galactosamine (GalNAc) units could be obtained through the two-pots route, whereas the one-pot pathway allowed an additional sulfation at position 3 of some glucuronic acid (GlcA) units. This difference was ascribed to the stabilization of a labile interglycosidic benzylidene acetal involving positions O-3 and O-6 of some GlcA and GalNAc, respectively, when the benzylidene-acetylation reactions were conducted in a one-pot fashion. Isolation and characterization of a polysaccharide intermediate showing interglycosidic acetal moieties was accomplished. Copyright © 2014 Elsevier Ltd. All rights reserved.

Solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae SK1.

PubMed

Cescutti, P; Paoletti, S; Navarini, L; Flaibani, A

1993-08-01

The solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae SK1, SK1-CPS, were investigated by various methods. The SK1-CPS repeating unit is a branched pentasaccharide containing one glucuronic acid as single unit side chain; acetyl groups are present as non-carbohydrate substituents on the uronic acid residue in non-stoichiometric amounts. Chiro-optical, potentiometric, viscometric and rheological measurements have been performed in order to characterize the conformational behaviour of the polymer in water and in aqueous salt solutions. Under the investigated experimental conditions, changes of temperature, ionic strength and pH were shown not to induce any cooperative conformational transition. All the results obtained suggest that the solution conformation of SK1-CPS is a random coil with a certain degree of chain flexibility. The removal of the acetyl substituents apparently does not modify the overall conclusions drawn for the native polymer, except for an incipient tendency to aggregation revealed for high salt conditions.

Optimization of polysaccharides extraction from watermelon rinds: Structure, functional and biological activities.

PubMed

Romdhane, Molka Ben; Haddar, Anissa; Ghazala, Imen; Jeddou, Khawla Ben; Helbert, Claire Boisset; Ellouz-Chaabouni, Semia

2017-02-01

In the present work, optimization of hot water extraction, structural characteristics, functional properties, and biological activities of polysaccharides extracted from watermelon rinds (WMRP) were investigated. The physicochemical characteristics and the monosaccharide composition of these polysaccharides were then determined using chemical composition analysis, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and gas chromatography-flame ionization detection (GC-FID). SEM images showed that extracted polysaccharides had a rough surface with many cavities. GC-FID results proved that galactose was the dominant sugar in the extracted polysaccharides, followed by arabinose, glucose, galacturonic acid, rhamnose, mannose, xylose and traces of glucuronic acid. The findings revealed that WMRP displayed excellent antihypertensive and antioxidant activities. Those polysaccharides had also a protection effect against hydroxyl radical-induced DNA damage. Functional properties of extracted polysaccharides were also evaluated. WMRP showed good interfacial dose-dependent proprieties. Overall, the results suggested that WMRP presents a promising natural source of antioxidants and antihypertensive agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hot-compressed water extraction of polysaccharides from soy hulls.

PubMed

Liu, Hua-Min; Wang, Fei-Yun; Liu, Yu-Lan

2016-07-01

The polysaccharides of soy hulls were extracted by hot-compressed water at temperatures of 110 from 180°C and various treatment times (10-150min) in a batch system. It was determined that a moderate temperature and short time are suitable for the preparation of polysaccharides. The structure of xylan and the inter- and intra-chain hydrogen bonding of cellulose fibrils in the soy hulls were not significantly broken down. The polysaccharides obtained were primarily composed of α-L-arabinofuranosyl units, 4-O-methyl-glucuronic acid units and α-D-galactose units attached with substituted units. A sugar analysis indicated that arabinose was the major component, constituting 35.6-46.9% of the polysaccharide products extracted at 130°C, 140°C, and 150°C. This investigation contributes to the knowledge of the polysaccharides of soy by-products, which can reduce the environmental impact of waste from the food industries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Production of 5'-phosphodiesterase by Catharanthus roseus cells promoted by heat-degraded products generated from uronic acid.

PubMed

Akimoto-Tomiyama, Chiharu; Aoyagi, Hideki; Ozawa, Tetsuo; Tanaka, Hideo

2002-01-01

Polyalginate was autoclaved at 121 degrees C for 20 min and its molecular weight distribution was analyzed. The autoclaved alginate yielded alginate polymer, oligomer and heat degraded products (HDPs). Each of the separated substances promoted 5'-phosphodiesterase (5'-PDase) production in suspension culture of Catharanthus roseus cells. HDPs could also be generated from other uronic acids (galacturonic acid and glucuronic acid) by autoclave treatment. The most effective substance in the HDPs was isolated and characterized as trans-4,5-dihydroxy-2-cyclopenten-1-one (DHCP). The optimal conditions for DHCP production were also established (autoclaving 1 mg/ml monogalacturonic acid [pH 2] at 121 degrees C for 2 h). A combination of oligo-alginate (below 4 kDa) and HDPs significantly promoted the production of 5'-PDase in C. roseus. Based on the above results, a novel alginate complex that gave a 44-fold increase in 5'-PDase production by C. roseus was developed.

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

PubMed Central

de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.

2015-01-01

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.

PubMed

Thevis, Mario; Dib, Josef; Thomas, Andreas; Höppner, Sebastian; Lagojda, Andreas; Kuehne, Dirk; Sander, Mark; Opfermann, Georg; Schänzer, Wilhelm

2015-01-01

Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2)  = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves. Copyright © 2015 John Wiley & Sons, Ltd.

Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover.

PubMed

Gunawan, Christa; Xue, Saisi; Pattathil, Sivakumar; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

2017-01-01

Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans. In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4- O -methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues. To our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4- O -methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future.

The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma

PubMed Central

Preston, B. N.; Davies, M.; Ogston, A. G.

1965-01-01

1. Materials containing hyaluronic acid have been prepared by filtration (Ogston & Stanier, 1950) from ox synovial fluid and from a protein-rich human mesothelioma fluid. The ox material has been deproteinized by treatment with chloroform and pentanol and by gradient elution on DEAE-Sephadex; several fractions were obtained by the latter method. These materials can be stored in solution at −20° without change of properties. The ox material contained 21% of protein; all other preparations contained less than 6% of protein. 2. The two materials have been compared by sedimentation and viscosity and shown to be closely similar. Treatment of the ox material with neuraminidase caused no change in its viscosity behaviour. 3. Information about the molecular configuration of the ox material has been obtained from measurements of light-scattering and viscosity. The results, though consistent with a highly extended configuration, are not consistent with a linear random-coil configuration. It is tentatively suggested that the structure may have some degree of branching and of cross-linking, which give it a rigidity with respect to expansion of the molecular domain that would not be possessed by a random coil. 4. The deproteinized material recovered from DEAE-Sephadex, though polydisperse, showed unchanged average molecular weight; however, the average radius of gyration was greater than before this treatment. 5. Acidification to approx. pH3 resulted in a contraction of the structure, with only a slight degree of expansion when the pH was restored to 6·8–7·0. 6. Measurements of optical rotatory dispersion qualitatively support a structure less simple than a linear random coil. 7. Colloid osmotic pressures of mixed solutions of bovine serum albumin and of hyaluronic acid prepared by filtration from ox synovial fluid have been measured. The results agree approximately with those of Laurent & Ogston (1963) but are in quantitative disagreement with the partition measurements of Ogston & Phelps (1960). The relationships between thermodynamic quantities in a quaternary system of electrolytes are discussed in Appendix 2. 8. Refractometric measurements have been made in connexion with light-scattering measurements, as the basis for a convenient method of determining the concentrations of solutions of hyaluronic acids, and to measure the partition of sodium chloride in dialysis experiments. The theory of the last use is discussed in Appendix 1. 9. Sedimentation measurements on the ox preparation have been made up to a concentration of 1·4×10−2g./ml. The form of the sedimentation coefficient–concentration relationship is discussed. The value of the sedimentation coefficient at higher concentration is the basis of an illustration of the likely effect of hyaluronic acid on the flow of water through narrow channels in connective tissue. 10. Available colorimetric methods have been shown to give low estimates for glucuronic acid when applied to highly polymerized materials, as compared with estimates by decarboxylation. A spectrophotometric titration with cetylpyridinium bromide has been shown to give estimates of carboxyl groups that agree well with those of decarboxylation when applied to preparations of hyaluronic acid under suitable conditions; the results are not affected by the presence of protein. 11. Estimates of glucosamine (Ogston, 1964) have been found to be low compared with those of total acetyl, independently of the presence of protein. The magnitude of the discrepancy is characteristically different for preparations from ox synovial fluid and from mesothelioma. 12. Sialic acid was estimated in several preparations. It is likely that this forms part of the protein. 13. Analyses of preparations for total nitrogen, amino acids, total acetyl, glucuronic acid (by decarboxylation) and ash account for at least 95·7% of the dry weight in terms of N-acetylglucosaminyl, glucuronyl, protein and metal ions. Previously published analyses of hyaluronic acids are reviewed. 14. The estimated molar ratios of glucuronic acid to glucosamine were all significantly greater than unity. 15. The analytical results are interpreted as agreeing with the physicochemical measurements in suggesting a more complex structure, for at least some hyaluronic acids, than that of an alternate linear copolymer in random-coil configuration. PMID:5837786

Use of liquid chromatography hybrid triple-quadrupole mass spectrometry for the detection of emodin metabolites in rat bile and urine.

PubMed

Wu, Songyan; Zhang, Yaqing; Zhang, Zunjian; Song, Rui

2017-10-01

Emodin is the representative form of rhubarb, which is widely used in traditional Chinese medicine for the treatment of purgative, anti-inflammatory, antioxidative and antiviral, etc. Previous reports demonstrated that emodin glucuronide was the major metabolite in plasma. Owing to the extensive conjugation reactions of polyphenols, the aim of this study was to identify the metabolites of emodin in rat bile and urine. Neutral loss and precursor ion scan methods of triple-quadrupole mass spectrometer revealed 13 conjugated metabolites in rat bile and 22 metabolites in rat urine, which included four phase I and 18 phase II metabolites. The major metabolites in rat biosamples were emodin glucuronoconjugates. Moreover, rhein monoglucuronide, chrysophanol monoglucuronide and rhein sulfate were proposed for the first time after oral administration of emodin. Overall, liquid chromatography hybrid triple-quadrupole mass spectrometry analysis leads to the discovery of several novel emodin metabolites in rat bile and urine and underscores that conjugated with glucuronic acid is the main metabolic pathway. Copyright © 2017 John Wiley & Sons, Ltd.

Structural Elucidation and Biological Activity of a Highly Regular Fucosylated Glycosaminoglycan from the Edible Sea Cucumber Stichopus herrmanni.

PubMed

Li, Xiaomei; Luo, Lan; Cai, Ying; Yang, Wenjiao; Lin, Lisha; Li, Zi; Gao, Na; Purcell, Steven W; Wu, Mingyi; Zhao, Jinhua

2017-10-25

Edible sea cucumbers are widely used as a health food and medicine. A fucosylated glycosaminoglycan (FG) was purified from the high-value sea cucumber Stichopus herrmanni. Its physicochemical properties and structure were analyzed and characterized by chemical and instrumental methods. Chemical analysis indicated that this FG with a molecular weight of ∼64 kDa is composed of N-acetyl-d-galactosamine, d-glucuronic acid (GlcA), and l-fucose. Structural analysis clarified that the FG contains the chondroitin sulfate E-like backbone, with mostly 2,4-di-O-sulfated (85%) and some 3,4-di-O-sulfated (10%) and 4-O-sulfated (5%) fucose side chains that link to the C3 position of GlcA. This FG is structurally highly regular and homogeneous, differing from the FGs of other sea cucumbers, for its sulfation patterns are simpler. Biological activity assays indicated that it is a strong anticoagulant, inhibiting thrombin and intrinsic factor Xase. Our results expand the knowledge on structural types of FG and illustrate its biological activity as a functional food material.

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons

PubMed Central

González-Álvarez, Rafael; Pérez-Ibave, Diana Cristina; Garza-Rodríguez, María Lourdes; Lugo-Trampe, Ángel; Delgado-Enciso, Iván; Tejero-Barrera, María Elizabeth; Martínez-De-Villarreal, Laura Elia; Garza-Guajardo, Raquel; Sánchez-Chaparro, María Marisela; Ruiz-Ayma, Gabriel; Barboza-Quintana, Oralia; Barrera-Saldaña, Hugo Alberto; Rocha-Pizaña, María Del Refugio; Rodríguez-Sánchez, Irám Pablo

2017-01-01

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe2+/Fe3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces. PMID:29085625

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons.

PubMed

González-Álvarez, Rafael; Pérez-Ibave, Diana Cristina; Garza-Rodríguez, María Lourdes; Lugo-Trampe, Ángel; Delgado-Enciso, Iván; Tejero-Barrera, María Elizabeth; Martínez-De-Villarreal, Laura Elia; Garza-Guajardo, Raquel; Sánchez-Chaparro, María Marisela; Ruiz-Ayma, Gabriel; Barboza-Quintana, Oralia; Barrera-Saldaña, Hugo Alberto; Rocha-Pizaña, María Del Refugio; Rodríguez-Sánchez, Irám Pablo

2017-10-01

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe 2+ /Fe 3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces.

Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

PubMed Central

Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

2009-01-01

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

Preparation and characterization of iron oxide magnetic nanoparticles functionalized by nisin.

PubMed

Gruskiene, Ruta; Krivorotova, Tatjana; Staneviciene, Ramune; Ratautas, Dalius; Serviene, Elena; Sereikaite, Jolanta

2018-05-08

Nisin is a known bacteriocin approved as a food additive for food preservation. It exhibits a wide spectrum antimicrobial activity against Gram-positive bacteria. Iron oxide magnetic nanoparticles were synthesized and characterized by X-ray diffraction method. A main part of iron oxide nanoparticles was found to be maghemite though a small quantity of magnetite could also be present. Magnetic nanoparticles were stabilized by citric, ascorbic, gallic or glucuronic acid coating. Stable iron oxide magnetic nanoparticles were functionalized by nisin using a simple and low cost adsorption method. Nisin loading was confirmed by FT-IR spectra, thermogravimetric analysis, dynamic light scattering and atomic force microscopy methods. Nisin-loaded iron oxide magnetic nanoparticles were stable at least six weeks as judged by the measurements of zeta-potential and hydrodynamic diameter. The antimicrobial activity of nisin-loaded iron oxide magnetic nanoparticles was demonstrated toward Gram-positive bacteria. Functionalized nanoparticles could therefore find the application as antimicrobials in innovative and emerging technologies based on the magnetic field. Copyright © 2018 Elsevier B.V. All rights reserved.

5-Acetamido-3,5-dideoxy-L-glycero-L-manno-non-2-ulosonic acid-containing O-polysaccharide from marine bacterium Pseudomonas glareae KMM 9500T.

PubMed

Kokoulin, Maxim S; Kalinovsky, Anatoly I; Romanenko, Lyudmila A; Mikhailov, Valery V

2018-05-22

The O-polysaccharide was isolated from the lipopolysaccharide of a marine bacterium Pseudomonas glareae KMM 9500 T and studied by chemical methods along with 1D and 2D 1 H and 13 C NMR spectroscopy including 1 H, 1 H-TOCSY, 1 H, 1 H-COSY, 1 H, 1 H-ROESY, 1 H, 13 C-HSQC and 1 H, 13 C-HMBC experiments. The O-polysaccharide was found to consist of linear tetrasaccharide repeating units constituted by D-glucuronic acid (D-GlcA), L-rhamnose (L-Rha), D-glucose (D-Glc) and 5-acetamido-7,9-O-[(S)-1-carboxyethylidene]-3,5-dideoxy-L-glycero-L-manno-non-2-ulosonic acid (Sug7,9(S-Pyr)), partially O-acetylated at position 8 (∼70%): →4)-α-D-GlcpA-(1→3)-β-L-Rhap-(1→4)-β-D-Glcp-(1→4)-β-Sugp8Ac(∼70%)7,9(S-Pyr)-(2→. Copyright © 2018 Elsevier Ltd. All rights reserved.

Metabolism of a new positive inotropic agent, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (OPC-8212) in the rat, mouse, dog, monkey and human.

PubMed

Miyamoto, G; Sasabe, H; Tominaga, N; Uegaki, N; Tominaga, M; Shimizu, T

1988-10-01

1. After OPC-8212 was orally given to rats, mice, dogs, monkeys and humans, its metabolites were identified by n.m.r. and mass spectrometry, and their concentrations in the plasma, urine and faeces of these species were measured by high-performance liquid chromatography (h.p.l.c.). 2. Hydrolysis of the amide group, oxidation and cleavage of the piperazine ring, O-demethylation of the methoxy group, and conjugation were proposed as metabolic pathways of OPC-8212. 3. In rats, mice and monkeys given OPC-8212 orally, metabolites M-1 to M-6 were detected in the plasma, urine and faeces, while M-1, -4, -5 and M-6 were detected in dogs, and M-1, M-3, M-4, M-5 and M-6 were detected in humans. 4. Conjugates of metabolites M-6 and M-7, with glucuronic acid and sulphuric acid, were observed in the urine of rats and humans.

Characterization of Momordica charantia L. polysaccharide and its protective effect on pancreatic cells injury in STZ-induced diabetic mice.

PubMed

Zhang, Cong; Chen, Hongman; Bai, Weiqi

2018-04-10

A polysaccharide with a molecular weight of 13,029Da was isolated from Momordica charantia (MCP) fruit and purified by ion-exchange and size-exclusion chromatography. The isolated polysaccharide MCPIIa contained L-Rha, D-GalA, D-Gal, D-Xyl, L-Ara in a molar ratio of 12:3.05:19.89:5.95:56. IR spectrum and NMR studies indicated that the MCPIIa sugar units were linked, via β-glycosidic bonds, to a large number of arabinofuranose, glucuronic acid, and xylopyranosyl residues. In addition, the hypoglycemic effect of MCPIIa was investigated in streptozotocin (STZ)-induced diabetic mice. After STZ-induction, MCPIIa (100, 200, or 300mg/kg body weight) was administered orally, once daily, for 28days. Glycemia in STZ-diabetogenic mice was significantly reduced, and compared with diabetes mellitus (DM) mice, serum insulin concentration increased significantly, following MCPIIa administration. Transmission electron microscopy showed an alleviation of STZ-lesions in pancreatic tissue from mice treated with MCPIIa. These results indicate that MCPIIa may be useful as an anti-diabetic agent. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis of multivalent sialyllactosamine-carrying glyco-nanoparticles with high affinity to the human influenza virus hemagglutinin.

PubMed

Ogata, Makoto; Umemura, Seiichiro; Sugiyama, Naohiro; Kuwano, Natsuki; Koizumi, Ami; Sawada, Tadakazu; Yanase, Michiyo; Takaha, Takeshi; Kadokawa, Jun-Ichi; Usui, Taichi

2016-11-20

A series of multivalent sialoglyco-conjugated nanoparticles were efficiently synthesized by using highly-branched α-glucuronic acid-linked cyclic dextrins (GlcA-HBCD) as a backbone. The sialoglycoside-moieties, with varying degrees of substitution, could be incorporated onto the preformed nanoparticles. These synthesized particles, which are highly soluble in aqueous solution, were shown to have a spherical nanostructure with a diameter of approximately 15nm. The interactions of the sialoglyco-nanoparticles (Neu5Acα2,6LacNAc-GlcA-HBCDs) with human influenza virus strain A/Beijing/262/95 (H1N1) were investigated using a hemagglutination inhibition assay. The sialoglyco-nanoparticle, in which the number of sialic acid substitution is 30, acted as a powerful inhibitor of virus binding activity. We show that both distance and multiplicity of effective ligand-virus formation play important roles in enhancing viral inhibition. Our results indicate that the GlcA-HBCD backbone can be used as a novel spherical nanocluster material for preparing a variety of glyco-nanoparticles to facilitate molecular recognition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suitability of bovine bile compared to urine for detection of free, sulfate and glucuronate boldenone, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone by LC-MS/MS.

PubMed

Chiesa, Luca; Nobile, Maria; Panseri, Sara; Vigo, Daniele; Pavlovic, Radmila; Arioli, Francesco

2015-12-01

The administration of boldenone and androstadienedione to cattle is forbidden in the European Union, while prednisolone is permitted for therapeutic purposes. They are pseudoendogenous substances (endogenously produced under certain circumstances). The commonly used matrices in control analyses are urine or liver. With the aim of improving the residue controls, we previously validated a method for steroid analysis in bile. We now compare urine (a 'classic' matrix) to bile, both collected at the slaughterhouse, to understand whether the detection of steroids in the latter is easier. With the aim of having clearer results, we tested the presence of the synthetic corticosteroid dexamethasone. The results show that bile does not substantially improve the detection of boldenone, or its conjugates, prednisolone and prednisone. Dexamethasone, instead, was found in 10 out of 53 bovine bile samples, but only in one urine sample from the same animals. Bile could constitute a novel matrix for the analysis of residues in food-producing animals, and possibly not only of synthetic corticosteroids. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antioxidant and antitumor effects of polysaccharides from the fungus Pleurotus abalonus.

PubMed

Ren, Daoyuan; Jiao, Yadong; Yang, Xingbin; Yuan, Li; Guo, Jianjun; Zhao, Yan

2015-07-25

Dietary supplement of edible Pleurotus abalonus (P. abalonus) rich in fungal polysaccharides is associated with anticancer health benefit. We here isolated the polysaccharides (PAP) from the fruiting bodies of P. abalonus, and evaluated the antiproliferative activity of the polysaccharides in human colorectal carcinoma LoVo cells. HPLC analysis showed that PAP consisted of D-mannose, D-ribose, l-rhamnose, D-glucuronic acid, D-glucose and D-galactose, and their corresponding mole percentages were 3.4%, 1.1%, 1.9%, 1.4%, 87.9% and 4.4%, respectively. PAP was shown to exert a high antioxidant activity in vitro and a dose-dependent antiproliferative effect against LoVo cancer cells. Flow cytometry analysis demonstrated that PAP exhibited a stimulatory effect on apoptosis of LoVo cells, and induced the cell-cycle arrest at the S phase. We also found that PAP could increase the generation of intracellular ROS which was a critical mediator in PAP-induced cell growth inhibition. These findings suggest that PAP may serve as a potential novel dietary agent for human colon cancer chemoprevention. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain*

PubMed Central

Zhao, Xue; Yang, Bo; Solakylidirim, Kemal; Joo, Eun Ji; Toida, Toshihiko; Higashi, Kyohei; Linhardt, Robert J.; Li, Lingyun

2013-01-01

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences. PMID:23423381

Monosaccharide composition of acidic gum exudates from Indian Acacia tortilis ssp. raddiana (Savi) Brenan.

PubMed

Lakhera, Ajeet Kumar; Kumar, Vineet

2017-01-01

Acacia tortilis ssp. raddiana (Savi) Brenan commonly known as Israeli Babool has contributed immensely for sand dunes management in Indian desert leading to wind erosion control and increased biological productivity. The species is extensively used in traditional medicine system for a number of therapeutic applications and as nutraceutical. The polysaccharide was isolated in 43.6% yield from gum exudates. The monosaccharides, L-arabinose, D-galactose D-glucose, L-rhamnose and D-mannose were determined in molar ratio of 78.1%, 18.64%, 0.60%, 1.71% and 0.74% respectively. The molar ratio of uronic acids was studied using diverse spectrophotometric methods and compared with GLC. The content of D-galacturonic acid and D-glucuronic was determined as 3.88% and 4.35% respectively by GLC. The results were compared with the spectrophotometric methods. The results using DMP as chromogenic reagent are closer to that obtained by GLC. Structural analysis of the polysaccharide may provide scientific basis for nutraceutical, pharmaceutical and biological applications of gum exudates from A. tortilis, which is extensively planted in India. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptomic Analysis of Flower Blooming in Jasminum sambac through De Novo RNA Sequencing.

PubMed

Li, Yong-Hua; Zhang, Wei; Li, Yong

2015-06-10

Flower blooming is a critical and complicated plant developmental process in flowering plants. However, insufficient information is available about the complex network that regulates flower blooming in Jasminum sambac. In this study, we used the RNA-Seq platform to analyze the molecular regulation of flower blooming in J. sambac by comparing the transcript profiles at two flower developmental stages: budding and blooming. A total of 4577 differentially-expressed genes (DEGs) were identified between the two floral stages. The Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that the DEGs in the "oxidation-reduction process", "extracellular region", "steroid biosynthesis", "glycosphingolipid biosynthesis", "plant hormone signal transduction" and "pentose and glucuronate interconversions" might be associated with flower development. A total of 103 and 92 unigenes exhibited sequence similarities to the known flower development and floral scent genes from other plants. Among these unigenes, five flower development and 19 floral scent unigenes exhibited at least four-fold differences in expression between the two stages. Our results provide abundant genetic resources for studying the flower blooming mechanisms and molecular breeding of J. sambac.

A Comparative Study on Aflatoxin B1 Metabolism in Mice and Rats

PubMed Central

Steyn, M.; Pitout, M. J.; Purchase, I. F. H.

1971-01-01

In vivo metabolic studies on rats and mice revealed a marked difference in the fluorescent compounds produced after ingestion of aflatoxin B1. The mouse converted aflatoxin B1 to three unknown fluorescent compounds, designated x1, x2 and x3 and the known aflatoxin M1, while the rat was only capable of producing aflatoxin M1. The results suggested that metabolites x1, x2, x3 and aflatoxin M1 were not part of a major metabolic pathway, but produced independently. These unknown yellowish-green fluorescent compounds did not seem to be conjugated with sulphate or glucuronic acid. In vitro incubations of various mouse liver cell fractions with aflatoxin B1 showed that metabolites x1, x2, x3 and aflatoxin M1, could only be produced by the microsomal fraction and that NADPH was needed as a co-factor. The differences in aflatoxin metabolism by mice and rats are discussed in relation to the apparent resistance of the mouse to the carcinogenic effects of this toxin. PMID:4398926

Microbial Glucuronoyl Esterases: 10 Years after Discovery

PubMed Central

2016-01-01

A carbohydrate esterase called glucuronoyl esterase (GE) was discovered 10 years ago in a cellulolytic system of the wood-rotting fungus Schizophyllum commune. Genes coding for GEs were subsequently found in a number of microbial genomes, and a new family of carbohydrate esterases (CE15) has been established. The multidomain structures of GEs, together with their catalytic properties on artificial substrates and positive effect on enzymatic saccharification of plant biomass, led to the view that the esterases evolved for hydrolysis of the ester linkages between 4-O-methyl-d-glucuronic acid of plant glucuronoxylans and lignin alcohols, one of the crosslinks in the plant cell walls. This idea of the function of GEs is further supported by the effects of cloning of fungal GEs in plants and by very recently reported evidence for changes in the size of isolated lignin-carbohydrate complexes due to uronic acid de-esterification. These facts make GEs interesting candidates for biotechnological applications in plant biomass processing and genetic modification of plants. This article is a brief summary of current knowledge of these relatively recent and unexplored esterases. PMID:27694239

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.

Investigation of bacterial resistance to the immune system response: cepacian depolymerisation by reactive oxygen species.

PubMed

Cuzzi, Bruno; Cescutti, Paola; Furlanis, Linda; Lagatolla, Cristina; Sturiale, Luisa; Garozzo, Domenico; Rizzo, Roberto

2012-08-01

Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

PubMed

Zenger, Katharina; Agnolet, Sara; Schneider, Bernd; Kraus, Birgit

2015-07-22

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

Cyclic changes in hormones, carbohydrates and indole metabolism in cervical mucus of normal, fertilizing cows and the relationship with non-fertility.

PubMed

Zaaijer, D; van der Horst, C J

1983-01-01

In the cervical mucus of 'normal' cows the cholesterol content was very low at D--0; at D + 12 it was about fifty times as high; then it decreased to about half its value at D + 17 and then to the low value at D--0. Usually small amounts of oestrogen, testosterone, pregnanedione and progesterone were found at D--0; on D + 12 more pregnanedione was found and less oestrogen, while at D + 17 more oestrogen occurred and less pregnanedione. The fructose and glucose content was very low at D--0; then it increased until D + 12, when glucose was dominant, while at D + 17 it had decreased and rather large amounts of glucuronic acid and of sorbitol occurred. On D + 12 a blue fluorescing indole metabolite was sometimes found. Deviations from these patterns, were found particularly in the winter months, and coincided with lowered fertility. Indole metabolism was stronger in the winter months than in the summer months and occurred more in cows than in heifers.

Endogenous nandrolone metabolites in human urine: preliminary results to discriminate between endogenous and exogenous origin.

PubMed

Le Bizec, Bruno; Bryand, Fabrice; Gaudin, Isabelle; Monteau, Fabrice; Poulain, Frédéric; Andre, François

2002-02-01

When administered to human subjects, nandrolone is metabolized into two main products, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Recent studies demonstrated the endogenous production of these compounds in man at concentrations very close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml. Because the possibility of reaching or exceeding this fateful limit is difficult to exclude, a complementary biochemical parameter is necessary for the differentiation of endogenous 19-NA and 19-NE production from residues resulting from nandrolone consumption. We measured the endogenous concentrations of 19-NA and 19-NE in 385 urine samples from professional football players, and we studied the phase II metabolite composition in individuals excreting the highest concentrations. The results showed that around 30% of endogenous 19-norandrosterone was sulfo-conjugated, whereas 100% of 19-norandrosterone was excreted conjugated to a glucuronic acid when nandrolone was administered. This significant qualitative difference appears to be a promising complementary criterion to more definitively conclude about an athlete's culpability, especially when nandrolone metabolites are found in the low ng/ml range.

The structural features of hemicelluloses dissolved out at different cooking stages of active oxygen cooking process.

PubMed

Shi, Jianbin; Yang, Qiulin; Lin, Lu

2014-04-15

This work described the morphologic changes of corn stalk and the structural characterization of its hemicelluloses dissolved in yellow liquor at different cooking stages. The results showed that active oxygen cooking process was an efficient method to depolymerize the corn stalk into cellulose, hemicelluloses, and lignin as a pretreatment of biomass conversion. This cooking process can also be divided into three phases: bulk delignification, extended delignification, and residual delignification. During the heating-up period 57.67% of hemicelluloses and 62.31% of lignin were removed from the raw material. However, only 15% of hemicelluloses and 23.21% of lignin were removed during at temperature' period. The hemicelluloses from the corn stalk and yellow liquor were composed of (1→4)-β-D-xylopyranose backbones substituted with α-l-arabinofuranosyl, 4-O-methyl-α-D-glucuronic acid, and some methoxyl residues. The backbones of hemicelluloses were gradually cleaved during the cooking process. The acetyl groups substituted with xylopyranosyl residues were completely cleaved during the cooking process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural characterization of hemicelluloses and topochemical changes in Eucalyptus cell wall during alkali ethanol treatment.

PubMed

Li, Han-Yin; Sun, Shao-Ni; Zhou, Xia; Peng, Feng; Sun, Run-Cang

2015-06-05

Eucalyptus was sequentially extracted with 70% ethanol containing 0.4, 1.0, 2.0, 3.0, and 5.0% NaOH for 2h at 80°C. The chemical composition and structural features of the hemicellulosic fractions obtained were comparatively characterized by the combination of high-performance anion-exchange chromatography, gel permeation chromatography, Fourier transform infrared, and nuclear magnetic resonance spectroscopies. Furthermore, the main component distribution and their changes in cell wall were investigated by confocal Raman microscopy. Based on the Fourier transform infrared and nuclear magnetic resonance analyses, the hemicelluloses extracted from Eucalyptus mainly have a linear backbone of (1→4)-linked-β-d-xylopyranosyl residues decorated with branch at O-2 of 4-O-methyl-α-glucuronic acid unit. Raman analysis revealed that the dissolution of hemicelluloses was different in the morphological regions, and the hemicelluloses released mainly originated from the secondary wall. The information obtained from the study conducted by combining chemical characterization with ultrastructure provides important basis for studying the mechanism of the alkali treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metabolism of fluoranthene in different plant cell cultures and intact plants

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

Kolb, M.; Harms, H.

The metabolism of fluoranthene was investigated in 11 cell cultures of different plant species using a [{sup 14}C]-labeled standard. Most species metabolized less than 5% of fluoranthene to soluble metabolites and formed less than 5% nonextractable residues during the standardized 48-h test procedure. Higher metabolic rates were observed in lettuce (Lactuca sativa, 6%), wheat (Tricitum aestivum, 9%), and tomato (Lycopersicon esculentum, 15%). A special high metabolic rate of nearly 50% was determined for the rose species Paul's Scarlet. Chromatographic analysis of metabolites extracted from aseptically grown tomato plants proved that the metabolites detected in the cell cultures were also formedmore » in the intact plants. Metabolites produced in tomato and rose cells from [{sup 14}C]-fluoranthene were conjugated with glucose, glucuronic acid, and other cell components. After acid hydrolyses, the main metabolite of both species was 1-hydroxyfluoranthene as identified by gas chromatography-mass spectrometry and high-performance liquid chromatography with diode array detection. The second metabolite formed by both species was 8-hydroxyfluoranthene. A third metabolite in tomatoes was 3-hydroxyfluoranthene.« less

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

PubMed

Ishii, Tadashi; Matsuoka, Keita; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Yaoi, Katsuro; Nakano, Yoshimi; Ohtani, Misato; Demura, Taku; Iwai, Hiroaki; Satoh, Shinobu

2017-11-15

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of isochlorogenic acid A metabolites in rats using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

PubMed

Wang, Jing; Cao, Guoxiu; Wang, Hong; Ye, Hui; Zhong, Yunxi; Wang, Guangji; Hao, Haiping

2017-08-01

Isochlorogenic acid A is widely present in fruits, vegetables and herbal medicines, and is characterized by anti-inflammatory, hepatoprotective and antiviral properties. However, little is known about its metabolic fate and pharmacokinetic properties. This study is thus designed to investigate the metabolic fate of isochlorogenic acid A. An analytical method based on high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) was established to characterize the metabolites of isochlorogenic acid A in the plasma, urine and feces of rats. A total of 32 metabolites were identified. The metabolic pathways mainly include hydrolyzation, dehydroxylation, hydrogenation and conjugation with methyl, glucuronic acid, glycine, sulfate, glutathione and cysteine. Moreover, the pharmacokinetic profiles of all the circulating metabolites were investigated. M11 resulting from hydrolyzation, dehydroxylation and hydrogenation was the dominant circulating metabolite after the intragastric administration of isochlorogenic acid A. The results obtained will be useful for further study of elucidating potential bioactive metabolites which can provide better explanation of the pharmacological and/or toxicological effects of this compound. Copyright © 2017 John Wiley & Sons, Ltd.

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming.

PubMed

Saito, Tetsuya; Ichimura, Yoshinobu; Taguchi, Keiko; Suzuki, Takafumi; Mizushima, Tsunehiro; Takagi, Kenji; Hirose, Yuki; Nagahashi, Masayuki; Iso, Tetsuro; Fukutomi, Toshiaki; Ohishi, Maki; Endo, Keiko; Uemura, Takefumi; Nishito, Yasumasa; Okuda, Shujiro; Obata, Miki; Kouno, Tsuguka; Imamura, Riyo; Tada, Yukio; Obata, Rika; Yasuda, Daisuke; Takahashi, Kyoko; Fujimura, Tsutomu; Pi, Jingbo; Lee, Myung-Shik; Ueno, Takashi; Ohe, Tomoyuki; Mashino, Tadahiko; Wakai, Toshifumi; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Motohashi, Hozumi; Waguri, Satoshi; Soga, Tomoyoshi; Yamamoto, Masayuki; Tanaka, Keiji; Komatsu, Masaaki

2016-06-27

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

[Comparative studies on the characteristics of the Fourier-transform infrared spectra between sturgeon and shark chondroitin sulfates].

PubMed

Zheng, Jiang; Guan, Rui-Zhang; Huang, Shi-Yu

2008-01-01

The characteristics of the Fourier-transform infrared spectra of sturgeon and shark chondroitin sulfates (CHSs) were comparatively studied. The results show that sturgeon CHS exhibits special vibrations at the wavenumbers of 1 376, 1 344, 1 310, 1 157, 883 and 856 cm(-1). Further analysis shows that shark CHS contains 6-sulfated-CHS, while sturgeon CHS contains 4, 6-disulfated CHS, indicating that sturgeon CHS could have higher biological activities in decreasing the toxicity of medicine and in killing cancer cells. In general, the two kinds of CHSs have similar infrared spectra and groups of acylamino, carboxyl, sulfate, and saccharide ring. But the N-H variable-angle vibration of acylamino group of sturgeon CHS occurs at the higher wavenumber, and the vibration intensity of carboxyl group at 1 415 cm(-1) is also stronger than that of shark CHS. Chemical analysis shows that sturgeon CHS has a higher content of glucuronic acid, suggesting that it probably could be a better kind of medical materials for the bone mineralization.

Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

PubMed

Guo, Chang; Ma, Jingfan; Zhong, Qionghong; Zhao, Mengyuan; Hu, Tianxing; Chen, Tong; Qiu, Longxin; Wen, Longping

2017-08-01

Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy. Copyright © 2017. Published by Elsevier Inc.

A new method for the quantification of monosaccharides, uronic acids and oligosaccharides in partially hydrolyzed xylans by HPAEC-UV/VIS.

PubMed

Lorenz, Dominic; Erasmy, Nicole; Akil, Youssef; Saake, Bodo

2016-04-20

A new method for the chemical characterization of xylans is presented, to overcome the difficulties in quantification of 4-O-methyl-α-D-glucuronic acid (meGlcA). In this regard, the hydrolysis behavior of xylans from beech and birch wood was investigated to obtain the optimum conditions for hydrolysis, using sulfuric acid. Due to varying linkage strengths and degradation, no general method for complete hydrolysis can be designed. Therefore, partial hydrolysis was applied, yielding monosaccharides and small meGlcA containing oligosaccharides. For a new method by HPAEC-UV/VIS, these samples were reductively aminated by 2-aminobenzoic acid. By quantification of monosaccharides and oligosaccharides, as well as comparison with borate-HPAEC and (13)C NMR-spectroscopy, we revealed that the concentrations meGlcA are significantly underestimated compared to conventional methods. The detected concentrations are 85.4% (beech) and 76.3% (birch) higher with the new procedure. Furthermore, the quantified concentrations of xylose were 9.3% (beech) and 6.5% (birch) higher by considering the unhydrolyzed oligosaccharides as well. Copyright © 2015 Elsevier Ltd. All rights reserved.

The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal-plant arms-race and co-evolution.

PubMed

Bock, Karl Walter

2016-01-01

UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs. Copyright © 2015. Published by Elsevier Inc.

Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

PubMed

Franke, Lukáš; Čožíková, Dagmar; Smirnou, Dzianis; Hermannová, Martina; Hanová, Tereza; Růžičková, Andrea; Velebný, Vladimír

2015-08-01

Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Developmental control of apiogalacturonan biosynthesis and UDP-apiose production in a duckweed. [Spirodela polyrrhiza

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

Longland, J.M.; Fry, S.C.; Trewavas, A.J.

1989-07-01

Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, D-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed D-({sup 3}H)glucuronic acid for 30 minutes, the accumulated UDP-({sup 3}H)apiose pool accounted for about 27% of the total phosphorylated ({sup 3}H)pentose derivatives; in turions, the UDP({sup 3}H)apiose pool accounted for only about 4% of the total phosphorylated ({sup 3}H)pentose derivatives.more » They conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.« less

Tyrosol and its metabolites as antioxidative and anti-inflammatory molecules in human endothelial cells.

PubMed

Muriana, Francisco J G; Montserrat-de la Paz, Sergio; Lucas, Ricardo; Bermudez, Beatriz; Jaramillo, Sara; Morales, Juan C; Abia, Rocio; Lopez, Sergio

2017-08-01

Tyrosol (Tyr) is a phenolic compound found in virgin olive oil. After ingestion, Tyr undergoes extensive first pass intestinal/hepatic metabolism. However, knowledge about the biological effects of Tyr metabolites is scarce. We chemically synthesized Tyr glucuronate (Tyr-GLU) and sulphate (Tyr-SUL) metabolites and explored their properties against oxidative stress and inflammation in TNF-α-treated human umbilical vein endothelial cells (hECs). Tyr and Tyr-SUL prevented the rise of reactive oxygen species, the depletion of glutathione, and the down-regulation of glutathione peroxidase 1, glutamate-cysteine ligase catalytic subunit, and heme oxygenase-1 genes. Tyr-SUL and to a lower extent Tyr and Tyr-GLU prevented the phosphorylation of NF-κB signaling proteins. Tyr-GLU and Tyr-SUL also prevented the over-expression of adhesion molecules at gene, protein, and secretory levels, and the adhesion (Tyr-SUL > Tyr-GLU) of human monocytes to hECs. In vivo, Tyr, and most notably Tyr-SUL in a dose-dependent manner, ameliorated plantar and ear edemas in mice models of acute and chronic inflammation. This study demonstrates the antioxidant and/or anti-inflammatory properties of Tyr metabolites, with Tyr-SUL being the most effective.

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

PubMed

Yurist-Doutsch, Sophie; Magidovich, Hilla; Ventura, Valeria V; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

2010-02-01

Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

PubMed Central

Iverson, Sonya V.; Eriksson, Sofi; Xu, Jianqiang; Prigge, Justin R.; Talago, Emily A.; Meade, Tesia A.; Meade, Erin S.; Capecchi, Mario R.; Arnér, Elias S.J.; Schmidt, Edward E.

2013-01-01

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug-metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however their constitutive metabolic state supported more robust GSH biosynthesis-, glutathionylation-, and glucuronidation-systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage. PMID:23743293

Comparative study on the physicochemical and functional properties of the mucilage in the carpel of Nymphaea odorata using ultrasonic and classical heating extractions.

PubMed

Wu, WeiZhi; Tu, ChinWei; Yang, WenJen; Wang, HengLong; Chang, ChaoLin; Chung, JengDer; Lu, MeiKuang; Liao, WeiTung

2018-02-21

The cooked carpel of Nymphaea odorata has a large amount of transparent mucilage; however, the basic characteristics of this mucilage have not yet been reported. This study compared the physicochemical and functional properties of this mucilage obtained using conventional hot water extraction (HWM) and ultrasonic-assisted extraction (UAM). Neither HWM nor UAM affected the viability of mouse skin fibroblasts (NIH/3T3) below 100 μg/mL. UAM had a higher yield production, phenol concentration, and in vitro antioxidant activity, but lower viscosity and water-holding capacity than for HWM. The Fourier transform infrared spectra revealed that the dialyzed HWM and UAM, named HWMD and UAMD, respectively, appeared major spectral differences at 1730 cm -1 and 1605 cm -1 , implying that the degree of methylation was different between HWMD and UAMD. Compared to HWMD, UAMD in low-molecular weight polysaccharides increased. Evidently, the basic characteristics of native mucilage in the carpel of N. odorata were greatly changed by various extractions. Nevertheless, sugar analysis indicated that glucuronic acid was the mainly composition of HWMD and UAMD. Copyright © 2017. Published by Elsevier B.V.

OsMIOX, a myo-inositol oxygenase gene, improves drought tolerance through scavenging of reactive oxygen species in rice (Oryza sativa L.).

PubMed

Duan, Junzhi; Zhang, Minghui; Zhang, Hongliang; Xiong, Haiyan; Liu, Pengli; Ali, Jauhar; Li, Jinjie; Li, Zichao

2012-11-01

Myo-inositol oxygenase (MIOX), a unique monooxygenase, catalyzes the oxidation of myo-inositol to d-glucuronic acid. However, the protective role of MIOX in plants against oxidative stress or drought stress remains unknown. In this study, the functional characterization of MIOX obtained from the cDNA library of upland rice (Oryza sativa L. cv. IRAT109), was performed. OsMIOX was expressed predominantly in the roots and induced by drought, H₂O₂, salt, cold and abscisic acid. The transgenic rice lines overexpressing OsMIOX showed obviously improved growth performance in the medium containing 200 mM mannitol. Further, the survival rate of leaves from the transgenic rice lines was significantly higher than that of the wild type plants under polyethylene glycol treatment. It was discovered that the activity of ROS-scavenging enzymes and proline content, as well as the transcript levels of many ROS scavenging genes were significantly increased in transgenic plants compared to the wild type plants under drought stress conditions. Together, these data suggest that OsMIOX has a specific function in drought stress tolerance by decreasing oxidative damage. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Mucilage processing and secretion in the green alga closterium. I. Cytology and biochemistry

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

Domozych, C.R.; Plante, K.; Blais, P.

1993-10-01

Placoderm desmids (Conjugales, Chlorophyta) such as Closterium exhibit a gliding locomotory behavior. This results from the forceful extrusion of an acidic polysaccharide from one pole of the cell causing the cell to glide in the opposite direction. A biochemical and cytological analysis of gliding behavior was performed. The mucilage is a high molecular weight polysaccharide rich in glucuronic acid and fucose. Under normal growth conditions, 3 [mu]g of mucilage is produced per cell in 30 days. Mucilage production increased 3-4 fold in cells challenged with low phosphate or nitrate conditions. A polyclonal antibody was raised against the mucilage and usedmore » in immunofluorescence studies. These results show that upon contact with another object Closterium aligns itself parallel to that object by a [open quotes]jack-knife[close quotes] motion. Subsequently, large amounts of mucilage are released to form elongate tubes enmeshing the cell with that object. In post-cytokinetic phases of the cell cycle, mucilage is extruded only through the pole of the developing semi-cell. Chlorotetracyclene-labeling of mucilage-secreting cells show a correlation between calcium-rich loci on the cell surface and sites of mucilage release. 20 refs., 25 figs., 1 tab.« less

Carbohydrate metabolism changes in Prunus persica gummosis infected with Lasiodiplodia theobromae.

PubMed

Li, Z; Gao, L; Wang, Y T; Zhu, W; Ye, J L; Li, G H

2014-05-01

Peach gummosis represents a significant global disease of stone fruit trees and a major disease in the south peach production area of the Yangtze River of China. In this study, the carbohydrate composition of peach shoots during infection by Lasiodiplodia theobromae was examined. The expression of genes related to metabolic enzymes was also investigated. Control wounded and noninoculated tissue, lesion tissue, and wounded and inoculated surrounding lesion tissue of peach shoots were analyzed. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff's reagent staining during infection. Analysis using quantitative reverse-transcription polymerase chain reaction revealed that the abundance of key transcripts on the synthesis pathway of uridine diphosphate (UDP)-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased but the synthesis of L-galactose and guanosine diphosphate-L-galactose were inhibited. After inoculation, the transcript levels of sugar transport-related genes (namely, SUT, SOT, GMT, and UGT) was induced. These changes in sugar content and gene expression were directly associated with peach gum polysaccharide formation and may be responsible for the symptoms of peach gummosis.

Medical Gains of Chondroitin Sulfate Upon Fucosylation.

PubMed

Pomin, Vitor H

2015-01-01

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating N-acetyl galactosamine and glucuronic acid units within disaccharide building blocks. CS is a key functional component in proteoglycans of cartilaginous tissues. Owing to its numerous biological roles, CS is widely explored in the pharmaceutical market as nutraceutical ingredient commonly utilized against arthritis, osteoarthrosis, and sometimes osteoporosis. Tissues like shark cartilage and bovine trachea are common sources of CS. Nonetheless, a new CS type has been introduced and investigated in the last few decades in what regards its medical potentials. It is named fucosylated chondroitin sulfate (FucCS). This less common CS type is isolated exclusively from the body wall of sea cucumbers. The presence of fucosyl branching units in the holothurian FucCS gives to this unique GAG, therapeutic properties in various pathophysiological systems which are inexistent in the common CS explored in the market. Examples of these systems are coagulation, thrombosis, hemodialysis, atherosclerosis, cellular growth, angiogenesis, fibrosis, tumor growth, inflammation, viral and protozoan infections, hyperglycemia, diabetes-related pathological events and tissue damage. This report aims at describing the medical benefits gained upon fucosylation of CS. Clinical prospects of these medical benefits are also discussed herein.

Metabolism of isotretinoin. Biliary excretion of isotretinoin glucuronide in the rat.

PubMed

Meloche, S; Besner, J G

1986-01-01

The biliary metabolites of isotretinoin were examined after iv administration of 4-20-mg/kg doses to vitamin A-normal bile duct-cannulated rats. Analysis of bile by reverse phase high performance liquid chromatography showed that injection of isotretinoin is followed by a rapid excretion of metabolites in bile. Isotretinoin glucuronide was identified as the major metabolite in bile. A specific high performance liquid chromatography method based on the assay of generated isotretinoin in beta-glucuronidase-treated bile was developed for the determination of isotretinoin glucuronide in bile samples. The excretion rate of isotretinoin glucuronide increased rapidly to reach a maximum 55 min after dosing and then declined exponentially. After 330 min of collection, biliary excretion of isotretinoin glucuronide was almost complete, and the metabolite accounted for 34.8-37.9% of the dose. These results indicate that conjugation with glucuronic acid represents a major pathway for the metabolism of pharmacological doses of isotretinoin. The maximum excretion rate of isotretinoin glucuronide in bile increased in a linear manner with the dose of isotretinoin, and no delay was observed after the larger doses. These data suggest that glucuronidation and biliary excretion are not saturated at high pharmacological doses of isotretinoin.

Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis.

PubMed

Ferrari, M D; Neirotti, E; Albornoz, C; Saucedo, E

1992-10-05

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y-7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. ( (c) 1992 John Wiley & Sons, Inc.

HEPATIC VITAMIN A IN THE RAT AS AFFECTED BY THE ADMINISTRATION OF DIBENZANTHRACENE

PubMed Central

Abels, Jules C.; Gorham, Alice T.; Eberlin, Shirley L.; Halter, Robert; Rhoads, C. P.

1942-01-01

1. The decreased concentrations of vitamin A in the livers of rats given dibenzanthracene probably are due to a particular effect of the carcinogen on the ability of the liver to store the vitamin and not to the production of general hepatic dysfunction. 2. The administration of dibenzanthracene to normal rats does not (a) increase significantly their hepatic content of total fat nor decrease that of phospholipid; (b) impair the ability of their livers to fabricate serum albumin; (c) impair the capacity of their livers to esterify cholesterol or phenol; (d) interfere with the hepatic synthesis and conjugation of glucuronic acid; or (e) interfere with the hepatic storage of riboflavin. 3. The simultaneous ingestion of yeast by the dibenzanthracene-treated rats further depletes their hepatic stores of vitamin A. This depletion conceivably is due to the fact that yeast alone also might deplete the liver of its vitamin A and thus a summation of two similar effects is attained. 4. The results suggest a competition between vitamin A and dibenzanthracene for some substance, possibly a protein, to which vitamin A may be bound in the liver. PMID:19871225

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

PubMed

Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

2016-08-10

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

Cation and anion sequences in dark-adapted Balanus photoreceptor

PubMed Central

1977-01-01

Anion and cation permeabilities in dark-adapted Balanus photoreceptors were determined by comparing changes in the membrane potential in response to replacement of the dominant anion (Cl-) or cation (Na+) by test anions or cations in the superfusing solution. The anion permeability sequence obtained was PI greater than PSO4 greater than PBr greater than PCl greater than Pisethionate greater than Pmethanesulfonate. Gluconate, glucuronate, and glutamate generally appeared more permeable and propionate less permeable than Cl-. The alkali-metal cation permeability sequence obtained was PK greater than PRb greater than PCx greater than PNa approximately PLi. This corresponds to Eisenman's IV which is the same sequencethat has been obtained for other classes of nerve cells in the resting state. The values obtained for the permeability ratios of the alkali-metal cations are considered to be minimal. The membrane conductance measured by passing inward current pulses in the different test cations followed the sequence, GK greater than GRb greater than GCs greater than GNa greater than GLi. The conductance ratios obtained for a full substitution of the test cation agreed quite well with permeability ratios for all the alkali-metal cations except K+ which was generally higher. PMID:199688

Structural characterization of anti-complementary polysaccharides from the leaves of Artemisia princeps.

PubMed

Yamada, H; Otsuka, Y; Omura, S

1986-08-01

Structural characterizations of the anti-complementary acidic heteroglycans, AAF IIb-2 and IIb-3, obtained from the leaves of Artemisia princeps pamp have been studied. AAF IIb-2 consists of rhamnose, xylose, arabinose, galactose, glucose and uronic acids (glucuronic acid and galacturonic acid) in the molar ratio of 7.6:7.6:13.0:10.9:3.0:57.9, and AAF IIb-3 consists of the same sugars in the ratio of 3.9:2.6:24.7:19.7:2.6:46.5. Methylation analysis including carboxyl-reduction and also selective enzymolysis using EXO-alpha- L-arabinofuranosidase suggested that AAF IIb-3 has a main chain consisting of (1-->4)-linked galacturonic acid and (1-->2)-linked rhamnose mostly substituted at the O-4 position. AAF IIb-3 also contained arabino-3,6-galactan moiety and most of the arabinose was present as an alpha- L-furanosyl residue in the non-reducing terminals and highly branched side chains which mostly attached to the O-3 position of (1-->6)-linked galactopyranosyl residue. The basic structure of AAF IIb-2 is similar to that of AAF IIb-3, but IIb-3 has a higher arabinogalactan content than IIb-2.

Purification, characterization and anti-proliferation activities of polysaccharides extracted from Viscum coloratum (Kom.) Nakai.

PubMed

Chai, Yangyang; Zhao, Min

2016-09-20

Three polysaccharides, VCP1, VCP2 and VCP3 were isolated from Viscum coloratum (Kom.) Nakai using DEAE-cellulose chromatography. VCP1 (32KDa) was composed of glucose, galactose, arabinose, rhamnose and mannose, while VCP2 (280KDa) and VCP3 (21KDa) were consisted of glucose, galactose, arabinose, rhamnose, mannose, glucuronic acid and galacturonic acid. The optical rotation was measured at 20+1°C. The characteristic absorptive bands of purified fraction were determined by FT-IR. (13)C NMR spectroscopy analysis showed that VCP1 was a neutral polysaccharide, and VCP2 and VCP3 were RG-I type pectin. The linkage patterns of VCP2 were evaluated by methylation analysis: 1,5-linked Araf, 1,4-linked Galp, 1,2-linked Rhap, and 1,2,4-linked Rhap. The degree of esterification was 50%. The anti-proliferation ability against HepG2 cells and HepG2.2.15 cells of VCP2 was stronger than VCP1 and VCP3. So the polysaccharides from Viscum coloratum (Kom.) Nakai could be used as potential natural sources with inhibiting tumor cells proliferation. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Analysis of thickening polysaccharides by the improved diethyldithioacetal derivatization method].

PubMed

Akiyama, Takumi; Yamazaki, Takeshi; Tanamoto, Kenichi

2011-01-01

The identification test for thickening polysaccharides containing neutral saccharides and uronic acids was investigated by GC analysis of constituent monosaccharides. The reported method, in which monosaccharides were converted to diethyldithioacetal derivatives with ethanethiol followed by trimethylsilylation, was improved in terms of operability and reproducibility of GC/MS analysis. The suitability of the improved diethyldithioacetal derivatization method was determined for seven thickening polysaccharides, i.e., carob bean gum, guar gum, karaya gum, gum arabic, gum ghatti, tragacanth gum and peach gum. The samples were acid-hydrolyzed to form monosaccharides. The hydrolysates were derivatized and analyzed with GC/FID. Each sugar derivative was detected as a single peak and was well separated from others on the chromatograms. The amounts of constituent monosaccharides in thickening polysaccharides were successfully estimated. Seven polysaccharides were distinguished from each other on the basis of constituent monosaccharides. Further examination of the time period of hydrolysis of polysaccharides using peach gum showed that the optimal times were not the same for all monosaccharides. A longer time was needed to hydrolyze glucuronic acid than neutral saccharides. The findings suggest that hydrolysis time may sometimes affect the analytical results on composition of constituent monosaccharides in polysaccharides.

The butanol fraction of guava (Psidium cattleianum Sabine) leaf extract suppresses MMP-2 and MMP-9 expression and activity through the suppression of the ERK1/2 MAPK signaling pathway.

PubMed

Im, Inhwan; Park, Kyung-Ran; Kim, Sung-Moo; Kim, Chulwon; Park, Jeong Ha; Nam, Dongwoo; Jang, Hyeung-Jin; Shim, Bum Sang; Ahn, Kyoo Seok; Mosaddik, Ashik; Sethi, Gautam; Cho, Somi K; Ahn, Kwang Seok

2012-01-01

The leaf extract of guava (Psidium cattleianum Sabine) has traditionally been used for the treatment of diarrhea and diabetes in East Asia and other countries. Recently, the leaf extract has been employed in the therapy of cancer, bacterial infections, and inflammation in experimental models. However, the exact mechanisms of how guava leaf extract inhibits tumor metastasis and invasion are still unknown. In the present study, we investigated in detail the molecular mechanism(s) responsible for the potential antimetastatic and antiinvasive effects of the butanol fraction of guava leaf extract (GBF). Interestingly, we observed for the first time that GBF suppressed both matrix metalloproteinases (MMP)-9 and MMP-2 expression and activity in part through the downregulation of the ERK1/2 activation in lung cancer cells. Also, importantly, the major components of the GBF were identified as d-glucuronic acid, quercetin 3-glucuronide, loganin, and xanthyletin by LC-ESI-MS/MS. Collectively, our data indicate that the guava leaf could reduce the metastasis of lung cancer cells and therefore suggest that it could be advantageously used to control the metastatic process.

The monoclonal antibodies Elec-39, HNK-1 and NC-1 recognize common structures in the nervous system and muscles of vertebrates.

PubMed

Mailly, P; Younès-Chennouft, A B; Bon, S

1989-01-01

The IgM monoclonal antibodies, Elec-39, HNK-1 and NC-1, recognize the same subset of Torpedo electric organ acetylcholinesterase (AChE). We show that they react against a glycosphingolipid (SGPG) containing a sulfated glucuronic acid (SGA). The three antibodies appear essentially identical in their specificity but differ in their affinity for the antigens. We have examined their binding in the CNS, nerves and muscles of several vertebrate species, at the optical and in some cases at the electron microscope level. All three antibodies label the same structures: they show diffuse staining around neuromuscular endplates and label the plasma membrane of the Schwann cells, surrounding the outer layer of myelin sheaths. In the adult rat CNS, the antibodies label certain defined structures, notably extracellular material in the habenula and in the CA2 layer of the hippocampus. In the cortex and cerebellum, they label the surface of neural processes and terminals apposed to large multipolar neurons and Purkinje cells, as well as membranous material contained in inclusions dispersed in the cytoplasm of these neurons. These localizations are consistent with the suggestion that the SGA-antigens may be involved in cellular interactions.

Application of long-circulating liposomes to cancer photodynamic therapy.

PubMed

Oku, N; Saito, N; Namba, Y; Tsukada, H; Dolphin, D; Okada, S

1997-06-01

Photodynamic therapy (PDT) as a cancer treatment is notable for its quite low side effects in comparison with those of chemotherapy and radiotherapy. However, the accumulation of porphyrin derivatives used in PDT into tumor tissues is rather low. Since long-circulating liposomes are known to accumulate passively into tumor tissues, we liposomalized a porphyrin derivative, benzoporphyrin derivative monoacid ring A (BPD-MA), and used these liposomes to investigate the usefulness of PDT for tumor-bearing mice. BPD-MA was liposomalized into glucuronate-modified liposomes, which are known to be long-circulating. These liposomes were injected i.v. into Balb/c mice bearing Meth A sarcoma, and tumor regression and survival time were monitored after irradiation with laser light. Tumor regression and complete curing of tumor (80% cure rate by the treatment with 6 mg/kg BPD-MA) were observed when long circulating liposomalized BPD-MA was injected and laser-irradiated. In contrast, only a 20% cure rate was obtained when the animals were treated with BPD-MA solution or BPD-MA entrapped in conventional liposomes. These results suggest that a long-circulating liposomal formulation of photo-sensitive agents is useful for PDT.

Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.

PubMed

Senf, Deborah; Ruprecht, Colin; de Kruijff, Goswinus H M; Simonetti, Sebastian O; Schuhmacher, Frank; Seeberger, Peter H; Pfrengle, Fabian

2017-03-02

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production and properties of an exopolysaccharide synthesized by the extreme halophilic archaeon Haloterrigena turkmenica.

PubMed

Squillaci, Giuseppe; Finamore, Rosario; Diana, Paola; Restaino, Odile Francesca; Schiraldi, Chiara; Arbucci, Salvatore; Ionata, Elena; La Cara, Francesco; Morana, Alessandra

2016-01-01

We have isolated a novel exopolysaccharide (EPS) produced by the extreme halophilic archaeon Haloterrigena turkmenica. Some features, remarkable from an industrial point of view, such as emulsifying and antioxidant properties, were investigated. H. turkmenica excreted 20.68 mg of EPS per 100 ml of culture medium when grown in usual medium supplemented with glucose. The microorganism excreted the biopolymer mainly in the middle exponential growth phase and reached the maximal production in the stationary phase. Analyses by anion exchange chromatography and SEC-TDA Viscotek indicated that the EPS was composed of two main fractions of 801.7 and 206.0 kDa. It was a sulfated heteropolysaccharide containing glucose, galactose, glucosamine, galactosamine, and glucuronic acid. Studies performed utilizing the mixture of EPS anionic fractions showed that the biopolymer had emulsifying activity towards vegetable oils comparable or superior to that exhibited by the controls, moderate antioxidant power when tested with 2,2'-diphenyl-1-picrylhydrazyl (DPPH(·)), and moisture-retention ability higher than hyaluronic acid (HA). The EPS from H. turkmenica is the first exopolysaccharide produced by an archaea to be characterized in terms of properties that can have potential biotechnological applications.

Metabolism of methyltestosterone in the greyhound.

PubMed

Biddle, S T B; O'Donnell, A; Houghton, E; Creaser, C

2009-03-01

Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17alpha-methyl-5beta-androstane-3alpha-17beta-diol, 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol, and a further compound tentatively identified as 17alpha-methyl-5z-androstane-6z,17beta-triol. The most abundant of these was the 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide-TMS derivatisation techniques. There did not appear to be any evidence for 16beta-hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16alpha-hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here. John Wiley & Sons, Ltd

Production of FucoPol by Enterobacter A47 using waste tomato paste by-product as sole carbon source.

PubMed

Antunes, Sílvia; Freitas, Filomena; Sevrin, Chantal; Grandfils, Christian; Reis, Maria A M

2017-03-01

Out-of-specification tomato paste, a by-product from the tomato processing industry, was used as the sole substrate for cultivation of the bacterium Enterobacter A47 and production of FucoPol, a value-added fucose-rich extracellular polysaccharide. Among the different tested fed-batch strategies, pH-stat, DO-stat and continuous substrate feeding, the highest production (8.77gL -1 ) and overall volumetric productivity (2.92gL -1 d -1 ) were obtained with continuous substrate feeding at a constant flow rate of 11gh -1 . The polymer produced had the typical FucoPol composition (37mol% fucose, 27mol% galactose, 23mol% glucose and 12mol% glucuronic acid, with an acyl groups content of 13wt%). The average molecular weight was 4.4×10 6 Da and the polydispersity index was 1.2. This study demonstrated that out-of-specification tomato paste is a suitable low-cost substrate for the production of FucoPol, thus providing a route for the valorization of this by-product into a high-value microbial product. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrazinolysis of heparin and other glycosaminoglycans.

PubMed Central

Shaklee, P N; Conrad, H E

1984-01-01

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed. PMID:6421280

Mercaptursäure und Nukleosidaddukt im Harn als Biomarker in 1-Hydroxymethylpyren-exponierten Ratten

NASA Astrophysics Data System (ADS)

Ma, Lan

2002-01-01

1-Methylpyren (MP) ist hepatokanzerogen in neugeborenen männlichen Mäusen. Durch Hydroxylierung an der benzylischen Stelle und anschließende Sulfonierung wird MP zu DNA-reaktivem 1-Sulfooxymethylpyren (SMP) aktiviert. In der Ratte führt die Exposition des benzylischen Alkohols, 1-Hydroxymethylpyren (HMP), zur DNA-Adduktbildung in verschiedenen Geweben. Eventuelle Konsequenz der Toxifizierung ist die Ausscheidung entsprechender Mercaptursäure und Nukleosidaddukt im Harn, welche aufgrund ihrer Herkunft als Biomarker eignen könnten. In dieser Arbeit wird die Ausscheidung der Mercaptursäure und des N2-Desoxyguanosinadduktes in HMP-exponierten Ratten untersucht. Nach der Applikation von HMP bzw. MP wurden weniger als 1 % der Dosis als MPMA über Urin und Faeces ausgeschieden (0 - 48 h). Die Ausscheidung erfolgt hauptsächlich in den ersten 24 h nach der Applikation. MPdG konnte weder in Urin noch in Faeces der HMP-behandelten Tieren identifiziert werden. Nach direkter SMP-Applikation wurde MPdG nur in sehr geringe Menge (weniger als 0,9 ppm in 12 h) im Urin gefunden. Aufgrund der geringen Menge eignet sich MPdG nicht als Biomarker. MPMA dagegen, lässt sich analytisch gut erfassen. Es sollte daher untersucht werden, ob MPMA die Toxifizierung des HMP wiederspiegelt. Die Voraussetzung dafür ist die Kenntnisse über das Metabolismusmuster von HMP. Es wurde daher umfassende Untersuchungen zum Metabolismus des HMP durchgeführt. Die Ergebnisse zeigten, dass mehr als 80 % der Metaboiten in ihrer oxidierten Form (PCS, deren Glucuronsäure-Konjugate sowie phenolische Sulfatester der PCS) ausgeschieden wurden. Demnach spielt die Oxidation des HMP zu PCS eine sehr wichtige Rolle bei der Detoxifizierung und Ausscheidung von HMP. Ferne konnte nachgewiesen werden, dass die Enzyme Alkohol- und Aldehyd-Dehydrogenase an der Oxidation von HMP beteiligt waren. Die Inhibitoren Disulfiram und Ethanol der o. g. Enzyme wurde daher zur Modulation der Detoxifizierung in vivo eingesetzt. Die Veränderungen in der Toxifizierung von HMP zu SMP wurden durch die SMP-Konzentration im Plasma, die DNA-Addukthäufigkeit und die MPMA-Ausscheidung erfasst. Die Vorbehandlung von Disulfiram und Ethanol führte zu tendentielle Erhöhung der SMP-Konzentration im Plasma, DNA-Addukthäufigkeit in der Leber und die MPMA-Ausscheidung. Bemerkenswert ist jedoch, dass bereits eine Dosis von 0,2 g Ethanol/kg Körpermasse bereits zu statistisch signifikanten Erhöhungen der MPMA-Ausscheidung bei weiblichen Ratten. 1-Methylpyrene is hepatocarcinogenic in rodents. It is metabolized primarily to 1-hydroxymethylpyrene (HMP) by various cDNA-expressed rat and human cytochromes P450. HMP is activated to a highly reactive sulfuric acid ester, 1-sulfooxymethylpyrene (SMP), by sulphotransferases. In the rat, this activation pathway leads to the formation of DNA adducts in various tissues. Possible consequences of the toxification could be the excretion of the corresponding mercapturic acid and nucleosidadduct in urine and feces. Because of their origin, these substances should reflex the toxification process may be used as biomarkers. We investigate the excretion of 1-methylpyrenyl-mercapturic acid (MPMA) and the excretion of N2-(1-methylpyrenyl)-desoxyguanosin (MPdG) in urine and feces of HMP-treated rats. These studies showed that only a minor portion (< 1 %) of the administered dose of 1-HMP was excreted as mercapturic acid. MPdG could not be identified in urine and feces of HMP-treated rats. Treating rats with the active spieces sulfooxymethylpyrene, 0.9 ppm of the dose was found excreted within 12 h. I now investigated the alternative metabolic pathways of HMP. More than 50 % of the dose (administered intraperitoneally) was excreted as free 1-pyrenyl carboxylic acid and its glucuronic acid conjugate primarily in the urine. Other major urinary metabolites were phenolic sulpho conjugates of ring-oxidized 1-pyrenyl carboxylic acid (> 30 %). Minor metabolites were phenolic sulpho conjugates of HMP (< 5 %). The glucuronic acid conjugate of HMP was found in very small amounts. In total, > 80 % of the metabolites excreted were oxidized at the exocyclic carbon. This side-chain oxidation, probably catalyzed by alcohol and aldehyde dehydrogenases, appears to represent a detoxification pathway. Indeed, administration of ethanol shortly before the administration of HMP to rats increased the levels of SMP detected in blood, of DNA adducts formed in tissues and of mercapturic acid excreted. These effects were observed even at very low dose levels of ethanol (0.2 g per kg body weight). Similar effects were shown after administration of Disulfiram, an inhibitor of aldehyde dehydrogenase.

Development and validation of a colorimetric assay for simultaneous quantification of neutral and uronic sugars.

PubMed

Rondel, Caroline; Marcato-Romain, Claire-Emmanuelle; Girbal-Neuhauser, Elisabeth

2013-05-15

A colorimetric assay based on the conventional anthrone reaction was investigated for specific quantification of uronic acids (UA) in the presence of neutral sugars and/or proteins. Scanning of glucose (Glu) and glucuronic acid (GlA) was performed after the reaction with anthrone and a double absorbance reading was made, at 560 nm and at 620 nm, in order to quantify the UA and neutral sugars separately. The assay was implemented on binary or ternary solutions containing Glu, GlA and bovine serum albumin (BSA) in order to validate its specificity towards sugars and check possible interference with other biochemical components such as proteins. Statistical analysis indicated that this assay provided correct quantification of uronic sugars from 50 to 400 mg/l and of neutral sugars from 20 to 80 mg/l, in the presence of proteins with concentrations reaching 600 mg/l. The proposed protocol can be of great interest for simultaneous determination of uronic and neutral sugars in complex biological samples. In particular, it can be used to correctly quantify the Extracellular Polymeric Substances (EPS) isolated from the biological matrix of many bacterial aggregates, even in the presence of EPS extractant such as EDTA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide

PubMed Central

Acosta-Jurado, Sebastián; Soto, María J.; Margaret, Isabel; Crespo-Rivas, Juan C.; Sanjuan, Juan; Temprano, Francisco; Gil-Serrano, Antonio; Ruiz-Sainz, José E.; Vinardell, José M.

2014-01-01

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum. PMID:25521500

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography.

PubMed

Ganzera, Markus; Egger, Christoph; Zidorn, Christian; Stuppner, Hermann

2008-05-05

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability (sigma(rel) < or = 4.4%), precision (inter-day sigma(rel) < or = 8.13%, intra-day sigma(rel) < or = 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) < or = 4.5 microg mL(-1)) and linearity (R(2) > or = 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3-O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.

Production and Rheological Properties of Welan Gum Produced by Sphingomonas sp. ATCC 31555 with Different Nitrogen Sources.

PubMed

Xu, Xiaopeng; Nie, Zuoming; Zheng, Zhiyong; Zhu, Li; Zhan, Xiaobei

2017-01-01

This study aimed to investigate the effect of nitrogen sources on the production and rheological properties of welan gum produced by Sphingomonas sp. ATCC 31555. Six different nitrogen sources were used for ATCC 31555 fermentation, and 2 of these were further analyzed due to their more positive influence on welan gum production and bacterial biomass. Bacterial biomass, welan gum yield, welan viscosity, molecular weight, monosaccharide composition, acyl content, and welan structure were analyzed. Welan gum production and the biomass concentration of ATCC 31555 were higher in media containing NaNO3 and beef extract. Welan viscosity decreased at higher temperatures of 30-90°C, and it increased with a higher welan concentration. In the media containing NaNO3 (3 g·L-1), welan viscosity was higher at 30-70°C and a welan solution concentration of 6-10 g·L-1. With a reduced NaNO3 concentration, the molecular weight of welan gum and the molar ratio of mannose decreased, but the molar ratio of glucuronic acid increased. With different nitrogen sources, the acetyl content of welan gum differed but its structure was similar. NaNO3 and beef extract facilitated welan production. A reduced NaNO3 concentration promoted welan viscosity. © 2017 S. Karger AG, Basel.

Synergism and foaming properties in binary mixtures of a biosurfactant derived from Camellia oleifera Abel and synthetic surfactants.

PubMed

Jian, Hong-lei; Liao, Xiao-xia; Zhu, Li-wei; Zhang, Wei-ming; Jiang, Jian-xin

2011-07-15

A biosurfactant, named tea saponin (TS), was isolated and purified from the defatted seed of Camellia oleifera Abel. The characterization of TS including molecular weight, glycosyl composition, and thermal behavior as well as the surface and foaming properties was conducted. The synergistic interactions of binary systems of CTAB-TS, SDS-TS, and Brij35-TS were investigated. The results show that TS had a weight-average molecular weight of 809.12 g mol(-1) and contained four aglycones of L-rhamnose, D-galactose, D-glucose, and D-glucuronic acid. The critical micelle concentration (cmc) of 2.242 mmol L(-1) and the minimum surface tension (γ(cmc)) of 43.5 mN m(-1) were determined for TS. Synergisms in surface tension reduction efficiency, in mixed micelle formation, and in surface tension reduction effectiveness were observed in CTAB-TS and SDS-TS systems, whereas that was not shown in Brij35-TS mixtures. The mixtures of TS with CTAB and SDS showed synergism in foaming efficiency, but this synergism did not exist in Brij35-TS system with respect to the surface properties. Nevertheless, there appears to be no significant correlation between foam stability and the surface properties. Copyright © 2011 Elsevier Inc. All rights reserved.

High Resolution Crystal Structure of Human β-Glucuronidase Reveals Structural Basis of Lysosome Targeting

PubMed Central

Hassan, Md. Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H.; Klei, Herbert E.; Korolev, Sergey; Sly, William S.

2013-01-01

Human β-glucuronidase (GUS) cleaves β-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene. PMID:24260279

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

PubMed Central

Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

2016-01-01

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation. PMID:27681920

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.

PubMed

Ikushiro, Shinichi; Nishikawa, Miyu; Masuyama, Yuuka; Shouji, Tadashi; Fujii, Miharu; Hamada, Masahiro; Nakajima, Noriyuki; Finel, Moshe; Yasuda, Kaori; Kamakura, Masaki; Sakaki, Toshiyuki

2016-07-05

Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides.

Preparation, characterization and antiglycation activities of the novel polysaccharides from Boletus snicus.

PubMed

Liping, Sun; Xuejiao, Su; Yongliang, Zhuang

2016-11-01

Boletus snicus (BS) is one of the commercially important mushroom species. Two polysaccharides (BSP-1b and BSP-2b) were extracted and purified from the body of BS by DEAE-cellulose and Sephadex G-100 column chromatography. The average of molecular weight of BSP-1b and BSP-2b were 59.21kDa and 128.74kDa. BSP-1b is a heteropolysaccharide with a large number of glucose and a small amount of mannose, glucosamine hydrochloride and arabinose. The monosaccharide compositions of BSP-2b contain mannose, glucuronic acid, glucosamine hydrochloride, glucose, galactose, arabinose with the molar ratio of 10.70:6.95:12.05:12.57:1.83:1.00. The FTIR spectra and NMR analysis demonstrated that BSP-1b and BSP-2b existed pyranose ring structure and BSP-2b had high content of uronic acid. The antiglycation activities of BSP-1b and BSP-2b were investigated. The results showed BSP-1b and BSP-2b had high inhibitory effects on glycation and exhibited dose-dependent responses. BSP-2b showed stronger antiglycation activity than BSP-1b. This study indicated that the BSP-2b could effectively inhibit the formation of advanced glycation end-products. Copyright © 2016 Elsevier B.V. All rights reserved.

Cellulase-assisted extraction of polysaccharides from Malva sylvestris: Process optimization and potential functionalities.

PubMed

Rostami, Hosein; Gharibzahedi, Seyed Mohammad Taghi

2017-08-01

Enzyme-assisted extraction process of the water-soluble Malva sylvestris polysaccharides (MSPs) was optimized using response surface methodology (RSM). The highest yield (10.40%) of MSPs was achieved at 5.64% cellulase, 55.65°C temperature, 3.4h time, and 5.22 pH. Three homogeneous polysaccharide fractions (MSP-1, MSP-2, MSP-3) were purified by DEAE-cellulose and Sephadex G-100 chromatography, which were composed of galactose, glucuronic acid, arabinose, rhamnose and mannose in different molar ratios with molecular weight range of 2.6×10 5 -8.8×10 5 Da. The fractions could significantly increase antioxidant, antitumor and antimicrobial activities in a dose-dependent pattern. MSP-2 revealed stronger antioxidant activities than MSP-1 and MSP-3, including reducing power and scavenging activity of DPPH and OH radicals. The antiproliferative activity of MSP-2 (1.0mg/mL) on the growth of A549 and HepG2 cells was 45.1% and 53.2%, respectively. The Gram-positive bacteria (Bacillus cereus PTCC 1015 and Staphylococcus aureus PTCC 1112) compared with Gram-negative ones (Escherichia coli PTCC 1763 and Salmonella typhimurium PTCC 1709) showed less sensitivity against the various MSPs (3-15mg/mL). Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

PubMed Central

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Analysis of anabolic androgenic steroids as sulfate conjugates using high performance liquid chromatography coupled to tandem mass spectrometry.

PubMed

Rzeppa, S; Heinrich, G; Hemmersbach, P

2015-01-01

Improvements in doping analysis can be effected by speeding up analysis time and extending the detection time. Therefore, direct detection of phase II conjugates of doping agents, especially anabolic androgenic steroids (AAS), is proposed. Besides direct detection of conjugates with glucuronic acid, the analysis of sulfate conjugates, which are usually not part of the routine doping control analysis, can be of high interest. Sulfate conjugates of methandienone and methyltestosterone metabolites have already been identified as long-term metabolites. This study presents the synthesis of sulfate conjugates of six commonly used AAS and their metabolites: trenbolone, nandrolone, boldenone, methenolone, mesterolone, and drostanolone. In the following these sulfate conjugates were used for development of a fast and easy analysis method based on sample preparation using solid phase extraction with a mixed-mode sorbent and detection by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Validation demonstrated the suitability of the method with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA). In addition, suitability has been proven by successful detection of the synthesized sulfate conjugates in excretion urines and routine doping control samples. Copyright © 2015 John Wiley & Sons, Ltd.

Structural characterisation of a water-soluble polysaccharide from tissue-cultured Dendrobium huoshanense C.Z. Tang et S.J. Cheng.

PubMed

Si, Hua-Yang; Chen, Nai-Fu; Chen, Nai-Dong; Huang, Cheng; Li, Jun; Wang, Hui

2018-02-01

A water-soluble polysaccharide TC-DHPA4 with a molecular weight of 8.0 × 10 5  Da was isolated from tissue-cultured Dendrobium huoshanense by anion exchange and gel permeation chromatography. Monosaccharide analysis revealed that the homogeneous polysaccharide was made up of rhamnose, arabinose, mannose, glucose, galactose and glucuronic acid with a molar ratio of 1.28:1:1.67:4.71:10.43:1.42. The sugar residue sequence analysis based on the GC-MS files and NMR spectra indicated that the backbone of TC-DHPA4 consisted of the repeated units:→6)-β-Galp-(1→6)-β-Galp-(1→4)-β-GlcpA-(1→6)-β-Glcp-(1→6)-β-Glcp-(→. The sugar residue sequences β-Glcp-(1→)-α-Rhap-(1→3)-β-Galp-(1→, β-Glcp-(1→4)-α-Rhap-(1→3)-β-Galp-(1→, β-Galp-(1→6)-β-Manp-(1→3)-β-Galp-(1→, and α-l-Araf-(1→2)-β-Manp-(1→3)-β-Galp-(1→ were identified as the branches attached to the C-3 position of (1→6)-linked galactose in the backbone.

Metabolite characterization of a novel anti-cancer agent, icotinib, in humans through liquid chromatography/quadrupole time-of-flight tandem mass spectrometry.

PubMed

Liu, Dongyang; Jiang, Ji; Zhang, Li; Tan, Fenlai; Wang, Yingxiang; Hu, Pei

2011-08-15

Icotinib is a novel anti-cancer drug that has shown promising clinical efficacy and safety in patients with non-small-cell lung cancer (NSCLC). At this time, the metabolic fate of icotinib in humans is unknown. In the present study, a liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF MS) method was established to characterize metabolites of icotinib in human plasma, urine and feces. In addition, nuclear magnetic resonance (NMR) detection was utilized to determine the connection between side-chain and quinazoline groups for some complex metabolites. In total, 29 human metabolites (21 isomer metabolites) were characterized, of which 23 metabolites are novel compared to the metabolites in rats. This metabolic study revealed that icotinib was extensively metabolized at the 12-crown-4 ether moiety (ring-opening and further oxidation), carbon 15 (hydroxylation) and an acetylene moiety (oxidation) to yield 19 oxidized metabolites and to further form 10 conjugates with sulfate acid or glucuronic acid. To our knowledge, this is the first report of the human metabolic profile of icotinib. Study results indicated that significant attention should be paid to the metabolic profiles of NSCLC patients during the development of icotinib. Copyright © 2011 John Wiley & Sons, Ltd.

Ultrasmall lanthanide-doped nanoparticles as multimodal platforms

NASA Astrophysics Data System (ADS)

Yust, Brian G.; Pedraza, Francisco J.; Sardar, Dhiraj K.

2014-03-01

Recently, there has been a great amount of interest in nanoparticles which are able to provide a platform with high contrast for multiple imaging modalities in order to advance the tools available to biomedical researchers and physicians. However, many nanoparticles do not have ideal properties to provide high contrast in different imaging modes. In order to address this, ultrasmall lanthanide doped oxide and fluoride nanoparticles with strong NIR to NIR upconversion fluorescence and a strong magnetic response for magnetic resonance imaging (MRI) have been developed. Specifically, these nanoparticles incorporate gadolinium, dysprosium, or a combination of both into the nano-crystalline host to achieve the magnetic properties. Thulium, erbium, and neodymium codopants provide the strong NIR absorption and emission lines that allow for deeper tissue imaging since near infrared light is not strongly absorbed or scattered by most tissues within this region. This also leads to better image quality and lower necessary excitation intensities. As a part of the one pot synthesis, these nanoparticles are coated with peg, pmao, or d-glucuronic acid to make them water soluble, biocompatible, and bioconjugable due to the available carboxyl or amine groups. Here, the synthesis, morphological characterization, magnetic response, NIR emission, and the quantum yield will be discussed. Cytotoxicity tested through cell viability at varying concentrations of nanoparticles in growth media will also be discussed.

Toward Understanding the Outer Membrane Uptake of Small Molecules by Pseudomonas aeruginosa*

PubMed Central

Eren, Elif; Parkin, Jamie; Adelanwa, Ayodele; Cheneke, Belete; Movileanu, Liviu; Khalid, Syma; van den Berg, Bert

2013-01-01

Because small molecules enter Gram-negative bacteria via outer membrane (OM) channels, understanding OM transport is essential for the rational design of improved and new antibiotics. In the human pathogen Pseudomonas aeruginosa, most small molecules are taken up by outer membrane carboxylate channel (Occ) proteins, which can be divided into two distinct subfamilies, OccD and OccK. Here we characterize substrate transport mediated by Occ proteins belonging to both subfamilies. Based on the determination of the OccK2-glucuronate co-crystal structure, we identify the channel residues that are essential for substrate transport. We further show that the pore regions of the channels are rigid in the OccK subfamily and highly dynamic in the OccD subfamily. We also demonstrate that the substrate carboxylate group interacts with central residues of the basic ladder, a row of arginine and lysine residues that leads to and away from the binding site at the channel constriction. Moreover, the importance of the basic ladder residues corresponds to their degree of conservation. Finally, we apply the generated insights by converting the archetype of the entire family, OccD1, from a basic amino acid-specific channel into a channel with a preference for negatively charged amino acids. PMID:23467408

Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates.

PubMed

Sorich, Michael J; Smith, Paul A; Miners, John O; Mackenzie, Peter I; McKinnon, Ross A

2008-01-01

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

TMEM2: A missing link in hyaluronan catabolism identified?

PubMed

Yamaguchi, Yu; Yamamoto, Hayato; Tobisawa, Yuki; Irie, Fumitoshi

2018-03-27

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 10 6  Da and sometimes reaches 10 7  Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Cell and Tissue Imaging with Molecularly Imprinted Polymers.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-01-01

Advanced tools for cell imaging are of particular interest as they can detect, localize and quantify molecular targets like abnormal glycosylation sites that are biomarkers of cancer and infection. Targeting these biomarkers is often challenging due to a lack of receptor materials. Molecularly imprinted polymers (MIPs) are promising artificial receptors; they can be tailored to bind targets specifically, be labeled easily, and are physically and chemically stable. Herein, we demonstrate the application of MIPs as artificial antibodies for selective labeling and imaging of cellular targets, on the example of hyaluronan and sialylation moieties on fixated human skin cells and tissues. Thus, fluorescently labeled MIP nanoparticles templated with glucuronic acid (MIPGlcA) and N-acetylneuraminic acid (MIPNANA) are respectively applied. Two different fluorescent probes are used: (1) MIPGlcA particles, ~400 nm in size are labeled with the dye rhodamine that target the extracellular hyaluronan on cells and tissue specimens and (2) MIP-coated InP/ZnS quantum dots (QDs) of two different colors, ~125 nm in size that target the extracellular and intracellular hyaluronan and sialylation sites. Green and red emitting QDs are functionalized with MIPGlcA and MIPNANA respectively, enabling multiplexed cell imaging. This is a general approach that can also be adapted to other target molecules on and in cells.

Box-Behnken design for extraction optimization of crude polysaccharides from Tunisian Phormidium versicolor cyanobacteria (NCC 466): Partial characterization, in vitro antioxidant and antimicrobial activities.

PubMed

Belhaj, Dalel; Frikha, Donyez; Athmouni, Khaled; Jerbi, Bouthaina; Ahmed, Mohammad Boshir; Bouallagui, Zouhaier; Kallel, Monem; Maalej, Sami; Zhou, John; Ayadi, Habib

2017-12-01

In this study, response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to optimize the aqueous extraction of crude polysaccharides from Tunisian cyanobacteria Phormidium versicolor (NCC 466). The optimal extraction conditions with an extraction yield of 21.56±0.92% were as follows: extraction temperature at 81.05°C, extraction time of 3.99h, and water to raw material ratio of 21.52mLg -1 . Crude Phormidium versicolor polysaccharides (CPv-PS) are found to be a hetero-sulfated-anionic polysaccharides that contained carbohydrate (79.37±1.58%), protein (0.45±0.11%), uronic acids (4.37±0.19%) and sulfate (6.83±0.28%). The carbohydrate fraction was composed of arabinose, xylose, ribose, rhamnose, N-acetyl glucosamine, galactose, glucose, mannose, glucuronic acid and saccharose with corresponding mole percentages of 2.41, 14.58, 2.18, 6.23, 7.04, 28.21, 26.04, 3.02, 0.86 and 5.07, respectively. Evaluation of the antioxidant activity in vitro suggested that CPv-PS strongly scavenged radicals, prevented bleaching of β-carotene and reduced activity. Furthermore, the CPv-PS exhibited effective antimicrobial properties. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Frost Grape Polysaccharide (FGP), an Emulsion-Forming Arabinogalactan Gum from the Stems of Native North American Grape Species Vitis riparia Michx.

PubMed

Price, Neil P J; Vermillion, Karl E; Eller, Fred J; Vaughn, Steven F

2015-08-19

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified βGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at >15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a β1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

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

Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

Characteristics of fucose-containing polysaccharides from submerged fermentation of Agaricus blazei Murill.

PubMed

Wang, Hsueh-Ting; Yang, Li-Chan; Yu, Hui-Ching; Chen, Miaw-Ling; Wang, Huei-Ju; Lu, Ting-Jang

2018-04-01

Fucose is one of important residues of recognition pattern for many immune cells. In this study, we characterized bioactive fucose-containing acidic polysaccharides from submerged fermentation of Agaricus blazei Murill. We obtained the polysaccharides through a cell-based activity-guided strategy, and used carbohydrate recognition monoclonal antibodies based Enzyme-Linked Immuno Sorbent Assay (ELISA) along with methylation and NMR analyses to investigate the structural characteristics of the polysaccharides. The polysaccharides had Mw of 3.5 × 10 5  Da. The major sugars were l-fucose, l-arabinose, d-galactose, d-xylose, and d-galacturonic acid in the molar ratio of 6.4, 15.5, 28.5, 14.7, and 25.0% with a small amount of d-glucose, d-mannose, l-rhamnose, and d-glucuronic acid. Results indicated that the bioactive polysaccharides consisted of a (1,4)-Galp and (1,4)-GalAp back bone; (1,2)-Xyl and (1,2)-Rha might also comprise backbone or constitute side chain; linkage (1,5)-Ara and terminal fucosyl residues were also involved in the polysaccharides. Regarding bioactivity, removal of the terminal l-fucosyl residues reduced the TNF-α cytokine stimulating activity of the polysaccharides in a RAW 264.7 macrophage cell-line test, whereas NF-κB and TLR4 affected the polysaccharide-induced TNF-α production. Copyright © 2017. Published by Elsevier B.V.

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships.

PubMed

Freas, Nicholas; Newton, Peter; Perozich, John

2016-01-01

UDP-glucose dehydrogenase (UDPGDH), UDP-N-acetyl-mannosamine dehydrogenase (UDPNAMDH) and GDP-mannose dehydrogenase (GDPMDH) belong to a family of NAD (+)-linked 4-electron-transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP-SDHs). UDPGDH is an enzyme responsible for converting UDP-d-glucose to UDP-d-glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid and GDP-mannose to GDP-mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP-SDH group, including residue positions critical to NDP-sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research. UDP-glucose dehydrogenase (UDPGDH, EC 1.1.1.22).

Biotransformation of vinclozolin in rat precision-cut liver slices: comparison with in vivo metabolic pattern.

PubMed

Bursztyka, Julian; Debrauwer, Laurent; Perdu, Elisabeth; Jouanin, Isabelle; Jaeg, Jean-Philippe; Cravedi, Jean-Pierre

2008-06-25

Vinclozolin is a dicarboxymide fungicide that presents antiandrogenic properties through its two hydrolysis products M1 and M2, which bind to the androgen receptor. Because of the lack of data on the biotransformation of vinclozolin, its metabolism was investigated in vitro in precision-cut rat liver slices and in vivo in male rat using [ (14)C]-vinclozolin. Incubations were performed using different concentrations of substrate, and the kinetics of formation of the major metabolites were studied. Three male Wistar rats were fed by gavage with [ (14)C]-VZ. Urine was collected for 24 h and analyzed by radio-HPLC for metabolic profiling. Metabolite identification was carried out on a LCQ ion trap mass spectrometer. In rat liver slices and in vivo, the major primary metabolite has been identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (M5) and was mainly present as glucuronoconjugates. M5 is produced by dihydroxylation of the vinyl group of M2. Other metabolites have been identified as 3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione (M4), a dihydroxylated metabolite of vinclozolin, which undergoes further conjugation to glucuronic acid, and 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3,4-dihydroxy-butanoic acid (M6), a dihydroxylated metabolite of M1.

Metabolism study of boldenone in human urine by gas chromatography-tandem mass spectrometry.

PubMed

Wu, Xinchen; Gao, Feng; Zhang, Wenxin; Ni, Jian

2015-11-10

Boldenone (BOLD), an anabolic steroid, is likely to be abused in livestock breeding and in sports. Although some of BOLD metabolites in human urine, such as 5β-adrost-1-en-17β-ol-3-one (BM1), have been detected, investigations on their excretion patterns for both genders are insufficient. Moreover, little research on 17α-BOLD glucuronide as a metabolite in human urine has been reported. The aim of this study is to make a contribution to the knowledge of 17β-BOLD metabolism in humans. Three male and three female volunteers were orally administrated with 30mg 17β-BOLD. Urine samples were collected and analyzed with gas chromatography-tandem mass spectrometry. The data proved that 17β-BOLD, BM1, and 17α-BOLD were excreted in urine in both free and glucuronic conjugated forms after administration of 17β-BOLD. For most subjects, the urinary concentrations of BM1 were higher than that of 17β-BOLD. 17α-BOLD was excreted in small amounts. 17α-BOLD, 17β-BOLD, and BM1 were present naturally in urine with low concentrations. Administration of 30mg 17β-BOLD could not influence the excretion profiles of urinary androsterone, etiocholanolone, and testosterone/epitestosterone ratio. There were no differences in BOLD metabolic patterns between man and woman. Copyright © 2015 Elsevier B.V. All rights reserved.

Anticancer and immunoregulatory activity of Gynostemma pentaphyllum polysaccharides in H22 tumor-bearing mice.

PubMed

Liu, Jun; Zhang, Lihua; Ren, Yingang; Gao, Yanli; Kang, Li; Qiao, Qing

2014-08-01

A neutral polysaccharide fraction (CGPP) was extracted from Gynostemma pentaphyllum by water extraction and ethanol precipitation. Gas chromatography (GC) analysis showed that the CGPP was mainly composed of mannose, glucose, arabinose, rhamnose, galactose and glucuronic acid in molar ratios of 2.0:2.2:1.3:2.2:1.2:2.5. The present study aimed at evaluating the antitumor potentials of CGPP on the growth of H22 tumor transplanted in mice and the underlying mechanism. The results showed that CGPP (50 and 200mg/kg) could effectively inhibit the solid tumor growth of H22 hepatocarcinoma transplanted in ICR mice. Besides, the body weight, spleen/thymus indexes and splenocytes proliferation of H22 tumor bearing mice were also improved in CGPP-treated groups. Furthermore, the level of the cytokines, such as IL-2, TNF-α and IFN-γ, as well as the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) in tumor-bearing mice were markedly promoted by CGPP oral administration. In addition, CGPP treatment greatly prolonged the survival period in H22 ascites tumor-bearing mice. Taken together, these findings indicate that CGPP has antitumor activity in vivo at least partly via improving immune responses of host organism, and seems to be a safe and effective supplementary agent for the treatment of hepatocellular carcinoma (HCC). Copyright © 2014 Elsevier B.V. All rights reserved.

The Effects of Different Purifying Methods on the Chemical Properties, in Vitro Anti-Tumor and Immunomodulatory Activities of Abrus cantoniensis Polysaccharide Fractions

PubMed Central

Wu, Shaowei; Fu, Xiong; Brennan, Margaret A.; Brennan, Charles S.; Chun, Chen

2016-01-01

Abrus cantoniensis (Hance) is a popular Chinese vegetable consumed as a beverage, soup or folk medicine. To fully exploit the potential of the polysaccharide in Abrus cantoniensis, nine polysaccharide fractions of Abrus cantoniensis were isolated and purified (AP-AOH30-1, AP-AOH30-2, AP-AOH80-1, AP-AOH80-2, AP-ACl-1, AP-ACl-2, AP-ACl-3, AP-H and AP-L). Fourier-transform infrared spectroscopy (FT-IR) and gas chromatography (GC) were used to characterize these Abrus polysaccharides fractions (APF). In vitro anti-tumor and immunomodulatory activities were also investigated and compared using the rank-sum ratio (RSR) method. Results demonstrated significant differences in the structure and bioactivities among APF, which were associated to the process used for their purification. Among the APF, AP-ACl-3 yield was 613.5 mg/kg of product and consisted of rhamnose (9.8%), arabinose (8.9%), fructose (3.0%), galactose (9.9%), glucose (4.3%), galacturonic acid (3.0%) and glucuronic acid (61.1%) with a molecular weight of 4.4 × 104 Da. Furthermore, AP-ACl-3 exhibited considerable bioactivities significantly preventing the migration of MCF-7 cells and stimulating lymphocyte proliferation along with nitric oxide (NO) production of peritoneal macrophages. AP-ACl-3 could be explored as a novel potential anti-tumor and immunomodulatory agent. PMID:27058538

Preparation, characterization, and α-glycosidase inhibition activity of a carboxymethylated polysaccharide from the residue of Sarcandra glabra (Thunb.) Nakai.

PubMed

Liu, Wei; Hu, Cheng; Liu, Yameng; Dai, Shijie; Lu, Weisheng; Lv, Xing; Yao, Wenbing; Gao, Xiangdong

2017-06-01

A carboxymethylated polysaccharide (CMSERP) was prepared from the residue of Sarcandra glabra (Thunb.) Nakai. CMSERP was mainly composed of galacturonic acid (GalA), glucose (Glc), galactose (Gal), glucuronic acid (GlcA), arabinose (Ara), rhamnose (Rha), xylose (Xyl), ribose (Rib), and fucose (Fuc) at the ratio of 29.79:19.30:11.92:6.32:4.68:3.95:3.39:2.31:1.00. The primary structure features of CMSERP were determined to be a pectin like polysaccharide according to FT-IR, NMR, and HPAEC-PAD. The results of HPSEC-MALLS-RID and DLS indicated the Mw, Mn, Mz, and S2Z1/2 of CMSERP were 5.515×10 4 g/mol, 1.566×10 4 g/mol, 1.510×10 6 g/mol, and 62.8 (±1.2%) nm, respectively. TEM and AFM revealed CMSERP was dispersed in 0.05M sodium sulfate but aggregated in water. Moreover, a high α-glucosidase inhibition activity (83.38%±2.30% at 1000μg/mL) of CMSERP which is higher than that of acarbose was observed. The results proved the effects of carboxymethylation on poor water-soluble polysaccharides and explore a potential α-glucosidase inhibitor which from abandoned extracted residue for the functional foods and pharmaceutical industries. Copyright © 2017 Elsevier B.V. All rights reserved.

[3,4-methylene-dioxy-pyrovalerone (MDPV) epidemic?].

PubMed

Kalapos, Miklós Péter

2011-12-11

Little is known about 3,4-methylene-dioxy-pyrovalerone (MDPV), a new designer drug that has become popular in Hungary in the last couple of months. At the same time, its consumption, as a consequence of its low street-price, rises so fast that the event can be considered an epidemic. This paper reviews the chemistry, biochemistry and metabolism of MDPV. Then, on the basis of a few international reports and the author's own clinical observations, it discusses MDPV intoxication and withdrawal. In the metabolism of MDPV, the most important catalyst is the CYP2C19 isoenzyme, but the CYP1A2 and the CYP2D6 isoenzymes also play a crucial role. The formed catechols are conjugated with either glucuronic acid or sulfate. It is important to note that MDPV is consumed either together or in a sequence with other illicit drugs of abuse. As far as it can be established, MDPV use increases the activity and vigilance, decreases appetite and claim to sleep, but it can also provoke cardiac sensations and disturbance of perception. In the course of coming down, withdrawal after MDPV use, bone and muscle pain, hypersomnia, disturbance of vision are experienced, but panic attack may also occur. The appearance of new designer drugs on the market draws attention to a need of paradigm changing in spiritual field. Unless it happens these negative trends likely will speed up.

Commercial Fucoidans from Fucus vesiculosus Can Be Grouped into Antiadipogenic and Adipogenic Agents.

PubMed

Oliveira, Ruth Medeiros; Câmara, Rafael Barros Gomes; Monte, Jessyka Fernanda Santiago; Viana, Rony Lucas Silva; Melo, Karoline Rachel Teodosio; Queiroz, Moacir Fernandes; Filgueira, Luciana Guimarães Alves; Oyama, Lila Missae; Rocha, Hugo Alexandre Oliveira

2018-06-04

Fucus vesiculosus is a brown seaweed used in the treatment of obesity. This seaweed synthesizes various bioactive molecules, one of them being a sulfated polysaccharide known as fucoidan (FF). This polymer can easily be found commercially, and has antiadipogenic and lipolytic activity. Using differential precipitation with acetone, we obtained four fucoidan-rich fractions (F0.5/F0.9/F1.1/F2.0) from FF. These fractions contain different proportions of fucose:glucuronic acid:galactose:xylose:sulfate, and also showed different electrophoretic mobility and antioxidant activity. Using 3T3-L1 adipocytes, we found that all samples had lipolytic action, especially F2.0, which tripled the amount of glycerol in the cellular medium. Moreover, we observed that FF, F1.0, and F2.0 have antiadipogenic activity, as they inhibited the oil red staining by cells at 40%, 40%, and 50%, respectively. In addition, they decreased the expression of key proteins of adipogenic differentiation (C/EBPα, C/EBPβ, and PPARγ). However, F0.5 and F0.9 stimulated the oil red staining at 80% and increased the expression of these proteins. Therefore, these fucoidan fractions have an adipogenic effect. Overall, the data show that F2.0 has great potential to be used as an agent against obesity as it displays better antioxidant, lipolytic and antiadipogenic activities than the other fucoidan fractions that we tested.

A Liver-Centric Multiscale Modeling Framework for Xenobiotics.

PubMed

Sluka, James P; Fu, Xiao; Swat, Maciej; Belmonte, Julio M; Cosmanescu, Alin; Clendenon, Sherry G; Wambaugh, John F; Glazier, James A

2016-01-01

We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics.

A Liver-Centric Multiscale Modeling Framework for Xenobiotics

PubMed Central

Swat, Maciej; Cosmanescu, Alin; Clendenon, Sherry G.; Wambaugh, John F.; Glazier, James A.

2016-01-01

We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics. PMID:27636091

Metabolism of norethisterone in the greyhound.

PubMed

Biddle, S T B; O'Donnell, A; Houghton, E; Creaser, C S

2013-10-30

Norethisterone has been used as a successful oral contraceptive in humans for many years. It was recently permitted for use as an oestrus suppressant in racing greyhounds. To monitor the use of norethisterone as part of a routine drug surveillance programme, knowledge of its metabolism was required to enable detection. Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of norethisterone following oral administration to the greyhound. Metabolites were extracted using solid-phase and liquid-liquid extraction techniques. Several metabolites were identified, including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17α-ethynyl-5β-estrane-3α,17β-diol, 17α-ethynyl-5α-estrane-3β,17β-diol, three 17α-ethynylestranetriol stereoisomers and two 17α-ethynylestranetetrol stereoisomers. The major metabolites were predominantly excreted as glucuronic acid conjugates and detection of the administration of norethisterone was possible for up to 8 days post-dose using the methods described. The nandrolone metabolites, 19-norepiandrosterone, estranediol and 19-noretiocholanolone, were also identified in the post-administration samples collected up to 8 h after dosing the treated animals. The urinary metabolites identified in this study have further increased the knowledge of steroid metabolism in the greyhound, providing information to support routine drug testing programmes for greyhound racing. Copyright © 2013 John Wiley & Sons, Ltd.

Synthesis of the oligosaccharides related to branching sites of fucosylated chondroitin sulfates from sea cucumbers.

PubMed

Ustyuzhanina, Nadezhda E; Fomitskaya, Polina A; Gerbst, Alexey G; Dmitrenok, Andrey S; Nifantiev, Nikolay E

2015-02-02

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.

Differentiating chondroitin sulfate glycosaminoglycans using collision-induced dissociation; uronic acid cross-ring diagnostic fragments in a single stage of tandem mass spectrometry.

PubMed

Kailemia, Muchena J; Patel, Anish B; Johnson, Dane T; Li, Lingyun; Linhardt, Robert J; Amster, I Jonathan

2015-01-01

The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision-induced dissociation (CID) or EDD has been shown to correlate with the type of hexuronic acid present in chondroitin sulfate GAGs. The present work examines the effect of charge state and degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A and dermatan sulfate chains. The cross-ring fragments (2,4)A(n) and (0,2)X(n) formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and number of ionizable protons. The ions with the appropriate characteristics display diagnostic properties for all the chondroitin sulfate and dermatan sulfate chains (degree of polymerization of 4-10) studied.

Synthesis of the Oligosaccharides Related to Branching Sites of Fucosylated Chondroitin Sulfates from Sea Cucumbers

PubMed Central

Ustyuzhanina, Nadezhda E.; Fomitskaya, Polina A.; Gerbst, Alexey G.; Dmitrenok, Andrey S.; Nifantiev, Nikolay E.

2015-01-01

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS. PMID:25648510

Safety assessment of non-animal chondroitin sulfate sodium: Subchronic study in rats, genotoxicity tests and human bioavailability.

PubMed

Miraglia, Niccolò; Bianchi, Davide; Trentin, Antonella; Volpi, Nicola; Soni, Madhu G

2016-07-01

Chondroitin sulfate, an amino sugar polymer made of glucuronic acid and N-acetyl-galactosamine, is used in dietary supplements to promote joint health. Commonly used chondroitin sulfate is of animal origin and can pose potential safety problems including bovine spongiform encephalopathy (BSE). The objective of the present study was to investigate potential adverse effects, if any, of microbial derived chondroitin sulfate sodium (CSS) in subchronic toxicity, genotoxicity and bioavailability studies. In the toxicity study, Sprague Dawley rats (10/sex/group) were gavaged with CSS at dose levels of 0, 250, 500 and 1000 mg/kg body weight (bw)/day for 90-days. No mortality or significant changes in clinical signs, body weights, body weight gain or feed consumption were noted. Similarly, no toxicologically relevant treatment-related changes in hematological, clinical chemistry, urinalysis and organ weights were noted. Macroscopic and microscopic examinations did not reveal treatment-related abnormalities. In vitro mutagenic and clastogenic potentials as evaluated by Ames assay, chromosomal aberration test and micronucleus assay did not reveal genotoxicity of CSS. In pharmacokinetic study in human, CSS showed higher absorption as compared to chondroitin sulfate of animal origin. The results of subchronic toxicity study supports the no-observed-adverse-effect level (NOAEL) for CSS as 1000 mg/kg bw/day, the highest dose tested. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure-activity relationships for chloro- and nitrophenol toxicity in the pollen tube growth test

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

Schueuermann, G.; Somashekar, R.K.; Kristen, U.

Acute toxicity of 10 chlorophenols and 10 nitrophenols with identical substitution patterns is analyzed with the pollen tube growth (PTG) test. Concentration values of 50% growth inhibition (IC50) between 0.1 and 300 mg/L indicate that the absolute sensitivity of this alternative biotest is comparable to conventional aquatic test systems. Analysis of quantitative structure-activity relationships using lipophilicity (log K{sub ow}), acidity (pK{sub a}), and quantum chemical parameters to model intrinsic acidity, solvation interactions, and nucleophilicity reveals substantial differences between the intraseries trends of log IC50. With chlorophenols, a narcotic-type relationship is derived, which, however, shows marked differences in slope and interceptmore » when compared to reference regression equations for polar narcosis. Regression analysis of nitrophenol toxicity suggests interpretation in terms of two modes of action: oxidative uncoupling activity is associated with a pK{sub a} window from 3.8 to 8.5, and more acidic congeners with diortho-substitution show a transition from uncoupling to a narcotic mode of action with decreasing pK{sub a} and log K{sub ow}. Model calculations for phenol nucleophilicity suggest that differences in the phenol readiness for glucuronic acid conjugation as a major phase-II detoxication pathway have no direct influence on acute PTG toxicity of the compounds.« less

Acute UV irradiation increases heparan sulfate proteoglycan levels in human skin.

PubMed

Jung, Ji-Yong; Oh, Jang-Hee; Kim, Yeon Kyung; Shin, Mi Hee; Lee, Dayae; Chung, Jin Ho

2012-03-01

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

UPLC-Q-TOF/MS-based metabolomic studies on the toxicity mechanisms of traditional Chinese medicine Chuanwu and the detoxification mechanisms of Gancao, Baishao, and Ganjiang.

PubMed

Dong, Hui; Yan, Guang-Li; Han, Ying; Sun, Hui; Zhang, Ai-Hua; Li, Xian-Na; Wang, Xi-Jun

2015-09-01

Chuanwu (CW), a famous traditional Chinese medicine (TCM) from the mother roots of Aconitum carmichaelii Debx.. (Ranunculaceae), has been used for the treatment of various diseases. Unfortunately, its toxicity is frequently reported because of its narrow therapeutic window. In the present study, a metabolomic method was performed to characterize the phenotypically biochemical perturbations and potential mechanisms of CW-induced toxicity. Meanwhile, the expression level of toxicity biomarkers in the urine were analyzed to evaluate the detoxification by combination with Gancao (Radix Glyeyrrhizae, CG), Baishao (Radix Paeoniae Alba, CS) and Ganjiang (Rhizoma Zingiberis, CJ), which were screened from classical TCM prescriptions. Urinary metabolomics was performed by UPLC-Q-TOF-HDMS, and the mass spectra signals of the detected metabolites were systematically analyzed using pattern recognition methods. As a result, seventeen biomarkers associated with CW toxicity were identified, which were associated with pentose and glucuronate interconversions, alanine, aspartate, and glutamate metabolism, among others. The expression levels of most toxicity biomarkers were effectively modulated towards the normal range by the compatibility drugs. It indicated that the three compatibility drugs could effectively detoxify CW. In summary, our work demonstrated that metabolomics was vitally significant to evaluation of toxicity and finding detoxification methods for TCM. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Screening and comparison of antioxidant activities of polysaccharides from Coriolus versicolor.

PubMed

Sun, Xiaowen; Sun, Yanping; Zhang, Qingbo; Zhang, Hongwei; Yang, Bingyou; Wang, Zhibin; Zhu, Weiguo; Li, Bin; Wang, Qiuhong; Kuang, Haixue

2014-08-01

Six polysaccharide fractions (Coriolus versicolor polysaccharides: CVPS-1, CVPS-2, CVPS-3, CVPS-4, CVPS-5 and CVPS-6) were isolated and purified from the fruiting bodies of C. versicolor by ion exchange chromatography and gel chromatography. Their chemical and physical characteristics were determined by chemical methods, high performance liquid chromatography, and high-performance gel-permeation chromatography. Finally, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, superoxide radical assay, and hydroxyl radical assay were carried out to test the antioxidant activities of CVPS in vitro. The results indicated that the six CVPS fractions were acidic heteropolysaccharides, composed of mannose, rhamnose, glucuronic acid, glucose and fructose with different ratios. The molecular weights of CVPS-1, CVPS-2, CVPS-3, CVPS-4, CVPS-5 and CVPS-6 were 1740, 1480, 568, 880, 1260 and 1840kDa and the protein contents were 4.2%, 6.4%, 8.5%, 7.8%, 6.5% and 3.9%, respectively. Among the six fractions, CVPS with lower molecular weight, higher protein content and larger uronic acid amount, basically exhibited higher radical scavenging effects at the same concentration. Compared with other fractions, CVPS-3 exhibited the highest antioxidant activities. The effects of the molecular weight, protein content and uronic acid amount of the polysaccharides appeared to be significant on the improvement of the bioactivities. Copyright © 2014 Elsevier B.V. All rights reserved.

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

DOE PAGES

Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan; ...

2015-06-04

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40T*

PubMed Central

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.; Vuong, Thu V.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Gatenholm, Paul; Toriz, Guillermo; Tenkanen, Maija; Savchenko, Alexei; Master, Emma R.

2016-01-01

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C+ domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C+ in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes. PMID:27129264

Effect of pretreatments and endo-1,4-β-xylanase hydrolysis of canola meal and mustard bran for production of oligosaccharides.

PubMed

Yuan, Lin; Scanlon, Martin G; Eskin, N A Michael; Thiyam-Hollander, Usha; Aachary, Ayyappan A

2015-01-01

Alkali/acid-pretreated canola meal and mustard bran were subjected to endo-1,4-β-xylanase (T. longibrachiatum) hydrolysis for oligosaccharide production. Pretreatments significantly (α = 0.05) increased the relative content of pentose sugars, especially in alkali-pretreated canola meal (∼44 %) and mustard bran (∼72 %). The amounts of pentosan (g/100 g) in acid- and alkali-pretreated canola meal were 7.50 and 8.21 and in corresponding mustard bran were 8.67 and 10.39, respectively. These pretreated substrates produced a pentose content (g/100 g) of 2.10 ± 0.14 (18 h) and 2.95 ± 0.10 (24 h), respectively, during hydrolysis. As per UPLC-MS data, the main oligosaccharides in the hydrolyzates of alkali-pretreated substrates are xylo-glucuronic acid and xylobiose. The release of total phenolics of the hydrolyzates increased until 18 h irrespective of the type of substrate or pretreatment. Hydrolyzates of acid-pretreated substrates indicated more total antioxidant activity than alkali-pretreated substrates, attributed to its high phenolic content. The study suggests the potential of canola meal and mustard bran for the production of oligosaccharides, wherein the use of various combinations of cell-wall-degrading enzymes and its optimization may result in a better yield, with simultaneous production of endogenous phenolics.

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40 T

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

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as -glucuronidases that release the - (132)-linked (Me)GlcAp side groups. Herein, a family GH115 enzyme from the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domainmore » architecture, with an additional insertion C domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.« less

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

PubMed

Gu, Bin; Laborda, Pedro; Wei, Shuang; Duan, Xu-Chu; Song, Hui-Bo; Liu, Li; Voglmeir, Josef

2016-01-01

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.

Studies on the disturbance of glucuronide formation in infectious hepatitis

PubMed Central

Vest, M. F.; Fritz, E.

1961-01-01

The ability of the liver to form glucuronides was measured in 10 patients with infectious hepatitis. One test was done at the onset and another about four weeks later after the clinical symptoms had disappeared. N-acetyl-p-aminophenol (N.A.P.A.) or acetanilide was administered in doses ranging from 10 to 20 mg. per kg. body weight, either orally or by intravenous injection. N.A.P.A. is conjugated by the liver at the hydroxyl group and excreted in the urine as sulphuric and glucuronic acid conjugates. Total conjugated p-aminophenol, free N.A.P.A., and N.A.P.A. glucuronide were estimated in the urine of our patients. In the blood the disappearance of N.A.P.A. (free form) and the formation of N.A.P.A. glucuronide were traced. During the acute phase of hepatitis the excretion of total conjugated p-aminophenol and of N.A.P.A. glucuronide in the urine is lower than after recovery from the disease. Likewise free N.A.P.A. disappears more slowly from the circulation and the peak concentration of N.A.P.A. glucuronide in the serum remains lower at the onset of hepatitis than after clinical cure. These results indicate that glucuronide formation during the acute stage of infectious hepatitis is depressed, as are other transformation mechanisms, i.e., of hippuric acid. PMID:13925655

Optimization of hyaluronic acid production and its cytotoxicity and degradability characteristics.

PubMed

Gedikli, Serap; Güngör, Gökhan; Toptaş, Yağmur; Sezgin, Dilber Ece; Demirbilek, Murat; Yazıhan, Nuray; Aytar Çelik, Pınar; Denkbaş, Emir Baki; Bütün, Vural; Çabuk, Ahmet

2018-06-14

In the present study, culture conditions of Streptococcus equi was optimized through Box-Behnken experimental design for hyaluronic acid production. About 0.87 gL -1 of hyaluronic acid was produced under the determined conditions and optimal conditions were found as 38.42 °C, 24 hr and 250 rpm. The validity and practicability of this statistical optimization strategy were confirmed relation between predicted and experimental values. The hyaluronic acid obtained under optimal conditions was characterized. The effects of different conditions such as ultraviolet light, temperature and enzymatic degradation on hyaluronic acid produced under optimal conditions were determined. 118 °C for 32 min of autoclaved HA sample included 63.09 µg mL -1 of d-glucuronic acid, which is about two-fold of enzymatic effect. Cytotoxicity of hyaluronic acid on human dermal cells (HUVEC, HaCaT), L929 and THP-1 cells was studied. In vitro effect on pro or anti-inflammatory cytokine release of THP-1 cells was determined. Although it varies depending on the concentration, cytotoxicity of hyaluronic acid is between 5 and 30%. However, it varies depending on the concentration of hyaluronic acid, TNF-α release was not much increased compared to control study. Consequently, purification procedure is necessary to develop and it is worth developing the bacterial hyaluronic acid.

Anatomy, nutritional value and cell wall chemical analysis of foliage leaves of Guadua chacoensis (Poaceae, Bambusoideae, Bambuseae), a promising source of forage.

PubMed

Panizzo, Cecilia C; Fernández, Paula V; Colombatto, Darío; Ciancia, Marina; Vega, Andrea S

2017-03-01

The present study combines morphological and anatomical studies, cell wall chemical composition analysis, as well as assessment of the nutritional value of Guadua chacoensis foliage leaves. Foliage leaves of G. chacoensis are a promising source of forage because: (a) as a native woody bamboo, it is adapted to and helps maintain environmental conditions in America; (b) leaf anatomical studies exhibit discontinuous sclerenchyma, scarcely developed, while pilose indumentum, silica cells, prickles and hooks are also scarce; (c) it has a high protein content, similar to that of Medicago sativa, while other nutritional parameters are similar to those of common forages; and (d) glucuronoarabinoxylan, the major extracted polysaccharide, has one-third of the 4-linked β-d-xylopyranosyl units of the backbone substituted mainly with α-l-arabinofuranose as single stubs or non-reducing end of short chains, but also 5-linked α-l-arabinofuranose units, terminal β-d-xylopyranose and d-galactopyranose units, as well as α-d-glucuronic acid residues and small amounts of its 4-O-methylated derivative. These results constitute the first report on this species, and as culms are utilized in constructions and crafts, the remaining leaves, when used as forage, constitute a byproduct that allows an additional income opportunity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Bisphenol A glucuronidation in patients with Parkinson's disease.

PubMed

Landolfi, Annamaria; Troisi, Jacopo; Savanelli, Maria Cristina; Vitale, Carmine; Barone, Paolo; Amboni, Marianna

2017-12-01

Bisphenol A (BPA) is a widely distributed estrogen-mimetic molecule, with well-established effects on the dopaminergic system. It can be found in canned food, dental sealants, thermal paper, etc. BPA undergoes liver conjugation with glucuronic acid and is subsequently excreted in the urine. In the present study we quantified the concentration of free and conjugated Bisphenol A in blood of patients affected by Parkinson Disease, using their spouses as controls. An interview was performed to determine possible confounders in BPA exposure. Free and conjugated BPA were quantified by gas chromatography coupled with mass spectrometry. Parkinson's Disease patients carried a statistically significant lower amount of conjugated Bisphenol A compared to controls. The two populations were mostly homogeneous in terms of exposure to possible Bisphenol A sources. The only exceptions were exposure to canned tuna and canned tomatoes PD patients consumed significantly more of both (p<0.05). Moreover, no difference in Bisphenol A glucuronidation was found after stratification by typology of anti-Parkinson's drug taken and after conversion to the Levodopa Equivalent Daily Dose. BPA glucuronidation was decreased in patients with Parkinson disease. The possible unique mechanisms underlying Bisphenol A metabolism in PD patients deserve further elucidation. Moreover, further study is needed to assess a possible BPA role in Parkinson's Disease pathogenesis, due to its documented dopaminergic toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and monkeys.

PubMed

Reddy, V B G; Doss, G A; Karanam, B V; Samuel, K; Lanza, T J; Lin, L S; Yu, N X; Zhang, A S; Raab, C E; Stearns, R A; Kumar, S

2010-09-01

The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹⁴C]taranabant, the majority of the radioactivity was excreted within 72 h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

Adeno-associated virus type 2 binding study on model heparan sulfate surface

NASA Astrophysics Data System (ADS)

Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

2003-11-01

Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

Physicochemical properties of water-soluble polysaccharides from black cumin seeds.

PubMed

Trigui, Ines; Yaich, Héla; Sila, Assaâd; Cheikh-Rouhou, Salma; Bougatef, Ali; Blecker, Christophe; Attia, Hamadi; Ayadi, M A

2018-06-01

In the present work, water-soluble polysaccharides were isolated from black cumin seeds. Polysaccharides were characterized by their carbohydrate composition, molecular weight, thermal stability and by FTIR, NMR spectroscopy and X-ray diffraction. The surface, the functional and the antioxidant properties of black cumin water-soluble polysaccharides (BCWSP) were also investigated. BCWSP consisted mainly of galacturonic acid (30.20%), glucuronic acid (17.66%) and neutral sugar (22.99%). BCWSP was composed of high peak molecular weight. The FTIR spectrum obtained for BCWSP showed two most important absorptions, at 1659 and 1085 cm -1 , which corresponded to COO - of uronic acids and pyranose form, respectively. NMR spectroscopy data suggested that the BCWSP is probably a rhamnogalacturonan backbone with galactan and arabinan side chains. X-ray pattern revealed the semi-crystalline behavior of BCWSP. WHC and OHC of BCWSP were relatively high and varied with temperatures. The polysaccharide zeta potential was greatly affected by pH. Results indicated that the decrease of surface tension has influenced foaming and emulsifying capacities. The DPPH radical scavenging activity of the BCWSP was 63.25% at 1 mg/mL. The BCWSP displayed moderate reductive, β carotene bleaching and chelating abilities. Overall, our results suggested that BCWSP could be used as alternative additives in food and non-food products. Copyright © 2017. Published by Elsevier B.V.

Dual-mode T1 and T2 magnetic resonance imaging contrast agent based on ultrasmall mixed gadolinium-dysprosium oxide nanoparticles: synthesis, characterization, and in vivo application

NASA Astrophysics Data System (ADS)

Tegafaw, Tirusew; Xu, Wenlong; Wasi Ahmad, Md; Baeck, Jong Su; Chang, Yongmin; Bae, Ji Eun; Chae, Kwon Seok; Kim, Tae Jeong; Lee, Gang Ho

2015-09-01

A new type of dual-mode T1 and T2 magnetic resonance imaging (MRI) contrast agent based on mixed lanthanide oxide nanoparticles was synthesized. Gd3+ (8S7/2) plays an important role in T1 MRI contrast agents because of its large electron spin magnetic moment resulting from its seven unpaired 4f-electrons, and Dy3+ (6H15/2) has the potential to be used in T2 MRI contrast agents because of its very large total electron magnetic moment: among lanthanide oxide nanoparticles, Dy2O3 nanoparticles have the largest magnetic moments at room temperature. Using these properties of Gd3+ and Dy3+ and their oxide nanoparticles, ultrasmall mixed gadolinium-dysprosium oxide (GDO) nanoparticles were synthesized and their potential to act as a dual-mode T1 and T2 MRI contrast agent was investigated in vitro and in vivo. The D-glucuronic acid coated GDO nanoparticles (davg = 1.0 nm) showed large r1 and r2 values (r2/r1 ≈ 6.6) and as a result clear dose-dependent contrast enhancements in R1 and R2 map images. Finally, the dual-mode imaging capability of the nanoparticles was confirmed by obtaining in vivo T1 and T2 MR images.

Modulation of hyaluronan synthase activity in cellular membrane fractions.

PubMed

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

2009-10-30

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

Metabolite profiling of sex developmental steroid conjugates reveals an association between decreased levels of steroid sulfates and adiposity in obese girls.

PubMed

Lee, Su Hyeon; Kim, Shin Hye; Lee, Won-Yong; Chung, Bong Chul; Park, Mi Jung; Choi, Man Ho

2016-09-01

Free and conjugated steroids coexist in a dynamic equilibrium due to complex biosynthetic and metabolic processes. This may have clinical significance related to various physiological conditions, including sex development involving the reproductive system. Therefore, we performed quantitative profiling of 16 serum steroids conjugated with glucuronic and sulfuric acids using liquid chromatography-mass spectrometry (LC-MS). All steroid conjugates were purified by solid-phase extraction and then separated through a 3-μm particle size C18 column (150mm×2.1mm) at a flow rate of 0.3 mL/min in the negative ionization mode. The LC-MS-based analysis was found to be linear (r(2)>0.99), and all steroid conjugates had a limit-of-quantification (LOQ) of 10ng/mL, except for cholesterol sulfate and 17β-estradiol-3,17-disulfate (20ng/mL). The extraction recoveries of all steroid conjugates ranged from 97.9% to 110.7%, while the overall precision (% CV) and accuracy (% bias) ranged from 4.8% to 10.9% and from 94.4% to 112.9% at four different concentrations, respectively. Profiling of steroid conjugates corrected by adiposity revealed decreased levels of steroid sulfates (P<0.01) in overweight and obese girls compared to normal girls. The suggested technique can be used for evaluating metabolic changes in steroid conjugates and for understanding the pathophysiology and relative contributions of adiposity in childhood obesity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pharmacokinetics of table and Port red wine anthocyanins: a crossover trial in healthy men.

PubMed

Fernandes, I; Marques, C; Évora, A; Cruz, L; de Freitas, V; Calhau, C; Faria, A; Mateus, N

2017-05-24

This study was designed to evaluate the pharmacokinetics of Port and table red wine anthocyanins in healthy men. Volunteers were recruited to drink 250 mL of a table red wine (221 mg of anthocyanins) and 150 mL of young Port red wine (49 mg of anthocyanins). Venous blood was collected from participants at 0, 15, 30, 60 and 120 min after wine ingestion. Urine samples were collected at baseline and at 120 min. Anthocyanins and anthocyanin metabolites in plasma and urine samples were quantified by HPLC-DAD and tentatively identified by LC-MS. Red wine anthocyanins were detected in their intact forms in both plasma and urine samples, but the glucuronylated metabolites of peonidin and malvidin (PnGlucr and MvGlucr) were the two main derivatives detected after both red wine consumptions. For the first time, and supported by the synthesis of Mv3Glucr, the main pathway followed by Mv3glc after absorption was described and involves anthocyanidin conjugation with glucuronic acid after glucose removal. Despite the lower total content of anthocyanins ingested when volunteers drank Port wine, no differences were observed in the plasma C max of MvGlucr and PnGlucr after table and Port red wine consumption. The relative bioavailability of anthocyanins in Port wine was 96.58 ± 5.74%, compared to the anthocyanins present in red wine. In conclusion, both Port and table red wines are good sources of bioavailable anthocyanins.

Oleanane-type triterpene saponins from Calendula stellata.

PubMed

Lehbili, Meryem; Alabdul Magid, Abdulmagid; Kabouche, Ahmed; Voutquenne-Nazabadioko, Laurence; Abedini, Amin; Morjani, Hamid; Sarazin, Thomas; Gangloff, Sophie C; Kabouche, Zahia

2017-12-01

Five previously undescribed bisdesmosidic triterpenoid saponins named calendustellatosides A-E, along with fifteen known compounds were isolated from the 70% ethanol whole plant extract of Calendula stellata Cav. (Asteraceae). Their structures were determined by 1D- and 2D-NMR spectroscopy as well as high resolution mass spectrometry and acid hydrolysis. The saponins comprised oleanolic acid, echinocystic acid, morolic acid or mesembryanthemoidigenic acid as the aglycones and saccharide moieties at C-3 and C-28. Like most Calendula saponins, the sugar moiety linked at C-3 was either β-d-glucose or β-d-glucuronic acid which could be substituted at C-3 by a β-d-galactose and/or C-2 by a supplementary β-d-galactose or a β-d-glucose. The sugar moiety linked to C-28 was determined as β-d-glucose. The antibacterial evaluation of compounds 1-20 by bioautography on Staphylococcus aureus followed by the determination of MIC values of active compounds by serial dilution technique against 5 bacteria revealed that; calendustellatoside D was the most active against Enterococcus faecalis with an antibacterial effect comparable to antibiotics. The cytotoxic activities of isolated compounds were evaluated against fibrosarcoma cell line (HT1080) and human lung cancer cell line (A549). Calendustellatosides B and D exhibited a low cytotoxic activity against HT1080 cell line with IC 50 values of 47 ± 0.6 and 39 ± 0.5 μM, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

An HPLC Method for Microanalysis and Pharmacokinetics of Marine Sulfated Polysaccharide PSS-Loaded Poly Lactic-co-Glycolic Acid (PLGA) Nanoparticles in Rat Plasma

PubMed Central

Li, Peng-Li; Li, Chun-Xia; Xue, Yi-Ting; Li, Hai-Hua; Liu, Hong-Bing; He, Xiao-Xi; Yu, Guang-Li; Guan, Hua-Shi

2013-01-01

This study was aimed at developing a sensitive and selective HPLC method with postcolumn fluorescence derivatization for the detection of propylene glycol alginate sodium sulfate (PSS) in rat plasma. Plasma samples were prepared by a simple and fast ultrafiltration method. PSS was extracted from rat plasma with d-glucuronic acid as internal standard. Isocratic chromatographic separation was performed on a TSKgel G2500 PWxL column with the mobile phase of 0.1 M sodium sulfate at a flow rate of 0.5 mL/min. Analyte detection was achieved by fluorescence detection (FLD) at 250 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as postcolumn derivatizing reagent in an alkaline medium at 120 °C. The calibration curve was linear over a concentration range of 1–500 μg/mL, and the lower limit of detection (LLOD) was found to be 250 ng/mL. This validated method was applied successfully to the pharmacokinetic study of PSS and PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (PSS-NP) in rat plasma after a single intravenous (PSS only) and oral administration (PSS and PSS-NP). Significant differences in the main pharmacokinetic parameters of PSS and PSS-NP were observed. The relative bioavailability of PSS-NP was 190.10% compared with PSS which shows that PSS-NP can improve oral bioavailability. PMID:23549283

A polysaccharide from the stems of Rubus amabilis Focke and its immunological enhancement activity.

PubMed

Diao, Yu-Lin; Shan, Jun-Jie; Ma, Hao; Zhang, Tong; Liu, Bin

2016-09-01

A water-soluble polysaccharide (named RAP) was newly isolated from the stems of Rubus amabilis. Structural confirmation of the polysaccharide was provided by hydrolysis, periodate oxidation, Smith degradation, and methylation analysis, combined with nuclear magnetic resonance (NMR), capillary electrophoresis (CE), infrared spectroscopy (IR), and gas chromatography-mass spectra (GC-MS). In vitro immunological enhancement activity was characterized using the proliferative activity of spleen lymphocytes and phagocytic activity of peritoneal macrophages in mice. The polysaccharide was mainly composed of xylose, arabinose, glucose, rhamnose, galactose, mannose, glucuronic acid, and galactocuronic acid in the molar ratio of 1.0:6.9:0.8:1.1:6.9:0.3:0.5:3.3, with the average molecular weight of 26.2 kDa. The linkage types of netural monosaccharides were as follows: the arabinose was →2) Ara (1→ and galactose were Gal (1→, →3) Gal (1→, →3,6) Gal (1→, →2,3,6) Gal (1→ and →2,3,6) Galf (1→. Xyl (1→, →6) Glc (1→, →2) Glc (1→, →3) Rha (1→, Rha (1→ and Man (1→ were also found in the structure. RAP-B-2 could improve the proliferative activity of spleen T cells and B cells and boost phagocytic activity of peritoneal macrophages at the concentration of 50 μg/ml (p < 0.05, p < 0.01).

Chondroitin sulfate synthase-2 is necessary for chain extension of chondroitin sulfate but not critical for skeletal development.

PubMed

Ogawa, Hiroyasu; Hatano, Sonoko; Sugiura, Nobuo; Nagai, Naoko; Sato, Takashi; Shimizu, Katsuji; Kimata, Koji; Narimatsu, Hisashi; Watanabe, Hideto

2012-01-01

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2(-/-) mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2(-/-) chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ∼10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis.

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

PubMed

Joshi, Vikram; Umashankara, M; Ramakrishnan, Chandrasekaran; Nanjaraj Urs, Ankanahalli N; Suvilesh, Kanve Nagaraj; Velmurugan, Devadasan; Rangappa, Kanchugarakoppal S; Vishwanath, Bannikuppe Sannanaik

2016-05-15

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacokinetics and metabolism of radiolabelled SNI-2011, a novel muscarinic receptor agonist, in healthy volunteers. Comprehensive understanding of absorption, metabolism and excretion using radiolabelled SNI-2011.

PubMed

Washio, Takuo; Kohsaka, Kazuhiro; Arisawa, Hirohiko; Masunaga, Hiroaki; Nagatsuka, Shin-ichiro; Satoh, Yoshiaki

2003-01-01

The pharmacokinetics and metabolism of SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine]monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), a novel muscarinic acetylcholine receptor agonist developed for the treatment of Sjögen's syndrome, were investigated in six healthy volunteers after a single oral administration of 14C-SNI-2011. After administration, plasma concentrations of the radioactivity and SNI-2011 reached to Cmax at approximately 2 h, and then decreased with t 1/2 of 9 and 4 h, respectively. Cmax and AUC0-infinity of the radioactivity in plasma were 2.2 and 5.0 times higher than those of SNI-2011, respectively. The main excretion route of the radioactivity was urine, and 97.3% of the dose excreted in urine within 168 h, indicating that 14C-SNI-2011 was completely absorbed. The mean recoveries of the metabolites in urine at 24 h after administration were 16.0% for SNI-2011, 35.8% for SNI-2011 trans-sulfoxide (SNI-t-SO), 8.7% for SNI-2011 cis-sulfoxide, 4.1% for SNI-2011 N-oxide, furthermore, two unknown metabolites, UK-1 and UK-2, were detected 14.6% and 7.7%, respectively. LC/MS analysis and hydrolysis studies revealed that UK-1 and UK-2 were glucuronic acid conjugates of SNI-2011 and SNI-t-SO, respectively.

Copper removal by algal biomass: biosorbents characterization and equilibrium modelling.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Pinheiro, José P S; Domingos, Rute F; Boaventura, Rui A R

2009-04-30

The general principles of Cu(II) binding to algal waste from agar extraction, composite material and algae Gelidium, and different modelling approaches, are discussed. FTIR analyses provided a detailed description of the possible binding groups present in the biosorbents, as carboxylic groups (D-glucuronic and pyruvic acids), hydroxyl groups (cellulose, agar and floridean starch) and sulfonate groups (sulphated galactans). Potentiometric acid-base titrations showed a heterogeneous distribution of two major binding groups, carboxyl and hydroxyl, following the quasi-Gaussian affinity constant distribution suggested by Sips, which permitted to estimate the maximum amount of acid functional groups (0.36, 0.25 and 0.1 mmol g(-1)) and proton binding parameters (pK(H)=5.0, 5.3 and 4.4; m(H)=0.43, 0.37, 0.33), respectively for algae Gelidium, algal waste and composite material. A non-ideal, semi-empirical, thermodynamically consistent (NICCA) isotherm fitted better the experimental ion binding data for different pH values and copper concentrations, considering only the acid functional groups, than the discrete model. Values of pK(M) (3.2; 3.6 and 3.3), n(M) (0.98, 0.91, 1.0) and p (0.67, 0.53 and 0.43) were obtained, respectively for algae Gelidium, algal waste and composite material. NICCA model reflects the complex macromolecular systems that take part in biosorption considering the heterogeneity of the biosorbent, the competition between protons and metals ions to the binding sites and the stoichiometry for different ions.

Optimization of the microwave-assisted enzymatic extraction of Rosa roxburghii Tratt. polysaccharides using response surface methodology and its antioxidant and α-d-glucosidase inhibitory activity.

PubMed

Wang, Huizhu; Li, Yan; Ren, Zhihui; Cong, Zhongcheng; Chen, Mengjie; Shi, Lin; Han, Xu; Pei, Jin

2018-06-01

An extraction assay applying microwave-assisted enzymatic treatment for polysaccharides in Rosa roxburghii was developed using response surface methodology. The process parameters were optimized using Plackett-Burman (PB) design and central composite design to enhance the Rosa roxburghii polysaccharide extraction yield. Specific conditions (microwave power, 575W; microwave time, 18min; liquid-to-material ratio, 13.5:1mL/g; and enzyme dose, 6.5g/mL) generated an experimental yield of 36.21±0.62%, which closely agreed with the predicted value of 35.75%. Purification with a DEAE-52 cellulose column generated two fractions, PR-1 (from 6.2×10 3 to 7.4KDa) and PR-2 (from 559.8 to 106.6KDa). Subsequently, the antioxidant activity and α-d-glucosidase inhibitory activity of the two polysaccharide fractions were assessed; PR-1 exhibited stronger antioxidant activity and α-d-glucosidase inhibitory activity than PR-2. Finally, the monosaccharide composition of PR-1 was determined by HPLC using a 1-phenyl-3-methyl-5-pyrazolone precolumn derivatization method. The result showed that PR-1 contained mannose, ribose, rhamnose, glucosamine hydrochloride, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and fucose with molar percentages of 2.1%, 0.54%, 2.1%, 0.26%, 1.5%, 22.7%, 24.0%, 26.4%, 19.6% and 0.89%, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

DOE PAGES

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; ...

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

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

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

Temporal and longitudinal biofilm matrix analysis of a biofilter treating ethyl acetate during ozonation.

PubMed

Covarrubias-García, Itzel; Aizpuru, Aitor; Arriaga, Sonia

2018-05-04

The present paper focuses on the biofilm composition and pattern of biomass in gas biofiltration of ethyl acetate working under continuous addition of ozone (O 3 ). Two biofilters were operated for 230 days, one under continuous addition of O 3 (90 ppb v ) and another one without. Throughout the operation time, the extracellular polymeric substances (EPS), the main components in the extracellular matrix (ECM), were extracted from the biofilm and characterized qualitatively using Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and quantitatively by analyzing its main constituents: carbohydrates, proteins, and glucuronic acid. To date, EPS characterization has been attempted mainly with biofilm aggregates related to water treatment, not air biofiltration. The results of this study may be helpful and provide more information about EPS structure when O 3 was added. O 3 addition only affected the amount of EPS and not its composition. The greater effect was observed on carbohydrate content since it is the main component in EPS. The EPS/biomass ratio measured was twice lower with O 3 addition. Higher removal efficiency (RE) and mineralization rates were obtained with the biofilter subjected to O 3 addition, and a smaller volume of a reactor would be necessary to treat all contaminant under this condition. EPS content is only quantitatively reduced by O 3 addition, and at the low O 3 concentration applied , no structural alteration is noted regarding the composition of the EPS.

High-performance liquid chromatography assay using ultraviolet detection for urinary quantification of milrinone concentrations in cardiac surgery patients undergoing cardiopulmonary bypass.

PubMed

Gavra, Paul; Nguyen, Anne Q N; Beauregard, Natasha; Denault, André Y; Varin, France

2014-08-01

An analytical assay using liquid-liquid extraction and high-performance liquid chromatography with ultraviolet detection was developed for the quantification of total (conjugated and unconjugated) urinary concentrations of milrinone after the inhalation of a 5 mg dose in 15 cardiac patients undergoing cardiopulmonary bypass. Urine samples (700 μL) were extracted with ethyl-acetate and subsequently underwent acid back-extraction before and after deconjugation by mild acid hydrolysis. Milrinone was separated on a strong cation exchange analytical column. The mobile phase consisted of a constant mixture of acetonitrile:tetrahydrofurane-NaH2 PO4 buffer (40:60 v/v, pH 3.0). Thirteen calibration curves were linear in the concentration range of 31.25-4000 ng/mL, using olprinone as the internal standard (r(2) range 0.9911-0.9999, n = 13). Mean milrinone recovery and accuracy were respectively 85.2 ± 3.1% and ≥93%. Intra- and inter-day precisions (coefficients of variation) were ≤5% and ≤8%, respectively. Over a 24 h collection period, the cumulative urinary milrinone recovered from 15 patients was 26.1 ± 7.7% of the nominal 5 mg dose administered. The relative amount of milrinone glucuronic acid conjugate was negligible in the urine of patients undergoing cardiopulmonary bypass This method proved to be reliable, specific and accurate to determine the cumulative amount of total milrinone recovered in urine after inhalation. Copyright © 2014 John Wiley & Sons, Ltd.

Sugar Metabolism of the First Thermophilic Planctomycete Thermogutta terrifontis: Comparative Genomic and Transcriptomic Approaches

PubMed Central

Elcheninov, Alexander G.; Menzel, Peter; Gudbergsdottir, Soley R.; Slesarev, Alexei I.; Kadnikov, Vitaly V.; Krogh, Anders; Bonch-Osmolovskaya, Elizaveta A.; Peng, Xu; Kublanov, Ilya V.

2017-01-01

Xanthan gum, a complex polysaccharide comprising glucose, mannose and glucuronic acid residues, is involved in numerous biotechnological applications in cosmetics, agriculture, pharmaceuticals, food and petroleum industries. Additionally, its oligosaccharides were shown to possess antimicrobial, antioxidant, and few other properties. Yet, despite its extensive usage, little is known about xanthan gum degradation pathways and mechanisms. Thermogutta terrifontis, isolated from a sample of microbial mat developed in a terrestrial hot spring of Kunashir island (Far-East of Russia), was described as the first thermophilic representative of the Planctomycetes phylum. It grows well on xanthan gum either at aerobic or anaerobic conditions. Genomic analysis unraveled the pathways of oligo- and polysaccharides utilization, as well as the mechanisms of aerobic and anaerobic respiration. The combination of genomic and transcriptomic approaches suggested a novel xanthan gum degradation pathway which involves novel glycosidase(s) of DUF1080 family, hydrolyzing xanthan gum backbone beta-glucosidic linkages and beta-mannosidases instead of xanthan lyases, catalyzing cleavage of terminal beta-mannosidic linkages. Surprisingly, the genes coding DUF1080 proteins were abundant in T. terrifontis and in many other Planctomycetes genomes, which, together with our observation that xanthan gum being a selective substrate for many planctomycetes, suggest crucial role of DUF1080 in xanthan gum degradation. Our findings shed light on the metabolism of the first thermophilic planctomycete, capable to degrade a number of polysaccharides, either aerobically or anaerobically, including the biotechnologically important bacterial polysaccharide xanthan gum. PMID:29163426

Beta-carotene-rich carotenoid-protein preparation and exopolysaccharide production by Rhodotorula rubra GED8 grown with a yogurt starter culture.

PubMed

Frengova, Ginka I; Simova, Emilina D; Beshkova, Dora M

2006-01-01

The underlying method for obtaining a beta-carotene-rich carotenoid-protein preparation and exopolysaccharides is the associated cultivation of the carotenoid-synthesizing lactose-negative yeast strain Rhodotorula rubra GED8 with the yogurt starter culture (Lactobacillus bulgaricus 2-11 + Streptococcus thermophilus 15HA) in whey ultrafiltrate (45 g lactose/l) with a maximum carotenoid yield of 13.37 mg/l culture fluid on the 4.5th day. The chemical composition of the carotenoid-protein preparation has been identified. The respective carotenoid and protein content is 497.4 microg/g dry cells and 50.3% per dry weight, respectively. An important characteristic of the carotenoid composition is the high percentage (51.1%) of beta-carotene (a carotenoid pigment with the highest provitamin A activity) as compared to 12.9% and 33.7%, respectively, for the other two individual pigments--torulene and torularhodin. Exopolysaccharides (12.8 g/l) synthesized by the yeast and lactic acid cultures, identified as acid biopolymers containing 7.2% glucuronic acid, were isolated in the cell-free supernatant. Mannose, produced exclusively by the yeast, predominated in the neutral carbohydrate biopolymer component (76%). The mixed cultivation of R. rubra GED8 with the yogurt starter (L. bulgaricus 2-11 + S. thermophilus 15HA) in ultrafiltrate under conditions of intracellular production of maximum amount of carotenoids and exopolysaccharides synthesis enables combined utilization of the culture fluid from the fermentation process.

Sublethal Effects of the Neonicotinoid Insecticide Thiamethoxam on the Transcriptome of the Honey Bees (Hymenoptera: Apidae).

PubMed

Shi, Teng-Fei; Wang, Yu-Fei; Liu, Fang; Qi, Lei; Yu, Lin-Sheng

2017-12-05

Neonicotinoid insecticides are now the most widely used insecticides in the world. Previous studies have indicated that sublethal doses of neonicotinoids impair learning, memory capacity, foraging, and immunocompetence in honey bees (Apis mellifera, Linnaeus) (Hymenoptera: Apidae). Despite these, few studies have been carried out on the molecular effects of neonicotinoids. In this study, we focus on the second-generation neonicotinoid thiamethoxam, which is currently widely used in agriculture to protect crops. Using high-throughput RNA-Seq, we investigated the transcriptome profile of honey bees after subchronic exposure to 10 ppb thiamethoxam over 10 d. In total, 609 differentially expressed genes (DEGs) were identified, of which 225 were upregulated and 384 were downregulated. Several genes, including vitellogenin, CSP3, defensin-1, Mrjp1, and Cyp6as5 were selected and further validated using real-time quantitative polymerase chain reaction assays. The functions of some DEGs were identified, and Gene Ontology-enrichment analysis showed that the enriched DEGs were mainly linked to metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that thiamethoxam affected biological processes including ribosomes, the oxidative phosphorylation pathway, tyrosine metabolism pathway, pentose and glucuronate interconversions, and drug metabolism. Overall, our results provide a basis for understanding the molecular mechanisms of the complex interactions between neonicotinoid insecticides and honey bees. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sulfation pattern of fucose branches affects the anti-hyperlipidemic activities of fucosylated chondroitin sulfate.

PubMed

Wu, Nian; Zhang, Yu; Ye, Xingqian; Hu, Yaqin; Ding, Tian; Chen, Shiguo

2016-08-20

Fucosylated chondroitin sulfates (fCSs) are glycosaminoglycans extracted from sea cucumbers, consisting of chondroitin sulfate E (CSE) backbones and sulfated fucose branches. The biological properties of fCSs could be affected by the sulfation pattern of their fucose branches. In the present study, two fCSs were isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg). Their monosaccharide compositions of glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), fucose (Fuc) and sulfate were at similar molar ratio with 1.0/0.7/0.9/3.1 for fCS-Ib and 1.0/0.8/1.5/2.6 for fCS-Pg. The two fCSs have different sulfation patterns on their fucose branches, fCS-Pg with 3,4-O-disulfation while fCS-Ib with 2,4-O-disulfation. Their antihyperlipidemic effects were compared using a high-fat high-fructose diet (HFFD)-fed C57BL/6J mice model. Both fCS-Ib and fCS-Pg had significant effects on lipid profile improvement, liver protection, blood glucose diminution and hepatic glycogen synthesis. Specifically, fCS-Pg with 3,4-O-disulfation fucose branches was more effective in reduction of blood cholesterol (TC), low density lipoprotein (LDL) and atherogenic index (AI). Our results indicate that both fCSs, especially fCS-Pg, could be used as a potential anti-hyperlipidemic drug. Copyright © 2016. Published by Elsevier Ltd.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

PubMed

Shioiri, Tatsumasa; Tsuchimoto, Jun; Watanabe, Hideto; Sugiura, Nobuo

2016-06-01

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genome-Wide Identification and Expression Analysis of the UGlcAE Gene Family in Tomato.

PubMed

Ding, Xing; Li, Jinhua; Pan, Yu; Zhang, Yue; Ni, Lei; Wang, Yaling; Zhang, Xingguo

2018-05-27

The UGlcAE has the capability of interconverting UDP-d-galacturonic acid and UDP-d-glucuronic acid, and UDP-d-galacturonic acid is an activated precursor for the synthesis of pectins in plants. In this study, we identified nine UGlcAE protein-encoding genes in tomato. The nine UGlcAE genes that were distributed on eight chromosomes in tomato, and the corresponding proteins contained one or two trans-membrane domains. The phylogenetic analysis showed that SlUGlcAE genes could be divided into seven groups, designated UGlcAE1 to UGlcAE6 , of which the UGlcAE2 were classified into two groups. Expression profile analysis revealed that the SlUGlcAE genes display diverse expression patterns in various tomato tissues. Selective pressure analysis indicated that all of the amino acid sites of SlUGlcAE proteins are undergoing purifying selection. Fifteen stress-, hormone-, and development-related elements were identified in the upstream regions (0.5 kb) of these SlUGlcAE genes. Furthermore, we investigated the expression patterns of SlUGlcAE genes in response to three hormones (indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA)). We detected firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. Here, we systematically summarize the general characteristics of the SlUGlcAE genes in tomato, which could provide a basis for further function studies of tomato UGlcAE genes.

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

PubMed

Fett, W F; Wells, J M; Cescutti, P; Wijey, C

1995-02-01

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.

Saccharides enhance iron bioavailability to Southern Ocean phytoplankton

PubMed Central

Hassler, Christel S.; Nichols, Carol Mancuso; Butler, Edward C. V.; Boyd, Philip W.

2011-01-01

Iron limits primary productivity in vast regions of the ocean. Given that marine phytoplankton contribute up to 40% of global biological carbon fixation, it is important to understand what parameters control the availability of iron (iron bioavailability) to these organisms. Most studies on iron bioavailability have focused on the role of siderophores; however, eukaryotic phytoplankton do not produce or release siderophores. Here, we report on the pivotal role of saccharides—which may act like an organic ligand—in enhancing iron bioavailability to a Southern Ocean cultured diatom, a prymnesiophyte, as well as to natural populations of eukaryotic phytoplankton. Addition of a monosaccharide (>2 nM of glucuronic acid, GLU) to natural planktonic assemblages from both the polar front and subantarctic zones resulted in an increase in iron bioavailability for eukaryotic phytoplankton, relative to bacterioplankton. The enhanced iron bioavailability observed for several groups of eukaryotic phytoplankton (i.e., cultured and natural populations) using three saccharides, suggests it is a common phenomenon. Increased iron bioavailability resulted from the combination of saccharides forming highly bioavailable organic associations with iron and increasing iron solubility, mainly as colloidal iron. As saccharides are ubiquitous, present at nanomolar to micromolar concentrations, and produced by biota in surface waters, they also satisfy the prerequisites to be important constituents of the poorly defined “ligand soup,” known to weakly bind iron. Our findings point to an additional type of organic ligand, controlling iron bioavailability to eukaryotic phytoplankton—a key unknown in iron biogeochemistry. PMID:21169217

Network pharmacology-based virtual screening of natural products from Clerodendrum species for identification of novel anti-cancer therapeutics.

PubMed

Gogoi, Barbi; Gogoi, Dhrubajyoti; Silla, Yumnam; Kakoti, Bibhuti Bhushan; Bhau, Brijmohan Singh

2017-01-31

Plant-derived natural products (NPs) play a vital role in the discovery of new drug molecules and these are used for development of novel therapeutic drugs for a specific disease target. Literature review suggests that natural products possess strong inhibitory efficacy against various types of cancer cells. Clerodendrum indicum and Clerodendrum serratum are reported to have anticancer activity; therefore a study was carried out to identify selective anticancer agents from these plants species. In this report, we employed a docking weighted network pharmacological approach to understand the multi-therapeutics potentiality of C. indicum and C. serratum against various types of cancer. A library of 53 natural products derived from these plants was compiled from the literature and three dimensional space analyses were performed in order to establish the drug-likeness of the NPs library. Further, an NPs-cancer network was built based on docking. We predicted five compounds, namely apigenin 7-glucoside, hispidulin, scutellarein-7-O-beta-d-glucuronate, acteoside and verbascoside, to be potential binding therapeutics for cancer target proteins. Apigenin 7-glucoside and hispidulin were found to have maximum binding interactions (relationship) with 17 cancer drug targets in terms of docking weighted network pharmacological analysis. Hence, we used an integrative approach obtained from network pharmacology for identifying combinatorial drug actions against the cancer targets. We believe that our present study may provide important clues for finding novel drug inhibitors for cancer.

Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy

PubMed Central

Abayakoon, Palika; Lingford, James P.; Jin, Yi; Bengt, Christopher; Davies, Gideon J.; Yao, Shenggen; Goddard-Borger, Ethan D.

2018-01-01

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden–Meyerhof–Parnas-like or Entner–Doudoroff (ED)-like pathway harbor an uncharacterized gene (yihR in Escherichia coli; PpSQ1_00415 in Pseudomonas putida) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea (HsSQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. HsSQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d-galactose, d-glucose, d-glucose-6-phosphate (Glc-6-P), and d-glucuronic acid, but not d-mannose. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state. PMID:29535276

A family 30 glucurono-xylanase from Bacillus subtilis LC9: Expression, characterization and its application in Chinese bread making.

PubMed

Guo, Yalan; Gao, Zhen; Xu, Jiaxing; Chang, Siyuan; Wu, Bin; He, Bingfang

2018-05-22

A GH30-8 endoxylanase was identified from an environmental Bacillus subtilis isolate following growth selection on aspen wood glucuronoxylan. The putative endoxylanase was cloned for protein expression and characterization in the Gram-positive protease deficient protein expression host B. subtilis WB800. The extracellular activity obtained was 55 U/mL, which was 14.5-fold higher than that obtained with the native species. The apparent molecular mass of BsXyn30 was estimated as 43 kDa by SDS-PAGE. BsXyn30 showed an optimal activity at pH 7.0 and 60 °C. Recombinant BsXyn30 displayed maximum activity against aspen wood xylan, followed by beechwood xylan but showed no catalytic activity on arabinose-substituted xylans. Analysis of hydrolyzed products of beechwood xylan by thin-layer chromatography and mass spectroscopy revealed the presence of xylooligosaccharides with a single methyl-glucuronic acid residue. BsXyn30 exhibited very low activity for hydrolysis xylotetraose and xylopentaose, but had no detectable activity against xylobiose and xylotriose. Using BsXyn30 as an additive in breadmaking, a decrease in water-holding capacity, an increase in dough expansion as well as improvements in volume and specific volume of the bread were recorded. Thus, the present study provided the basis for the application of GH30 xylanase in breadmaking. Copyright © 2018 Elsevier B.V. All rights reserved.

Antioxidant and Antiproliferative Activities of Heterofucans from the Seaweed Sargassum filipendula

PubMed Central

Costa, Leandro Silva; Fidelis, Gabriel Pereira; Telles, Cinthia Beatrice Silva; Dantas-Santos, Nednaldo; Camara, Rafael Barros Gomes; Cordeiro, Sara Lima; Pereira Costa, Mariana Santana Santos; Almeida-Lima, Jailma; Melo-Silveira, Raniere Fagundes; Oliveira, Ruth Medeiros; Albuquerque, Ivan Rui Lopes; Andrade, Giulianna Paiva Viana; Rocha, Hugo Alexandre Oliveira

2011-01-01

Fucan is a term used to denominate a type of polysaccharide which contains substantial percentages of l-fucose and sulfate ester groups. We obtained five heterofucans from Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. These heterofucans are composed mainly of fucose, glucose, glucuronic acid, galactose and sulfate. These fucans did not show anticoagulant activity in PT and aPTT tests. Their antioxidant activity was evaluated using the follow tests; total antioxidant capacity, scavenging hydroxyl and superoxide radicals, reducing power and ferrous ion [Fe(II)] chelating. All heterofucans displayed considerable activity, especially SF-1.0v which showed the most significant antioxidant potential with 90.7 ascorbic acid equivalents in a total antioxidant capacity test and similar activity when compared with vitamin C in a reducing power assay. The fucan antiproliferative activity was performed with HeLa, PC3 and HepG2 cells using MTT test. In all tested conditions the heterofucans exhibited a dose-dependent effect. The strongest inhibition was observed in HeLa cells, where SF-1.0 and SF-1.5 exhibited considerable activity with an IC50 value of 15.69 and 13.83 μM, respectively. These results clearly indicate the beneficial effect of S. filipendula polysaccharides as antiproliferative and antioxidant. Further purification steps and additional studies on structural features as well as in vivo experiments are needed to test the viability of their use as therapeutic agents. PMID:21747741

Expression of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase involved in chondroitin sulfate synthesis is responsible for pulmonary metastasis.

PubMed

Mizumoto, Shuji; Watanabe, Moto; Yamada, Shuhei; Sugahara, Kazuyuki

2013-01-01

Chondroitin sulfate (CS) containing E-disaccharide units, glucuronic acid-N-acetylgalactosamine(4, 6-O-disulfate), at surfaces of tumor cells plays a key role in tumor metastasis. However, the molecular mechanism of the metastasis involving the CS chain-containing E-units is not fully understood. In this study, to clarify the role of E-units in the metastasis and to search for potential molecular targets for anticancer drugs, the isolation and characterization of Lewis lung carcinoma (LLC) cells stably downregulated by the knockdown for the gene encoding N-acetylgalactosamine 4-O-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which is responsible for the formation of E-units in CS chains, were performed. Knockdown of GalNAc4S-6ST in LLC cells resulted in a reduction in the proportion of E-units, in adhesiveness to extracellular matrix adhesion molecules and in proliferation in vitro. Furthermore, the stable downregulation of GalNAc4S-6ST expression in LLC cells markedly inhibited the colonization of the lungs by inoculated LLC cells and invasive capacity of LLC cells. These results provide clear evidence that CS chain-containing E-units and/or GalNAc4S-6ST play a crucial role in pulmonary metastasis at least through the increased adhesion and the invasive capacity of LLC cells and also provides insights into future drug targets for anticancer treatment.

Sensitive Carbohydrate Detection using Surface Enhanced Raman Tagging

PubMed Central

Vangala, Karthikeshwar; Yanney, Michael; Hsiao, Cheng-Te; Wu, Wells W.; Shen, Rong-Fong; Zou, Sige; Sygula, Andrzej; Zhang, Dongmao

2010-01-01

Glycomic analysis is an increasingly important field in biological and biomedical research as glycosylation is one of the most important protein post-translational modifications. We have developed a new technique to detect carbohydrates using surface enhanced Raman spectroscopy (SERS) by designing and applying a Rhodamine B derivative as the SERS tag. Using a reductive amination reaction, the Rhodamine-based tag (RT) was successfully conjugated to three model carbohydrates (glucose, lactose and glucuronic acid). SERS detection limits obtained with 632 nm HeNe laser were ~1 nM in concentration for all the RT-carbohydrate conjugates and ~10 fmol in total sample consumption. The dynamic range of the SERS method is about 4 orders of magnitude, spanning from 1 nM to 5 µM. Ratiometric SERS quantification using isotope-substituted SERS internal references also allows comparative quantifications of carbohydrates labeled with RT and deuterium/hydrogen substituted RT tags, respectively. In addition to enhancing the SERS detection of the tagged carbohydrates, the Rhodamine tagging facilitates fluorescence and mass spectrometric detection of carbohydrates. Current fluorescence sensitivity of RT-carbohydrates is ~ 3 nM in concentration while the mass spectrometry (MS) sensitivity is about 1 fmol that was achieved with linear ion trap electrospray ionization (ESI)-MS instrument. Potential applications that take advantage of the high SERS, fluorescence and MS sensitivity of this SERS tagging strategy are discussed for practical glycomic analysis where carbohydrates may be quantified with a fluorescence and SERS technique, and then identified with ESI-MS techniques. PMID:21082777

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

PubMed

de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

2002-09-01

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Metabolomics Study of Type 2 Diabetes Mellitus and the AntiDiabetic Effect of Berberine in Zucker Diabetic Fatty Rats Using Uplc-ESI-Hdms.

PubMed

Dong, Yu; Chen, Yi-Tao; Yang, Yuan-Xiao; Zhou, Xiao-Jie; Dai, Shi-Jie; Tong, Jun-Feng; Shou, Dan; Li, Changyu

2016-05-01

The present study aimed to evaluate the pathogenesis of type 2 diabetes mellitus (T2DM) and the anti-diabetic effect of berberine in Zucker diabetic fatty (ZDF) rats. A urinary metabolomics analysis was performed with ultra-performance liquid chromatography/electrospray ionization synapt high-definition mass spectrometry. Pattern recognition approaches were integrated to discover differentiating metabolites. We identified 29 ions (13 in negative mode and 16 in positive mode) as 'differentiating metabolites' with this metabolomic approach. A functional pathway analysis revealed that the alterations were mainly associated with glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions and sphingolipid metabolism. These results indicated that the dysfunctions of glycometabolism and lipometabolism are involved in the pathological process of T2DM. Berberine could decrease the serum levels of glycosylated hemoglobin, total cholesterol and triglyceride and increase the secretion of insulin. The urinary metabolomics analysis showed that berberine could reduce the concentrations of citric acid, tetrahydrocortisol, ribothymidine and sphinganine to a near-normal state. These results suggested that the anti-diabetic effect of berberine occurred mainly via its regulation of glycometabolism and lipometabolism and activation of adenosine 5'-monophosphate-activated protein kinase. Our work not only provides a better understanding of the anti-diabetic effect of berberine in ZDF rats but also supplies a useful database for further study in humans and for investigating the pharmacological actions of drugs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

PubMed Central

St John, Franz J.; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M.; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C.

2014-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyro­solvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

PubMed Central

2013-01-01

Background Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples. PMID:24074284

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

PubMed Central

Fett, W F; Wells, J M; Cescutti, P; Wijey, C

1995-01-01

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs. PMID:7574589

Characterization of a bioflocculant produced by the marine myxobacterium Nannocystis sp. NU-2.

PubMed

Zhang, J; Liu, Z; Wang, S; Jiang, P

2002-08-01

The marine myxobacterium strain NU-2, which can grow on high concentrations (up to 7%) of NaCl, was isolated from a salt soil sample collected from the coast of the Huanghai Sea, China. Morphological properties and 16S rDNA sequence analysis indicated that the isolate is a novel species related to the genus Nannocystis. Nannocystis sp. NU-2 produced a new kind of flocculating substance in a starch medium with a yield of 14.8 g l(-1). The NU-2 flocculant was composed of 40.3% proteins and 56.5% polysaccharides, of which glucose, mannose and glucuronic acid were the principal constituents in the relative proportions of 5:4:1. The flocculation activity of the NU-2 flocculant depends strongly on cations such as Fe(3+) and Al(3+). When a 30 mg l(-1) FeCl(3) solution is present in kaolin clay suspension, 30 mg l(-1)of the flocculant produced a high flocculating activity value of 90%, which remained unchanged over an extensive pH range (pH 2.0-13.0). The flocculant was tested for its ability to bleach dyeing liquors, and the bleaching activities were 98.2% for acid red in 100 mg l(-1)of the flocculant and 99.0% for direct emerald blue in 50 mg l(-1)of the flocculant under test conditions. Use of the flocculant to bleach basic pink and cation emerald blue liquors was not effective.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage[OPEN

PubMed Central

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; Sanhueza, Dayan; Ejsmentewicz, Troy; Sandoval-Ibañez, Omar; Parra-Rojas, Juan Pablo; Ebert, Berit; Reyes, Francisca C.

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix. PMID:28062750

In vitro biosynthesis, isolation, and identification of predominant metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (RVX-208).

PubMed

Khmelnitsky, Yuri L; Mozhaev, Vadim V; Cotterill, Ian C; Michels, Peter C; Boudjabi, Sihem; Khlebnikov, Vladimir; Madhava Reddy, M; Wagner, Gregory S; Hansen, Henrik C

2013-06-01

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (∼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the β-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Semi-synthesis of unusual chondroitin sulfate polysaccharides containing GlcA(3-O-sulfate) or GlcA(2,3-di-O-sulfate) units.

PubMed

Bedini, Emiliano; De Castro, Cristina; De Rosa, Mario; Di Nola, Annalida; Restaino, Odile F; Schiraldi, Chiara; Parrilli, Michelangelo

2012-02-13

The extraction from natural sources of Chondroitin sulfate (CS), a polysaccharide used for management of osteoarthritis, leads to very complex mixtures. The synthesis of CS by chemical modification of other polysaccharides has seldom been reported due to the intrinsic complexity that arises from fine chemical modifications of the polysaccharide structure. In view of the growing interest in expanding the application of CS to pharmacological fields other than osteoarthritis treatment, we launched a program to find new sources of known or even unprecedented CS polysaccharides. As part of this program, we report herein on an investigation of the use of a cyclic orthoester group to selectively protect the 4,6-diol of N-acetyl-galactosamine residues in chondroitin (obtained from a microbial source), thereby facilitating its transformation into CSs. In particular, three CS polysaccharides were obtained and demonstrated to possess rare or hitherto unprecedented sulfation patterns by 2D NMR spectroscopy characterization. Two of them contained disaccharide subunits characterized by glucuronic acid residues selectively sulfated at position 3 (GlcA(3S)), the biological functions of which are known but have yet to be fully investigated. This first semi-synthetic access to GlcA(3S)-containing CS could greatly expedite such studies, since it can easily furnish considerable amounts of these polysaccharides, which are usually isolated with difficulty and in very low quantity from natural sources. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

PubMed

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

In vitro and in vivo genotoxicity assessment of the dopamine receptor antagonist molindone hydrochloride.

PubMed

Krishna, Gopala; Gopalakrishnan, Gopa; Goel, Saryu

2016-05-01

Molindone hydrochloride is a dihydroindolone neuroleptic with dopamine D2 and D5 receptor antagonist activity. As an integral component of its preclinical safety evaluation, molindone hydrochloride was evaluated in a series of in vitro and in vivo genetic toxicology assays. In the bacterial reverse gene mutation assays employing four Salmonella tester strains (TA98, TA100, TA1535, and TA1537) and the E. coli tester strain WP2uvrA, molindone hydrochloride was negative in all strains, except TA100, in which it induced a positive response (up to 3-fold) in the presence of rat liver S9. With human S9, a small (2-fold), but nonreproducible, increase in revertants was observed in TA100 at the highest concentration of molindone tested (5,000 µg/plate). The mutagenicity was completely abrogated by the addition of glutathione and UDP-glucuronic acid to rat liver S9, suggesting detoxification of the mutagenic metabolite(s) by Phase II conjugation reactions, pathways commonly operational in humans. Molindone hydrochloride did not induce chromosomal aberrations in human lymphocyte cultures, did not elicit a positive response in a rat bone marrow micronucleus test for clastogencity/aneugenicity, and did not give a positive response in the rat liver comet assay for DNA damage. Collectively, the weight of evidence from these studies, combined with a large margin of safety and efficient detoxification through Phase II conjugation supports the interpretation that molindone hydrochloride does not pose a genotoxic risk to humans at the anticipated clinical dose levels. © 2016 Wiley Periodicals, Inc.

Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu.

PubMed

Chen, Ruizhan; Liu, Zhiqiang; Zhao, Jimin; Chen, Ruiping; Meng, Fanlei; Zhang, Min; Ge, Wencheng

2011-07-15

A water-soluble polysaccharide obtained from Acanthopanax senticosus leaves (ASL), was fractionated by DEAE-Sepharose fast-flow column chromatography, and purified by Sephadex G-75 gel-permeation column chromatography. The characteristics of ASP-2-1 were determined by chemical analysis, high-performance capillary electrophoresis (HPCE), high-performance gel-permeation chromatography (HPGPC). The results show that ASP-2-1 contained 89.47% carbohydrate, 7.45% uronic acid, 2.16% protein and seven kinds of monosaccharides including rhamnose, xylose, glucose, mannose, arabinose, galactose and glucuronic acid in a molar ratio of 7.45:18.63:25.15:0.93:8.35:2.79:5.69, with an average molecular weight of about 14,573Da. Furthermore, the immunobiological and antioxidant activities, in vitro, of ASP-2-1 were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ferric-reducing antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH()), superoxide radical (()O(2)(-)) and hydroxyl radical (()OH) free radical-scavenging assay, respectively. The results showed that ASP-2-1 exhibited significantly higher immunomodulatory activities against the lymphocyte proliferation in vitro, pronounced reductive power (FRAP value: 785.1μM at 0.2mg/ml), strong hydroxyl radical (89.56% at 1mg/ml) scavenging activity, moderate superoxide radicals (65.32% at 1mg/ml) and DPPH radicals (68.9% at 1mg/ml) scavenging activities. ASP-2-1 should be explored as a novel and potential natural antioxidant and immunostimulating agent for use in functional foods or medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biological characterization of lead-enhanced exopolysaccharide produced by a lead resistant Enterobacter cloacae strain P2B.

PubMed

Naik, Milind Mohan; Pandey, Anju; Dubey, Santosh Kumar

2012-09-01

A lead resistant bacterial strain isolated from effluent of lead battery manufacturing company of Goa, India has been identified as Enterobacter cloacae strain P2B based on morphological, biochemical characters, FAME profile and 16S rDNA sequence data. This bacterial strain could resist lead nitrate up to 1.6 mM. Significant increase in exopolysaccharide (EPS) production was observed as the production increased from 28 to 108 mg/L dry weight when exposed to 1.6 mM lead nitrate in Tris buffered minimal medium. Fourier-transformed infrared spectroscopy of this EPS revealed presence of several functional groups involved in metal binding viz. carboxyl, hydroxyl and amide groups along with glucuronic acid. Gas chromatography coupled with mass spectrometry analysis of alditol-acetate derivatives of acid hydrolysed EPS produced in presence of 1.6 mM lead nitrate demonstrated presence of several neutral sugars such as rhamnose, arabinose, xylose, mannose, galactose and glucose, which contribute to lead binding hydroxyl groups. Scanning electron microscope coupled with energy dispersive X-ray spectrometric analysis of this lead resistant strain exposed to 1.6 mM lead nitrate interestingly revealed mucous EPS surrounding bacterial cells which sequestered 17 % lead (as weight %) extracellularly and protected the bacterial cells from toxic effects of lead. This lead resistant strain also showed multidrug resistance. Thus these results significantly contribute to better understanding of structure, function and environmental application of lead-enhanced EPSs produced by bacteria. This lead-enhanced biopolymer can play a very important role in bioremediation of several heavy metals including lead.

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.

Effect of ultrasonic degradation of hyaluronic acid extracted from rooster comb on antioxidant and antiglycation activities.

PubMed

Hafsa, Jawhar; Chaouch, Mohamed Aymen; Charfeddine, Bassem; Rihouey, Christophe; Limem, Khalifa; Le Cerf, Didier; Rouatbi, Sonia; Majdoub, Hatem

2017-12-01

Recently, low-molecular-weight hyaluronic acid (LMWHA) has been reported to have novel features, such as free radical scavenging activities, antioxidant activities and dietary supplements. In this study, hyaluronic acid (HA) was extracted from rooster comb and LMWHA was obtained by ultrasonic degradation in order to assess their antioxidant and antiglycation activities. Molecular weight (Mw) and the content of glucuronic acid (GlcA) were used as the index for comparison of the effect of ultrasonic treatment. The effects on the structure were determined by ultraviolet (UV) spectra and Fourier transform infrared spectra (FTIR). The antioxidant activity was determined by three analytical assays (DPPH, NO and TBARS), and the inhibitory effect against glycated-BSA was also assessed. The GlcA content of HA and LMWHA was estimated at about 48.6% and 47.3%, respectively. The results demonstrate that ultrasonic irradiation decreases the Mw (1090-181 kDa) and intrinsic viscosity (1550-473 mL/g), which indicate the cleavage of the glycosidic bonds. The FTIR and UV spectra did not significantly change before and after degradation. The IC 50 value of HA and LWMHA was 1.43, 0.76 and 0.36 mg/mL and 1.20, 0.89 and 0.17 mg/mL toward DPPH, NO and TBARS, respectively. Likewise LMWHA exhibited significant inhibitory effects on the AGEs formation than HA. The results demonstrated that the ultrasonic irradiation did not damage and change the chemical structure of HA after degradation; furthermore, decreasing Mw and viscosity of LMWHA after degradation may enhance the antioxidant and antiglycation activity.

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function

PubMed Central

Montanier, Cedric; van Bueren, Alicia Lammerts; Dumon, Claire; Flint, James E.; Correia, Marcia A.; Prates, Jose A.; Firbank, Susan J.; Lewis, Richard J.; Grondin, Gilles G.; Ghinet, Mariana G.; Gloster, Tracey M.; Herve, Cecile; Knox, J. Paul; Talbot, Brian G.; Turkenburg, Johan P.; Kerovuo, Janne; Brzezinski, Ryszard; Fontes, Carlos M. G. A.; Davies, Gideon J.; Boraston, Alisdair B.; Gilbert, Harry J.

2009-01-01

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-β-d-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Δ4,5-anhydrogalaturonic acid (Δ4,5-GalA). Δ4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands. PMID:19218457

Cytochrome P450s and molecular epidemiology

NASA Astrophysics Data System (ADS)

Gonzalez, Frank J.; Gelboin, Harry V.

1993-03-01

Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

Bioconcentration, Metabolism and Excretion of Triclocarban in larval Qurt Medaka (Oryzias latipes)

PubMed Central

Schebb, Nils Helge; Flores, Ida; Kurobe, Tomofumi; Franze, Bastian; Ranganathan, Anupama; Hammock, Bruce D.; Teh, Swee

2011-01-01

The antimicrobial triclocarban (TCC) is frequently found in personal care products and commonly observed in surface waters and sediments. Due to its long environmental persistence TCC accumulates in sewage sludge. It also shows a high unintended biological activity as a potent inhibitor of the soluble epoxide hydrolase (sEH) and may be an endocrine disruptor. In this study, we investigated bioconcentration, metabolism and elimination of TCC in fish using Medaka (Oryzias latipes) as a model. Medaka larvae (7±1 days post hatching) were exposed to 63 nM (20 µg/L) TCC water for 24 hours. The LC-MS/MS analysis of water and tissues provided bioconcentration of TCC and its metabolites in fish body and rapid excretion into culture water. Results from tissue samples showed a tissue concentration of 34 µmol/kg and a log bioconcentration factor (BCF) of 2.86. These results are slightly lower than previous findings in snails and algae. A significant portion of the absorbed TCC was oxidatively metabolized by the fish to hydroxylated products. These metabolites underwent extensive phase II metabolism to yield sulfate and glucuronic acid conjugates. The most abundant metabolite in fish tissue was the glucuronide of 2’-OH-TCC. Elimination of TCC after transferring the fish to fresh water was rapid, with a half-life of 1 hour. This study shows that larval medaka metabolize TCC similarly to mammals. The rapid rate of metabolism results in a lower bioconcentration than calculated from the n-octanol/water partition coefficient of TCC. PMID:21872556

Detection of early changes in lung cell cytology by flow-systems analysis techniques. Progress report, January 1-June 30, 1979

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

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1979-08-01

This report summarizes results of ongoing experiments designed to develop automated flow-analysis assay methods for discerning damage to exfoliated respiratory tract cells in model test animals exposed by inhalation to physical and chemical agents associated with the production of synthetic fuels from oil shale and coal, the specific goal being the determination of atypical changes in exposed alveolar macrophages and epithelial cells. Animals were exposed to oil shale particles (raw and spent), silica, and polystyrene latex spheres via intratracheal instillation. Respiratory tract cells were obtained by lavaging the lungs with normal saline, stained with mithramycin for DNA content, and analyzedmore » using flow cytometric analysis methods. In addition to measuring DNA content, differential and total cell counts were made on all samples analyzed. DNA content frequency distribution histograms and cytology showed definite atypical changes resulting from exposure to shale and silica particulates when compared to the controls. To continue development of fluorescence staining methods for measuring intracellular enzymes in alveolar macrophages, studies were initiated for determining ..beta..-glucuronidase using naphthol AS-BI-..beta..-d-glucuronic acid as a fluorogenic substrate. As this new technology becomes adapted to analyzing pulmonary macrophages and epithelial cells, the measurement of physical and biochemical properties as a function of exposure to particulate and gaseous toxic agents related to the production of synthetic fuels will be increased. This analytical approach is designed to assist in the establishment of future guideline for estimating the risks to exposed humans.« less

Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

PubMed

Brox, Stephan; Seiwert, Bettina; Haase, Nora; Küster, Eberhard; Reemtsma, Thorsten

2016-01-01

The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzyme-Assisted Extraction Optimization, Characterization and Antioxidant Activity of Polysaccharides from Sea Cucumber Phyllophorus proteus.

PubMed

Qin, Yujing; Yuan, Qingxia; Zhang, Yuexing; Li, Jialu; Zhu, Xinjiao; Zhao, Lingling; Wen, Jing; Liu, Jikai; Zhao, Longyan; Zhao, Jinhua

2018-03-06

Enzyme-assisted extraction optimization, characterization and in vitro antioxidant activity of polysaccharides from sea cucumber Phyllophorus proteus (PPP) were investigated in the present study. The optimal extraction conditions with a yield of 6.44 ± 0.06% for PPP were determined as follows: Extraction time of 2.89 h, ratio of extraction solvent to raw material of 16.26 mL/g, extraction pH of 6.83, exraction temperature of 50 °C and papain concentration of 0.15%. Three purified fractions, PPP-1a, PPP-1b and PPP-2 with molecular weights of 369.60, 41.73 and 57.76 kDa, respectively, were obtained from PPP by chromatography of FPA98Cl and Sepharose CL-6B columns. Analysis of monosaccharide compositions showed that PPP-1a consisted of N -acetyl-galactosamine (GalNAc), galactose (Gal) and fucose (Fuc), PPP-1b of Fuc as the only monosaccharide and PPP-2 of glucuronic acid, GalNAc and Fuc. Sulfate contents of PPP, PPP-1a, PPP-1b and PPP-2 were determined to be 21.9%, 20.6%, 25.2% and 28.0% ( w / w ), respectively. PPP and PPP-1a had higher molecular weight and intrinsic viscosity than those of the PPP-1b and PPP-2. PPP, PPP-1a, PPP-1b and PPP-2 exhibited obvious activities of scavenging 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, superoxide radical and ABTS radical in different extent, which suggested that the polysaccharides from Phyllophorus proteus may be novel agents having potential value for antioxidation.

Influence of the collection tube on metabolomic changes in serum and plasma.

PubMed

López-Bascón, M A; Priego-Capote, F; Peralbo-Molina, A; Calderón-Santiago, M; Luque de Castro, M D

2016-04-01

Major threats in metabolomics clinical research are biases in sampling and preparation of biological samples. Bias in sample collection is a frequently forgotten aspect responsible for uncontrolled errors in metabolomics analysis. There is a great diversity of blood collection tubes for sampling serum or plasma, which are widely used in metabolomics analysis. Most of the existing studies dealing with the influence of blood collection on metabolomics analysis have been restricted to comparison between plasma and serum. However, polymeric gel tubes, which are frequently proposed to accelerate the separation of serum and plasma, have not been studied. In the present research, samples of serum or plasma collected in polymeric gel tubes were compared with those taken in conventional tubes from a metabolomics perspective using an untargeted GC-TOF/MS approach. The main differences between serum and plasma collected in conventional tubes affected to critical pathways such as the citric acid cycle, metabolism of amino acids, fructose and mannose metabolism and that of glycerolipids, and pentose and glucuronate interconversion. On the other hand, the polymeric gel only promoted differences at the metabolite level in serum since no critical differences were observed between plasma collected with EDTA tubes and polymeric gel tubes. Thus, the main changes were attributable to serum collected in gel and affected to the metabolism of amino acids such as alanine, proline and threonine, the glycerolipids metabolism, and two primary metabolites such as aconitic acid and lactic acid. Therefore, these metabolite changes should be taken into account in planning an experimental protocol for metabolomics analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

PubMed Central

Sattelle, Benedict M.; Shakeri, Javad; Roberts, Ian S.; Almond, Andrew

2010-01-01

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without 15N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties. PMID:20022001

A characteristic chondroitin sulfate trisaccharide unit with a sulfated fucose branch exhibits neurite outgrowth-promoting activity: Novel biological roles of fucosylated chondroitin sulfates isolated from the sea cucumber Apostichopus japonicus.

PubMed

Shida, Miharu; Mikami, Tadahisa; Tamura, Jun-Ichi; Kitagawa, Hiroshi

2017-06-03

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). CS chains are found throughout the pericellular and extracellular spaces and contribute to the formation of functional microenvironments for numerous biological events. However, their structure-function relations remain to be fully characterized. Here, a fucosylated CS (FCS) was isolated from the body wall of the sea cucumber Apostichopus japonicus. Its promotional effects on neurite outgrowth were assessed by using isolated polysaccharides and the chemically synthesized FCS trisaccharide β-D-GalNAc(4,6-O-disulfate) (1-4)[α-l-fucose (2,4-O-disulfate) (1-3)]-β-D-GlcA. FCS polysaccharides contained the E-type disaccharide unit GlcA-GalNAc(4,6-O-disulfate) as a CS major backbone structure and carried distinct sulfated fucose branches. Despite their relatively lower abundance of E unit, FCS polysaccharides exhibited neurite outgrowth-promoting activity comparable to squid cartilage-derived CS-E polysaccharides, which are characterized by their predominant E units, suggesting potential roles of the fucose branch in neurite outgrowth. Indeed, the chemically synthesized FCS trisaccharide was as effective as CS-E tetrasaccharide in stimulating neurite elongation in vitro. In conclusion, FCS trisaccharide units with 2,4-O-disulfated fucose branches may provide new insights into understanding the structure-function relations of CS chains. Copyright © 2017 Elsevier Inc. All rights reserved.

Large scale analysis of the mutational landscape in β-glucuronidase: A major player of mucopolysaccharidosis type VII.

PubMed

Khan, Faez Iqbal; Shahbaaz, Mohd; Bisetty, Krishna; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Md Imtaiyaz

2016-01-15

The lysosomal storage disorders are a group of 50 unique inherited diseases characterized by unseemly lipid storage in lysosomes. These malfunctions arise due to genetic mutations that result in deficiency or reduced activities of the lysosomal enzymes, which are responsible for catabolism of biological macromolecules. Sly syndrome or mucopolysaccharidosis type VII is a lysosomal storage disorder associated with the deficiency of β-glucuronidase (EC 3.2.1.31) that catalyzes the hydrolysis of β-D-glucuronic acid residues from the non-reducing terminal of glycosaminoglycan. The effects of the disease causing mutations on the framework of the sequences and structure of β-glucuronidase (GUSBp) were analyzed utilizing a variety of bioinformatic tools. These analyses showed that 211 mutations may result in alteration of the biological activity of GUSBp, including previously experimentally validated mutations. Finally, we refined 90 disease causing mutations, which presumably cause a significant impact on the structure, function, and stability of GUSBp. Stability analyses showed that mutations p.Phe208Pro, p.Phe539Gly, p.Leu622Gly, p.Ile499Gly and p.Ile586Gly caused the highest impact on GUSBp stability and function because of destabilization of the protein structure. Furthermore, structures of wild type and mutant GUSBp were subjected to molecular dynamics simulation to examine the relative structural behaviors in the explicit conditions of water. In a broader view, the use of in silico approaches provided a useful understanding of the effect of single point mutations on the structure-function relationship of GUSBp. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn's Disease Patients and Unaffected First-Degree Relatives.

PubMed

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn's disease.

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.

PubMed

Ternes, T A; Kreckel, P; Mueller, J

1999-01-12

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.

Perineuronal nets labeled by monoclonal antibody VC1.1 ensheath interneurons expressing parvalbumin and calbindin in the rat amygdala

PubMed Central

McDonald, Alexander J.; Hamilton, Patricia G.; Barnstable, Colin J.

2018-01-01

Perineuronal nets (PNNs) are specialized condensations of extracellular matrix that ensheath particular neuronal subpopulations in the brain and spinal cord. PNNs regulate synaptic plasticity, including the encoding of fear memories by the amygdala. The present immunohistochemical investigation studied PNN structure and distribution, as well as the neurochemistry of their ensheathed neurons, in the rat amygdala using monoclonal antibody VC1.1, which recognizes a glucuronic acid 3-sulfate glycan associated with PNNs in the cerebral cortex. VC1.1+ PNNs surrounded the cell bodies and dendrites of a subset of nonpyramidal neurons in cortex-like portions of the amygdala (basolateral amygdalar complex, cortical nuclei, nucleus of the lateral olfactory tract, and amygdalohippocampal region). There was also significant neuropilar VC1.1 immunoreactivity whose density varied in different amygdalar nuclei. Cell counts in the basolateral nucleus revealed that virtually all neurons ensheathed by VC1.1+ PNNs were parvalbumin-positive (PV+) interneurons, and these VC1.1+/PV+ cells constituted 60% of all PV+ interneurons, including all of the larger PV+ neurons. Approximately 70% of VC1.1+ neurons were calbindin-positive (CB+), and these VC1.1+/CB+ cells constituted about 40% of all CB+ neurons. Colocalization of VC1.1 with Vicia villosa agglutinin (VVA) binding, which stains terminal N-acetylgalactosamines, revealed that VC1.1+ PNNs were largely a subset of VVA+ PNNs. This investigation provides baseline data regarding PNNs in the rat which should be useful for future studies of their function in this species. PMID:29094304

Structure Characterization of Honey-Processed Astragalus Polysaccharides and Its Anti-Inflammatory Activity In Vitro.

PubMed

Liao, Jingzhu; Li, Chanyi; Huang, Jing; Liu, Wuping; Chen, Hongce; Liao, Shuangye; Chen, Hongyuan; Rui, Wen

2018-01-15

Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which strengthens the tonic effect. Astragalus polysaccharide (APS), perform its immunomodulatory effects by relying on the tonic effect of Radix Astragalus , therefore, the improved pharmacological activity of honey-processed Astragalus polysaccharide (HAPS) might be due to structural changes during processing. The molecular weights of HAPS and APS were 1,695,788 Da, 2,047,756 Da, respectively, as determined by high performance gel filtration chromatography combined with evaporative light scattering detection (HPGFC-ELSD). The monosaccharide composition was determined by ultra-performance liquid chromatogram quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) after pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The results showed that the essential components were mannose, glucose, xylose, arabinose, glucuronic acid and rhamnose, is molar ratios of 0.06:28.34:0.58:0.24:0.33:0.21 and 0.27:12.83:1.63:0.71:1.04:0.56, respectively. FT-IR and NMR analysis of HAPS results showed the presence of uronic acid and acetyl groups. The anti-inflammatory activities of HAPS were more effective than those of APS according to the NO contents and the expression of IFN-γ, IL-1β, IL-22 and TNF-α in lipopolysaccharide (LPS)-induced RAW264.7 cells. This findings suggest that the anti-inflammatory and bioactivity improvement might be associated with molecular structure changes, bearing on the potential immunomodulatory action.

Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls1[OPEN

PubMed Central

Gorshkova, Tatyana; Mokshina, Natalia; Chernova, Tatyana; Ibragimova, Nadezhda; Salnikov, Vadim; Mikshina, Polina; Tryfona, Theodora; Banasiak, Alicja; Immerzeel, Peter; Dupree, Paul; Mellerowicz, Ewa J.

2015-01-01

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood. PMID:26378099

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

PubMed Central

Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

2014-01-01

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. PMID:24889696

Aspen Tension Wood Fibers Contain β-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

PubMed

Gorshkova, Tatyana; Mokshina, Natalia; Chernova, Tatyana; Ibragimova, Nadezhda; Salnikov, Vadim; Mikshina, Polina; Tryfona, Theodora; Banasiak, Alicja; Immerzeel, Peter; Dupree, Paul; Mellerowicz, Ewa J

2015-11-01

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood. © 2015 American Society of Plant Biologists. All Rights Reserved.

Identification of a sulfate metabolite of PCB 11 in human serum

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M.; Hornbuckle, Keri C.; Thorne, Peter S.; Robertson, Larry W.; Duffel, Michael W.

2016-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20 % of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. PMID:27816204

Identification of a sulfate metabolite of PCB 11 in human serum.

PubMed

Grimm, Fabian A; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M; Hornbuckle, Keri C; Thorne, Peter S; Robertson, Larry W; Duffel, Michael W

2017-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization and Biological Activity of Taishan Pinus massoniana Pollen Polysaccharide In Vitro

PubMed Central

Yang, Shifa; Wei, Kai; Jia, Fengjuan; Zhao, Xue; Cui, Guolin; Guo, Fanxia; Zhu, Ruiliang

2015-01-01

Taishan Pinus massoniana pollen polysaccharide (TPPPS) improves cellular and humoral immune responses of animals and is a novel potential immunomodulator. However, the components of TPPPS have not been recognized. To investigate the composition of TPPPS, crude polysaccharide was obtained from Taishan P. massoniana pollen through water extraction and ethanol precipitation. Three homogeneous polysaccharide fractions (TPPPS1, TPPPS2, and TPPPS3) were purified from TPPPS by DEAE-cellulose column chromatography. The average molecular weights of the three polysaccharides were 56, 25, and 128 kDa, respectively. Results of high-performance liquid chromatography (HPLC) showed that TPPPS comprised mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The biological activity assays showed that TPPPS2 and TPPPS3 significantly promoted spleen lymphocyte proliferation, and that TPPPS3 showed better effect than TPPPS2. TPPPS3 enhanced the secretion of cytokine IL-2 and TNF, whereas TPPPS2 mainly elevated IL-2 secretion. By contrast, TPPPS1 exhibited other effects, and it induced the highest amount of NO production, thereby indicating that TPPPS1 had the best antioxidant activity. TPPPS3 at 50 μg/mL significantly inhibited the proliferation of subgroup B Avian Leukosis virus (ALV-B) through virus adsorption interference in vitro. Results indicated that TPPPS comprised three main components, among which, TPPPS1 mainly showed antioxidant effects, whereas TPPPS2 and TPPPS3 played key roles in immunomodulation, especially TPPPS3. Further studies on the use of a reasonable proportion of TPPPS1-3 may facilitate the development of an effective immunomodulator. PMID:25782009

Single Stage Tandem Mass Spectrometry Assignment of the C-5 Uronic Acid Stereochemistry in Heparan Sulfate Tetrasaccharides using Electron Detachment Dissociation

NASA Astrophysics Data System (ADS)

Agyekum, Isaac; Zong, Chengli; Boons, Geert-Jan; Amster, I. Jonathan

2017-09-01

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B3 ´ -CO2, which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2- O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2- O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA. [Figure not available: see fulltext.

Polymorphisms of genes involved in polycyclic aromatic hydrocarbons’ biotransformation and atherosclerosis

PubMed Central

Marinković, Natalija; Pašalić, Daria; Potočki, Slavica

2013-01-01

Polycyclic aromatic hydrocarbons (PAHs) are among the most prevalent environmental pollutants and result from the incomplete combustion of hydrocarbons (coal and gasoline, fossil fuel combustion, byproducts of industrial processing, natural emission, cigarette smoking, etc.). The first phase of xenobiotic biotransformation in the PAH metabolism includes activities of cytochrome P450 from the CYP1 family and microsomal epoxide hydrolase. The products of this biotransformation are reactive oxygen species that are transformed in the second phase through the formation of conjugates with glutathione, glucuronate or sulphates. PAH exposure may lead to PAH-DNA adduct formation or induce an inflammatory atherosclerotic plaque phenotype. Several genetic polymorphisms of genes encoded for enzymes involved in PAH biotransformation have been proven to lead to the development of diseases. Enzyme CYP P450 1A1, which is encoded by the CYP1A1 gene, is vital in the monooxygenation of lipofilic substrates, while GSTM1 and GSTT1 are the most abundant isophorms that conjugate and neutralize oxygen products. Some single nucleotide polymorphisms of the CYP1A1 gene as well as the deletion polymorphisms of GSTT1 and GSTM1 may alter the final specific cellular inflammatory respond. Occupational exposure or conditions from the living environment can contribute to the production of PAH metabolites with adverse effects on human health. The aim of this study was to obtain data on biotransformation and atherosclerosis, as well as data on the gene polymorphisms involved in biotransformation, in order to better study gene expression and further elucidate the interaction between genes and the environment. PMID:24266295

Impact of storage conditions on the urinary metabolomics fingerprint.

PubMed

Laparre, Jérôme; Kaabia, Zied; Mooney, Mark; Buckley, Tom; Sherry, Mark; Le Bizec, Bruno; Dervilly-Pinel, Gaud

2017-01-25

Urine stability during storage is essential in metabolomics to avoid misleading conclusions or erroneous interpretations. Facing the lack of comprehensive studies on urine metabolome stability, the present work performed a follow-up of potential modifications in urinary chemical profile using LC-HRMS on the basis of two parameters: the storage temperature (+4 °C, -20 °C, -80 °C and freeze-dried stored at -80 °C) and the storage duration (5-144 days). Both HILIC and RP chromatographies have been implemented in order to globally monitor the urinary metabolome. Using an original data processing associated to univariate and multivariate data analysis, our study confirms that chemical profiles of urine samples stored at +4 °C are very rapidly modified, as observed for instance for compounds such as:N-acetyl Glycine, Adenosine, 4-Amino benzoic acid, N-Amino diglycine, creatine, glucuronic acid, 3-hydroxy-benzoic acid, pyridoxal, l-pyroglutamic acid, shikimic acid, succinic acid, thymidine, trigonelline and valeryl-carnitine, while it also demonstrates that urine samples stored at -20 °C exhibit a global stability over a long period with no major modifications compared to -80 °C condition. This study is the first to investigate long term stability of urine samples and report potential modifications in the urinary metabolome, using both targeted approach monitoring individually a large number (n > 200) of urinary metabolites and an untargeted strategy enabling assessing for global impact of storage conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 Is Necessary for Normal Endochondral Ossification and Aggrecan Metabolism*

PubMed Central

Sato, Takashi; Kudo, Takashi; Ikehara, Yuzuru; Ogawa, Hiroyasu; Hirano, Tomoko; Kiyohara, Katsue; Hagiwara, Kozue; Togayachi, Akira; Ema, Masatsugu; Takahashi, Satoru; Kimata, Koji; Watanabe, Hideto; Narimatsu, Hisashi

2011-01-01

Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1−/− mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1−/− cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152–4161). Histologically, the growth plate of Csgalnact1−/− mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1−/− cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan. PMID:21148564

Ion chromatography characterization of polysaccharides in ancient wall paintings.

PubMed

Colombin, Maria Perla; Ceccarini, Alessio; Carmignani, Alessia

2002-08-30

An analytical procedure for the characterisation of polysaccharides and the identification of plant gums in old polychrome samples is described. The procedure is based on hydrolysis with 2 M trifluoroacetic acid assisted by microwaves (20 min, 120 degrees C, 500 W), clean-up of the hydrolysate by an ion-exchange resin, and analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Using this method the hydrolysis time was reduced to 20 min and the chromatographic separation of seven monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose) and two uronic acids (galacturonic and glucuronic) was achieved in 40 min. The whole analytical procedure allows sugar determination in plant gums at picomole levels, with an average recovery of 72% with an RSD of 8% as tested on arabic gum. The analytical procedure was tested with several raw gums, watercolour samples and reference painting specimens prepared according to old recipes at the Opificio delle Pietre Dure of Florence (Italian Ministry of Cultural Heritage, Italy). All the data collected expressed in relative sugar percentage contents were submitted to principal components analysis for gum identification: five groups were spatially separated and this enabled the identification of arabic, tragacanth, karaya, cherry+ghatty, and guar+locust bean gum. Wall painting samples from Macedonian tombs (Greece) of the 4th-3rd Centuries B.C., processed by the suggested method, showed the presence of a complex paint media mainly consisting of tragacanth and fruit tree gums. Moreover, starch had probably been added to plaster as highlighted by the presence of a huge amount of glucose.

CD, MCD and VTVH MCD Studies of Biferrous and Mixed-Valent myo-Inositol Oxygenase: Insights into Substrate Activation of O2 Reactivity

PubMed Central

Snyder, Rae Ana; Bell, Caleb B.; Diao, Yinghui; Krebs, Carsten; Bollinger, J. Martin; Solomon, Edward I.

2013-01-01

Myo-inositol oxygenase (MIOX) catalyzes the 4e− oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10,000 cm−1, split by ~2,000 cm−1, characteristic of 6 coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe(II)Fe(III) state, MIOX shows two LF transitions in the ~10,000 cm−1 region, again implying a coordinatively saturated Fe(II) site. Upon MI binding, these split in energy to 5,200 cm−1 and 11,200 cm−1, showing that MI binding causes the Fe(II) to become coordinately unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe(II)Fe(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe(III)-μ-OH bond and strengthening the Fe(II)-μ-OH bond. This perturbation results in the release of a coordinated water from the Fe(II) that enables its O2 activation. PMID:24066857

The physostigmine depolarization potentiating effect of salicylate in frog skeletal muscle.

PubMed

Varga, E; Kovács, L; Szücs, G; Illés, B

1975-01-01

1) The frog's sartorius muscle was depolarized depending on the degree of concentration 2--4 times more intensely by physostigmine salicylate than by physostigmine sulphate. 2) In normal Ringer's solution, 1 mM physostigmine salicylate decreased the sensitivity of the membrane to potassium depolarization by about 90%. Under similar experimental conditions, physostigmine sulphate and Na salicylate, respectively, decrease the sensitivity of the membrane to potassium depolarization by about 30%. 3) The difference manifested in the depolarizing effect of salicylate and other physostigmine salts (chloride, sulphate, phosphate, formiate, acetate, monochloracetate, benzoate and para-oxy-benzoate) is expressed already at 1 mM concentration (about 10-fold), if the muscle had been equilibrated in chloride-free glucuronate or sulphate milieu. 4) The depolarization develops slowly. It takes 30--60 minutes for the new steady state to develop even in the superficial sartorius fibres. If depolarization has reached its maximum on an average 100 mV, the membrane potential remains unchanged for hours. 5) Depolarization ensues at an unchanged degree in the presence of Na-free (choline) Ringer as well as in the presence of 2X10(-8) g/ml tetrodotoxin; therefore, it is not a Na-dependent process. 6) Under the influence of 1 mM physostigmine salicylate the membrane's resistance to the inward potassium current increased about twofold, while the increase was only 15% to the outward potassium current. It is assumed that the salicylate anion is characteristically capable of potentiating the decreasing effect of physostigmine on potassium permeability, though the role of the metabolic effect of salicylate cannot be excluded.

Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

PubMed Central

Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

2014-01-01

Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

A urinary metabonomics analysis of long-term effect of acetochlor exposure on rats by ultra-performance liquid chromatography/mass spectrometry.

PubMed

Li, Longxue; Wang, Maoqing; Chen, Shuhong; Zhao, Wei; Zhao, Yue; Wang, Xu; Zhang, Yang

2016-03-01

The study was to assess the long-term toxic effects of acetochlor on rats. Two different doses (42.96 and 107.4 mg/kg body weight/day) of acetochlor were administered to Wistar rats through their food for over 24 weeks. Rat urine samples were collected at two time-points for the measurements of the metabonomics profiles with ultra-performance liquid chromatography-mass spectrometry (UPLC-MSMS). The results of clinical chemistry and histopathology suggested that long-term use of acetochlor in rats caused liver and kidney damage, and dysfunction of antioxidant system. The urinary metabonomics analysis indicated that the high and low-dose exposure of acetochlor could cause alterations of these metabonomics in urine in the rat. Significant changes of the levels of hippuric acid (0.403-fold decrease), citric acid (0.430-fold decrease), pantothenic acid (0.486-fold decrease), uracil (0.419-fold decrease), β-Alanine (0.325-fold decrease), nonanedioic acid (0.445-fold decrease), L-tyrosine (0.410-fold decrease), D-glucuronic acid (8.389-fold increase) and 2-ethyl-6-methyl-N-methyl-2-chloro-acetanilide in urine were observed. In addition, it may interfere with the fatty acid synthesis, the pyrimidine degradation and pantothenate biosynthesis. The level of 2-ethyl-6-methyl-N-methyl-2-chloro-acetanilide is detected in all treated groups which is not found in the control groups, indicating which can be used as an early, sensitive marker of acetochlor exposure in rat. This study illustrates the important utility of metabonomics approaches to understand the toxicity of long-term exposure of acetochlor. Copyright © 2015. Published by Elsevier Inc.

The lipoprotein LpqW is essential for the mannosylation of periplasmic glycolipids in Corynebacteria.

PubMed

Rainczuk, Arek K; Yamaryo-Botte, Yoshiki; Brammananth, Rajini; Stinear, Timothy P; Seemann, Torsten; Coppel, Ross L; McConville, Malcolm J; Crellin, Paul K

2012-12-14

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1-4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis*

PubMed Central

Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Karousou, Eugenia; Viola, Manuela; Bartolini, Barbara; Hascall, Vincent C.; Tammi, Markku; De Luca, Giancarlo; Passi, Alberto

2012-01-01

Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies. PMID:22887999

Antibiofilm Activity of an Exopolysaccharide from Marine Bacterium Vibrio sp. QY101

PubMed Central

Han, Feng; Duan, Gaofei; Lu, Xinzhi; Gu, Yuchao; Yu, Wengong

2011-01-01

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides. PMID:21490923

Novel hydroxyamides and amides containing D-glucopyranose or D-fructose units: Biological assays in MCF-7 and MDST8 cell lines.

PubMed

Carreiro, Elisabete P; Costa, Ana R; Cordeiro, Maria M; Martins, Rute; Pires, Tiago O; Saraiva, Mafalda; Antunes, Célia M; Burke, Anthony J

2016-02-01

A novel library of 15 compounds, hydroxyamides and amides containing a β-D-glucopyranose (D-Gluc) or a β-D-fructose (D-Fruc) units was designed and synthesized for antiproliferative assays in breast (MCF-7) and colon (MDST8) cancer cell lines. Twelve of them were hydroxyamides and were successfully synthesized from β-D-glucuronic acid (D-GluA). Six of these hydroxyamides which were acetylated hydroxy-β-D-glucopyranuronamide 2a-2f (1st Family) and the other six were their respective isomers, that is, hydroxy-β-D-fructuronamide 3a-3f (2nd Family), obtained by acid-base catalyzed isomerization. These compounds have the general structure, D-Gluc-C=ONH-CHR-(CH2)n-OH and D-Fruc-C=ONH-CHR-(CH2)n-OH, where R=an aromatic, alkyl or a hydrogen substituent, with n=0 or 1. Eight of these contained a chiral aminoalcohol group. Three compounds were amides containing a D-glucopyranose unit (3rd Family). SAR studies were conducted with these compounds. Antiproliferative studies showed that compound 4a, the bromo-amide containing the β-D-glucopyranose ring, potently inhibits the proliferation of the MDST8 cells. Five compounds (2e, 2f, 3d, 3e, and 3f) were shown to potently selectively inhibit the proliferation of the MCF-7 cells. Compound 4b was the only one showing inhibition in both cell lines. In general, the more active compounds were the amides and hydroxyamides containing the β-D-fructose moiety, and containing an alkyl group or hydrogen. Half-inhibitory concentrations (IC50) of between 0.01 and 10 μM, were observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Timeline and bibliography of early isolations of plant metabolites (1770-1820) and their impact to pharmacy: A critical study.

PubMed

Drobnik, Jacek; Drobnik, Elżbieta

2016-10-27

Plant metabolites became objects of chemical research for pharmaceutical and medicinal reasons. The period of pure plant substances in chemistry started 1770 with isolation of tartaric acid from wine (wine in pharmacy is a plant-derived preparation). Carl Scheele isolated 7 plant acids: tartaric, benzoic, citric, oxalic, malic, glucuronic and gallic. The era of alkaloids started 1803 when narcotine was discovered and published. Since that time, pharmacists and toxicologists began to recognize alkaloids (or substances regarded as such) as highly active principles responsible for their powerful, thus easily-observed actions to humans and test animals. By 1820 when solanine was isolated, pharmaceutical chemistry has dealt with increasing number of natural plant-derived substances as organic medicines or reagents. The following historical facts have been unknown: Scheele's tartaric acid was introduced officially as a medicinal substance as early as in 1775, benzoic, citric and oxalic acids became official by the end of the 18th century. Morphine was effectively published in 1806 (not 1804), hence the first alkaloid known in isolated state is narcotine (published 1803, official since 1827). Morphine became official in French pharmacy in 1818. And, 1814 is the year when 2 first toxicological accounts on plant-derived acids (oxalic and tartaric) appeared. Practical use in therapy, sometimes soon after discovery, inspired practical pharmacy and stimulated the progress of toxicology. We studied the earliest 50years of plant metabolites isolations era. A revised bibliography and a timeline chart for 24 plant substances from this period is provided. Plants from original publications are taxonomically identified. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt.

PubMed

Socas-Rodríguez, Bárbara; Lanková, Darina; Urbancová, Kateřina; Krtková, Veronika; Hernández-Borges, Javier; Rodríguez-Delgado, Miguel Ángel; Pulkrabová, Jana; Hajšlová, Jana

2017-07-01

Within this study, a new method enabling monitoring of various estrogenic substances potentially occurring in milk and dairy products was proposed. Groups of compounds fairly differing in physico-chemical properties and biological activity were analyzed: four natural estrogens, four synthetic estrogens, five mycoestrogens, and nine phytoestrogens. Since they may pass into milk mainly in glucuronated and sulfated forms, an enzymatic hydrolysis was involved prior to the extraction based on the QuEChERS methodology. For the purification of the organic extract, a dispersive solid-phase extraction (d-SPE) with sorbent C18 was applied. The final analysis was performed by ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). Method recovery ranged from 70 to 120% with a relative standard deviation (RSD) value lower than 20% and limits of quantification (LOQs) in the range of 0.02-0.60 μg/L (0.2-6.0 μg/kg dry weight) and 0.02-0.90 μg/kg (0.2-6.0 μg/kg dry weight) for milk and yogurt, respectively. The new procedure was applied for the investigation of estrogenic compounds in 11 milk samples and 13 yogurt samples from a Czech retail market. Mainly phytoestrogens were found in the studied samples. The most abundant compounds were equol and enterolactone representing 40-90% of all estrogens. The total content of phytoestrogens (free and bound) was in the range of 149-3870 μg/kg dry weight. This amount is approximately 20 times higher compared to non-bound estrogens.

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids*

PubMed Central

Sanematsu, Keisuke; Kusakabe, Yuko; Shigemura, Noriatsu; Hirokawa, Takatsugu; Nakamura, Seiji; Imoto, Toshiaki; Ninomiya, Yuzo

2014-01-01

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca2+]i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3. PMID:25056955

Association between UGT2B7 gene polymorphisms and fentanyl sensitivity in patients undergoing painful orthognathic surgery

PubMed Central

Muraoka, Wataru; Nishizawa, Daisuke; Fukuda, Kenichi; Kasai, Shinya; Hasegawa, Junko; Wajima, Koichi; Nakagawa, Taneaki

2016-01-01

Background Fentanyl is often used instead of morphine for the treatment of pain because it has fewer side effects. The metabolism of morphine by glucuronidation is known to be influenced by polymorphisms of the UGT2B7 gene. Some metabolic products of fentanyl are reportedly metabolized by glucuronate conjugation. The genes that are involved in the metabolic pathway of fentanyl may also influence fentanyl sensitivity. We analyzed associations between fentanyl sensitivity and polymorphisms of the UGT2B7 gene to clarify the hereditary determinants of individual differences in fentanyl sensitivity. Results This study examined whether single-nucleotide polymorphisms (SNPs) of the UGT2B7 gene affect cold pain sensitivity and the analgesic effects of fentanyl, evaluated by a standardized pain test and fentanyl requirements in healthy Japanese subjects who underwent uniform surgical procedures. The rs7439366 SNP of UGT2B7 is reportedly associated with the metabolism and analgesic effects of morphine. We found that this SNP is also associated with the analgesic effects of fentanyl in the cold pressor-induced pain test. It suggested that the C allele of the rs7439366 SNP may enhance analgesic efficacy. Two SNPs of UGT2B7, rs4587017 and rs1002849, were also found to be novel SNPs that may influence the analgesic effects of fentanyl in the cold pressor-induced pain test. Conclusions Fentanyl sensitivity for cold pressor-induced pain was associated with the rs7439366, rs4587017, and rs1002849 SNPs of the UGT2B7 gene. Our findings may provide valuable information for achieving satisfactory pain control and open to new avenues for personalized pain treatment. PMID:28256933

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

PubMed

Sugiura, Nobuo; Clausen, Thomas Mandel; Shioiri, Tatsumasa; Gustavsson, Tobias; Watanabe, Hideto; Salanti, Ali

2016-12-01

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans*†

PubMed Central

Tracy, Breca S.; Avci, Fikri Y.; Linhardt, Robert J.; DeAngelis, Paul L.

2014-01-01

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (~102–4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes. PMID:17099217

Tetrahydrocannabinols in clinical and forensic toxicology.

PubMed

Kochanowski, Maciej; Kała, Maria

2005-01-01

Cannabinoids are the natural constituents of marihuana (cannabis). The main of them are delta9-tetrahydrocannabinol (9THC)--psychoactive agent, cannabinol (CBN) and cannabidiol (CBD). Cannabis is administered either by smoking or orally. 9THC potency and duration of action as well as its and two of its major metabolites concentrations in organism highly depend on the route of administration. A single active dose of 9THC is estimated on 520 mg. 9THC is rapidly metabolised. It is hydroxylated to an active metabolite, I1 -hydroxy-delta9-tetrahydro-cannabinol (11-OH-THC), then oxidised to an inactive 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCCOOH), which is conjugated with glucuronic acid and predominantly excreted in the urine. The maximum psychological effect persists for 4-6 h after administration despite of very low 9THC blood concentrations. 9THC plasma concentration declined to values of 2-3 ng/ml during 3-4 h after smoking. Such a low concentration of the active compound in human organism create a demand for use of sensitive analytical methods for detection and determination of 9THC and its metabolites. The most effective techniques for 9THC and related compounds determination in biological material are chromatographic ones (gas and liquid) with mass spectrometric detection and different ionization modes. 9THC and its two metabolites (11-OH-THC and THCCOOH) are present in blood and hair, 9THC in saliva, and THCCOOH in urine. 9THC and related compounds are determined in autopsy material, although deaths by overdose of cannabis are exceptionally rare. Fatalities happen most often after intravenous injection of hashish oil. 9THC and its metabolites determination in different biological materials gives the basis for a wide interpretation of analytical results for clinical and forensic toxicology purposes.

The Inhibitory Effect of Ciprofloxacin on the β-Glucuronidase-mediated Deconjugation of the Irinotecan Metabolite SN-38-G.

PubMed

Kodawara, Takaaki; Higashi, Takashi; Negoro, Yutaka; Kamitani, Yukio; Igarashi, Toshiaki; Watanabe, Kyohei; Tsukamoto, Hitoshi; Yano, Ryoichi; Masada, Mikio; Iwasaki, Hiromichi; Nakamura, Toshiaki

2016-05-01

The enterohepatic recycling of a drug consists of its biliary excretion and intestinal reabsorption, which is sometimes accompanied by hepatic conjugation and intestinal deconjugation reactions. β-Glucuronidase, an intestinal bacteria-produced enzyme, can break the bond between a biliary excreted drug and glucuronic acid. Antibiotics such as ciprofloxacin can reduce the enterohepatic recycling of glucuronide-conjugated drugs. In this study, we established an in vitro system to evaluate the β-glucuronidase-mediated deconjugation of the irinotecan metabolite SN-38-G to its active SN-38 form and the effect of ciprofloxacin thereon. SN-38 formation increased in a time-dependent manner from 5 to 30 min. in the presence of β-glucuronidase. Ciprofloxacin and phenolphthalein-β-D-glucuronide (PhePG), a typical β-glucuronidase substrate, significantly decreased SN-38-G deconjugation and, hence SN-38 formation. Similarly, the antibiotics enoxacin and gatifloxacin significantly inhibited the conversion of SN-38-G to SN-38, which was not observed for levofloxacin, streptomycin, ampicillin and amoxicillin/clavulanate. Ciprofloxacin showed a dose-dependent inhibitory effect on the β-glucuronidase-mediated conversion of SN-38-G to SN-38 with a half-maximal inhibitory concentration (IC50 ) value of 83.8 μM. PhePG and ciprofloxacin afforded the inhibition in a competitive and non-competitive manner, respectively. These findings suggest that the reduction in the serum SN-38 concentration following co-administration of ciprofloxacin during irinotecan treatment is due, at least partly, to the decreased enterohepatic circulation of SN-38 through the non-competitive inhibition of intestinal β-glucuronidase-mediated SN-38-G deconjugation. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn’s Disease Patients and Unaffected First-Degree Relatives

PubMed Central

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn’s disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn’s disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn’s disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn’s disease. PMID:26824357

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.

PubMed

Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

2014-08-01

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan-cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. © 2014 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Liquid crystal-type assembly of native cellulose-glucuronoxylans extracted from plant cell wall.

PubMed

Reis, D; Vian, B; Chanzy, H; Roland, J C

1991-01-01

In numerous plant cell walls, the cellulose microfibrils are arranged in a helicoidal pattern which has been considered as an analog to a cholesteric order. Here, we report on the spontaneous helicoidal organization which occurs in acellular conditions from aqueous suspensions of cellulose. The cellulosic mucilage of mature seeds of quince (Cydonia oblonga L) was studied both in situ (pre-release mucilage) and after water extraction and in in vitro re-assembly (prolonged high speed ultracentrifugation, further progressive dehydration and embedding in LR White methacrylate or hydrosoluble melamine resin). The cellulosic component was characterized by the use of cellobiohydrolase (CBH1) bound to colloidal gold, and the glucuronic acid residues of the xylan matrix were characterized by the use of cationised gold. Inside the seeds, the pre-release mucilage is mostly helicoidal, with the occurrence of more or less ordered domains, which indicate a fluid organization relevant to an actual liquid crystal state. Cytochemical tests revealed the tight association between cellulose and glucuronoxylans, the latter constituting a charged coat around each microfibril. Following the hydration of the seed, a cellulosic suspension was extracted in which microfibrils were totally dispersed. The progressive dehydration of the suspension gave rise to concentrated viscous drops. Ultrastructural observations revealed the occurrence of multidomain organization, from non-ordered to cholesteric-like regions, revealing that the mucilage is at the same time crystalline and liquid. This constitutes the first demonstration that liquid crystal type assemblies can arise from crystalline and biological cellulose in aqueous suspension. It strengthens the hypothesis that a transient liquid crystal state must occur during the cellulose ordering. The possible morphogenetic role of the glucuronoxylans in the cholesteric organization of the cellulose is discussed.

Pentavalent Bismuth-Mediated Glycosylation Methods to Activate Sialic and Uronic Acids and the Incorporation of Sialic Acids Into Insulin

NASA Astrophysics Data System (ADS)

Kabotso, Daniel Elorm Kwame

The negative charge at physiological pH of carboxylic acid-containing monosaccharides modulate the properties of many natural biomolecules such as oligosaccharides and glycoconjugates. Unfortunately, these altered electronic properties also make the incorporation of such acidic sugars more challenging as compared to the more commonly studied neutral sugars. Herein are reported the first demonstration of glycosylation reactions mediated by triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth with thioglycosides containing carboxylic acid substituents protected as esters. Unlike with many neutral sugar substrates, the addition of 1-propanethiol to the reactions proved critical to obtaining good yields of the desired glycosylation products using sialic acid, galacturonic acid, and glucuronic acid. The protocol was demonstrated to be amenable to automation using a liquid-handling platform. The consequences of artificially incorporating carboxylic-acid-containing sugars into proteins were tested by the design of a linker containing 1 to 4 sialic acids--a sugar found in many human proteins and brain tissues--that was attached via reductive amination of trityl thiopropylaldehyde at the phenyl alanine terminal end of the protein insulin produced through solid-phase peptide synthesis. Removal of the trityl group with neat trifluoroacetic acid furnished the thiol-free modified insulin that was ligated via a disulfide bond to the peptide scaffold bearing acetyl protected sialic acids. A 14-15% ammonium hydroxide solution was found to be effective in deprotecting the acetyl groups without degradation of the disulfide bond. In addition to maintaining the potency and bioactivity of insulin, the sialic acid-containing linker rendered insulin more resistant to aggregation due to heat and mechanical agitation compared to the unmodified protein.

The urinary metabolites of DINCH® have an impact on the activities of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ.

PubMed

Engel, Anika; Buhrke, Thorsten; Kasper, Stefanie; Behr, Anne-Cathrin; Braeuning, Albert; Jessel, Sönke; Seidel, Albrecht; Völkel, Wolfgang; Lampen, Alfonso

2018-05-01

DINCH ® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH ® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH ® , there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH ® and five DINCH ® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ in vitro. DINCH ® itself did not have any effect on the activity of these receptors whereas DINCH ® metabolites were shown to activate all these receptors. In the case of AR, DINCH ® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH ® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH ® metabolites exert different effects at the molecular level compared to DINCH ® itself. All these in vitro effects of DINCH ® metabolites, however, were only observed at high concentrations such as 10 μM or above which is about three orders of magnitude above reported DINCH ® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH ® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels. Copyright © 2018 Elsevier B.V. All rights reserved.

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

PubMed

Sporck, Daniele; Reinoso, Felipe A M; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, José C; Ferraz, André; Milagres, Adriane M F

2017-01-01

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis

PubMed Central

Xu, Jialin; Kulkarni, Supriya R.; Li, Liya

2012-01-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lepob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance. PMID:22031624

UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

PubMed

Xu, Jialin; Kulkarni, Supriya R; Li, Liya; Slitt, Angela L

2012-02-01

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry.

PubMed

Wu, Xiaodan; Jiang, Wei; Lu, Jiajia; Yu, Ying; Wu, Bin

2014-02-15

Sargassum fusiforme (hijiki) is the well-known edible algae, whose polysaccharides have been proved to possess interesting bioactivities like antitumor, antioxidant, antimicrobial and immunomodulatory activities. A facile and sensitive method based on high-performance liquid chromatography method of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) coupled with electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been established for the analysis of the monosaccharide composition of polysaccharides in S. fusiforme. Monosaccharides have been converted into PMP-labelled derivatives with aqueous ammonia as a catalyst at 70 °C for 30 min. The optimisation of the pre-column derivatization process was studied. The LODs of the monosaccharides were in the range from 0.01 to 0.02 nmol. PMP-labelled mixture of monosaccharides has been well separated by a reverse-phase HPLC and detected by on-line ESI-MS method under optimised conditions. The mobile phase of elution system was chosen as acetonitrile (solvent A) and 20mM aqueous ammonium acetate (solvent B) (pH 3.0) with Zorbax XDB-C18 column at 30 °C for the separation of the monosaccharide derivatives. Identification of the monosaccharides composition was carried out by analysis with mass spectral behaviour and chromatography characteristics of 1-phenyl-3-methyl-5-pyrazolone (PMP) labelled monosaccharides. All PMP-labelled derivatives display high chemical stabilities, whose regular MS fragmentation is specific for reducing labelled sugars. The result showed that the S. fusiforme polysaccharide consisted of mannose, glucose, galactose, xylose, fucose and glucuronic acid or galacturonic acid, or both uronic acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of polysaccharides in medical plant and gels

NASA Astrophysics Data System (ADS)

Pielesz, A.

In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology. Of them, polysaccharides or their glycoconjugates were shown to exhibit multiple biological activities including anticarcinogenic, anticoagulant, immunostimulating, antioxidant, etc. Pharmacotherapy using plant-derived substances can be currently regarded as a very promising future alternative to conventional therapy. The advanced biotechnologies available today enable chemical investigation of well-defined bioactive plant components as sources of novel drugs. The need for safer drugs without side effects has led to the use of natural ingredients with proven safety. Special interest is focused on plant polysaccharides. This article attempts to review the current structural and conformational characterization of some importantly bioactive monosaccharides isolated from following plant cell-wall: Symphytum officinale (comfrey), Thymus pulegioides (thyme), Trigonella foenum-graecum L. (fenugreek), Tussilago farfara L. (coltsfoot), Hyssopus officinalis (hyssop), Althaea officinalis L. (marshmallow) and Equisetum arvense L. (horsetail). The chemical structures of monosaccharides were analysed using FTIR and Raman spectroscopies as well as cellulose acetate membrane electrophoresis (CAE). The dried plant samples were gently hydrolysed with sulphuric acid. The presence of glucuronic acid, galacturonic acid, alginic acid, glucose, mannose and xylose in the hydrolysates of reference substances and non-defatted plant films was proved. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed. Individual bands were selected to monitor the sugar content in medical plant cell walls and to confirm the identity of the analysed plants.

[Analysis of monosaccharides and uronic acids in polysaccharides by pre-column derivatization with p-aminobenzoic acid and high performance liquid chromatography].

PubMed

Hao, Guitang; Chen, Shangwei; Zhu, Song; Yin, Hongping; Dai, Jun; Cao, Yuhua

2007-01-01

An ion-pair reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of carbohydrate and uronic acids was developed. p-Aminobenzoic acid (p-AMBA) was used for pre-column derivatization of the analytes, enabling fluorescence (lambda(ex) = 313 nm, lambda(em) = 358 nm) or ultraviolet (UV at 303 nm) detection. Reaction conditions such as reaction temperature and reaction time were optimized. Atlantis dC18 column with hydrophilic end capping was selected for the separation of derivatives. Effects of mobile phase compositions such as ion pairs and their concentrations and pH on the retention behaviors and separation results of 9 monosaccharides and 2 uronic acids were investigated. Derivatives of fructose, galactose, glucose, mannose, xylose, arabinose, ribose, galacturonic acid, fucose, glucuronic acid and rhamnose were separated within 42 min, applying tetrabutyl ammonium hydrogen bisulfate (TBAHSO4) as the ion pair reagent. The detection limits were between 3.38 x 10(-8) mol/L and 176 x 10(-8) mol/L for fluorescence detection and between 2.55 x 10(-7) mol/L and 13.4 x 10(-7) mol/L for UV detection. Good linearities were obtained with correlation coefficients (r2) above 0.99. The relative standard deviations (RSDs) of the peak area of the derivatives in 12 - 51 h after derivatization were from 2.5% to 3.9%. This method has been applied for the determination of mono-/disaccharides and uronic acids in spirulina polysaccharide after dissolved in trifluoroacetic acid solution (2 mol/L). The results showed this method is suitable for the analysis of monosaccharide compositions in polysaccharides.

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

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

Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin

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

Synthesis of UDP-apiose in Bacteria: The marine phototroph Geminicoccus roseus and the plant pathogen Xanthomonas pisi.

PubMed

Smith, James Amor; Bar-Peled, Maor

2017-01-01

The branched-chain sugar apiose was widely assumed to be synthesized only by plant species. In plants, apiose-containing polysaccharides are found in vascularized plant cell walls as the pectic polymers rhamnogalacturonan II and apiogalacturonan. Apiosylated secondary metabolites are also common in many plant species including ancestral avascular bryophytes and green algae. Apiosyl-residues have not been documented in bacteria. In a screen for new bacterial glycan structures, we detected small amounts of apiose in methanolic extracts of the aerobic phototroph Geminicoccus roseus and the pathogenic soil-dwelling bacteria Xanthomonas pisi. Apiose was also present in the cell pellet of X. pisi. Examination of these bacterial genomes uncovered genes with relatively low protein homology to plant UDP-apiose/UDP-xylose synthase (UAS). Phylogenetic analysis revealed that these bacterial UAS-like homologs belong in a clade distinct to UAS and separated from other nucleotide sugar biosynthetic enzymes. Recombinant expression of three bacterial UAS-like proteins demonstrates that they actively convert UDP-glucuronic acid to UDP-apiose and UDP-xylose. Both UDP-apiose and UDP-xylose were detectable in cell cultures of G. roseus and X. pisi. We could not, however, definitively identify the apiosides made by these bacteria, but the detection of apiosides coupled with the in vivo transcription of bUAS and production of UDP-apiose clearly demonstrate that these microbes have evolved the ability to incorporate apiose into glycans during their lifecycles. While this is the first report to describe enzymes for the formation of activated apiose in bacteria, the advantage of synthesizing apiose-containing glycans in bacteria remains unknown. The characteristics of bUAS and its products are discussed.

Antioxidative roles of sesamin, a functional lignan in sesame seed, and it's effect on lipid- and alcohol-metabolism in the liver: a DNA microarray study.

PubMed

Kiso, Yoshinobu

2004-01-01

Sesamin was orally administered to rats, and blood, bile and urine were collected periodically. Over 40% of the dose of sesamin was detected in bile as glucuronides of 2-(3, 4-methylenedioxyphenyl)-6-(3, 4-dihydroxyphenyl)-cis-dioxabicyclo[3.3.0] octane and 2-(3, 4-dihydroxyphenyl)-6-(3, 4-dihydroxyphenyl)-cis-dioxabicyclo[3.3.0] octane by 24 hr after administration. Antioxidant activities of these metabolites were compared and catechol metabolites showed strong radical scavenging activities against not only superoxide anion radical but also hydroxyl radical. It was suggested that sesamin was absorbed by the route of portal vein and metabolized to mono- or di-catechol metabolite by drug metabolizing enzymes in the liver cells. Both metabolites exhibited antioxidant activity in the liver and were finally conjugated with glucuronic acid and to excrete in bile. Sesamin can be classified as a pro-antioxidant. The profiles of gene expression of the liver in rats given sesamin or vehicle were compared. The gene expression levels of the late stage enzymes of beta-oxidation including trifunctional enzyme, acyl-CoA oxidase, bifunctional enzyme and 3-ketoacyl-CoA thiolase were significantly increased by sesamin. On the other hand, the transcription of the genes encoding the enzymes for fatty acid synthesis was decreased. Moreover, in sesamin rats, the gene expression of aldehyde dehydrogenase was increased about 3-fold, whereas alcohol dehydrogenase, liver catalase and CYP2E1 were not changed. These results suggested that sesamin ingestion regulated the transcription levels of hepatic metabolizing enzymes for lipids and alcohol.

Ultrasound-assisted extraction, characterization, and antioxidant activity in vitro and in vivo of polysaccharides from Chestnut rose (Rosa roxburghii tratt) fruit.

PubMed

Chen, Guangjing; Kan, Jianquan

2018-03-01

In this study, the response surface methodology was utilized to determine optimum conditions for extracting the polysaccharides from Rosa roxburghii Tratt fruit (RRTPs) using ultrasonic-assisted extraction, and the characterization and antioxidant activities of the RRTPs were discussed. RRTPs yield was 6.59 ± 1.34%, which was well consistent with the predicted value of 6.716%, under the following optimum conditions: ratio of water to raw material 40.18 mL/g, extraction temperature 78.8 °C, ultrasonic power 148 W, and extraction time 32.8 min. The monosaccharide composition analysis indicated that RRTPs were composed of mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), arabinose (Ara) and xylose (Xyl). The molecular weight distribution analysis showed that RRTPs had four main components with molecular weights of 332.56, 183.96, 11.92 and 5.95 kDa, respectively. In vitro antioxidant studies revealed RRTPs exhibited significant antioxidant potential on hydroxyl, superoxide and DPPH radicals. In addition, antioxidant assays in vivo demonstrated that RRTPs can significantly increase the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities, and total antioxidant capacity (TAOC) to some extent, as well as decrease the level of malondialdehyde (MDA) in both serum and liver of d-Gal aging-induced mice. These data suggested that RRTPs could be a potential candidate of natural antioxidants for applications in functional food, pharmaceuticals or cosmetic industries. In summary, this work provided an effective method for the exploitation and utilization of value-added R. roxburghii Tratt fruit which would be useful to fully utilize this resource.

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition*

PubMed Central

Ishizuka, Shinya; Askew, Emily B.; Ishizuka, Naoko; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Depletion of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. This loss is often accompanied by a coordinate loss in another glycosaminoglycan, hyaluronan. Chondrocytes experimentally depleted of cell-associated hyaluronan respond by switching to a pro-catabolic metabolism that includes enhanced production of endogenous inflammatory mediators and increased synthesis of matrix metalloproteinases. Hyaluronan turnover is also increased. Together, such a response provides for possible establishment of a self-perpetuating spiral of events that maintains or prolongs the pro-catabolic state. Chondrocytes or cartilage can also be activated by treatment with pro-inflammatory cytokines and mediators such as IL-1β, TNFα, LPS, fibronectin fragments, and hyaluronan oligosaccharides. To determine the mechanism of chondrocyte activation due to hyaluronan loss, a depletion method was required that did not include degrading the hyaluronan. In recent years, several laboratories have used the coumarin derivative, 4-methylumbelliferone, as a potent inhibitor of hyaluronan biosynthesis, due in part to its ability to sequester intracellular UDP-glucuronic acid and inhibition of hyaluronan synthase transcription. However, contrary to our expectation, although 4-methylumbelliferone was indeed an inhibitor of hyaluronan biosynthesis, this depletion did not give rise to an activation of chondrocytes or cartilage. Rather, 4-methylumbelliferone directly and selectively blocked gene products associated with the pro-catabolic metabolic state of chondrocytes and did so through a mechanism preceding and independent of hyaluronan inhibition. These data suggest that 4-methylumbelliferone has additional useful applications to block pro-inflammatory cell activation events but complicates how it is used for defining functions related to hyaluronan. PMID:27129266

Characterization and Antiproliferative Effect of Novel Acid Polysaccharides from the Spent Substrate of Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Cultivation.

PubMed

Zhang, Yong; Liu, Wei; Xu, Chunping; Huang, Wei; He, Peixin

2017-01-01

In this study, a high yield of crude polysaccharide (16.73 ± 0.756%) was extracted from the spent mushroom substrate of Lentinus edodes using a hot alkali extraction method. Two groups of polysaccharides (designated as LSMS-1 and LSMS-2) were obtained from the crude extract by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index detector system. The weight-average molar masses of LSMS-1 and LSMS-2 were determined to be 6.842 × 106 and 2.154 × 106 g/mol, respectively. The SEC/MALLS analysis revealed that the molecular shapes of LSMS-1 and LSMS-2 were sphere-like forms in aqueous solution. Carbohydrate composition analysis using chromatography--mass spectrometry revealed that they were both acid heteropolysaccharides. LSMS-1 comprised mainly glucose and galacturonic acid, whereas LSMS-2 mainly consisted of xylose and glucuronic acid. Fourier transform infrared spectral analysis of the purified fractions revealed typical characteristic polysaccharide groups. In addition, MTT assays with refined polysaccharide doses of 25, 50, 100, 200, and 400 µg/mL suggested that both of the polysaccharide fractions exhibited antiproliferative activity against 6 tested human tumor cell lines in a concentration-dependent manner, and LSMS-2 had better anticancer capacity in vitro than LSMS-1. The inhibition ratio of LSMS-2 against A549 human lung cancer cells, the SGC7901 gastric cancer cell line, MCF-7 breast cancer cells, the U937 histiocytic lymphoma cell line, and the MG-63 human osteosarcoma cell line reached 43.55%, 29.97%, 19.63%, 18.24%, and 17.93%, respectively, at a concentration of 400 µg/mL.

Liver glucose metabolism in humans

PubMed Central

Adeva-Andany, María M.; Pérez-Felpete, Noemi; Fernández-Fernández, Carlos; Donapetry-García, Cristóbal; Pazos-García, Cristina

2016-01-01

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP–glucose. Minor amounts of UDP–glucose are used to form UDP–glucuronate and UDP–galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis). PMID:27707936

Characterization of in vivo metabolites in rat urine following an oral dose of masitinib by liquid chromatography tandem mass spectrometry.

PubMed

Kadi, Adnan A; Amer, Sawsan M; Darwish, Hany W; Attwa, Mohamed W

2018-05-15

Masitinib (MST) is an orally administered drug that targets mast cells and macrophages, important cells for immunity, by inhibiting a limited number of tyrosine kinases. It is currently registered in Europe and USA for the treatment of mast cell tumors in dogs. AB Science announced that the European Medicines Agency has accepted a conditional marketing authorization application for MST to treat amyotrophic lateral sclerosis. In our work, we focused on studying in vivo metabolism of MST in Sprague-Dawley rats. Single oral dose of MST (33 mg kg -1 ) was given to Sprague-Dawley rats (kept in metabolic cages) using oral gavage. Urine was collected and filtered at 0, 6, 12, 18, 24, 48, 72 and 96 h from MST dosing. An equal amount of ACN was added to urine samples. Both organic and aqueous layers were injected into liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect in vivo phase I and phase II MST metabolites. The current work reports the identification and characterization of twenty in vivo phase I and four in vivo phase II metabolites of MST by LC-MS/MS. Phase I metabolic pathways were reduction, demethylation, hydroxylation, oxidative deamination, oxidation and N-oxide formation. Phase II metabolic pathways were the direct conjugation of MST, N-demethyl metabolites and oxidative metabolites with glucuronic acid. Part of MST dose was excreted unchanged in urine. The literature review showed no previous articles have been made on in vivo metabolism of MST or detailed structural identification of the formed in vivo phase I and phase II metabolites.

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils

PubMed Central

1990-01-01

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl- /HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization. PMID:2280252

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries.

PubMed

Cobucci-Ponzano, Beatrice; Strazzulli, Andrea; Iacono, Roberta; Masturzo, Giuseppe; Giglio, Rosa; Rossi, Mosè; Moracci, Marco

2015-10-01

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications. Copyright © 2015 Elsevier Inc. All rights reserved.

A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium

PubMed Central

Dietrich, Diane; Crooks, Casey; Balogun, Peter; deSerrano, Vesna; Pozharski, Edwin; Smith, James Kennon; Bales, Elizabeth; Hurlbert, Jason

2018-01-01

Glycoside hydrolase family 30 subfamily 8 (GH30-8) β-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the −3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains. PMID:29626157

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.

PubMed

Gufford, Brandon T; Graf, Tyler N; Paguigan, Noemi D; Oberlies, Nicholas H; Paine, Mary F

2015-11-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-D-glucuronide (15.7 mg), silybin A-5-O-β-D-glucuronide (1.6 mg), and silybin A-4´´-O-β-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides

PubMed Central

Gufford, Brandon T.; Graf, Tyler N.; Paguigan, Noemi D.; Oberlies, Nicholas H.

2015-01-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-d-glucuronide (15.7 mg), silybin A-5-O-β-d-glucuronide (1.6 mg), and silybin A-4´´-O-β-d-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. PMID:26316643

Capsules from pathogenic and non-pathogenic Cryptococcus spp. manifest significant differences in structure and ability to protect against phagocytic cells.

PubMed

Araujo, Glauber de S; Fonseca, Fernanda L; Pontes, Bruno; Torres, Andre; Cordero, Radames J B; Zancopé-Oliveira, Rosely M; Casadevall, Arturo; Viana, Nathan B; Nimrichter, Leonardo; Rodrigues, Marcio L; Garcia, Eloi S; Souza, Wanderley de; Frases, Susana

2012-01-01

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.

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

DOE PAGES

Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; ...

2016-01-08

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

Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of polysaccharides in medical plant and gels.

PubMed

Pielesz, A

2012-07-01

In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology. Of them, polysaccharides or their glycoconjugates were shown to exhibit multiple biological activities including anticarcinogenic, anticoagulant, immunostimulating, antioxidant, etc. Pharmacotherapy using plant-derived substances can be currently regarded as a very promising future alternative to conventional therapy. The advanced biotechnologies available today enable chemical investigation of well-defined bioactive plant components as sources of novel drugs. The need for safer drugs without side effects has led to the use of natural ingredients with proven safety. Special interest is focused on plant polysaccharides. This article attempts to review the current structural and conformational characterization of some importantly bioactive monosaccharides isolated from following plant cell-wall: Symphytum officinale (comfrey), Thymus pulegioides (thyme), Trigonella foenum-graecum L. (fenugreek), Tussilago farfara L. (coltsfoot), Hyssopus officinalis (hyssop), Althaea officinalis L. (marshmallow) and Equisetum arvense L. (horsetail). The chemical structures of monosaccharides were analysed using FTIR and Raman spectroscopies as well as cellulose acetate membrane electrophoresis (CAE). The dried plant samples were gently hydrolysed with sulphuric acid. The presence of glucuronic acid, galacturonic acid, alginic acid, glucose, mannose and xylose in the hydrolysates of reference substances and non-defatted plant films was proved. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed. Individual bands were selected to monitor the sugar content in medical plant cell walls and to confirm the identity of the analysed plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1.

PubMed

Zhou, Jin; Tracy, Timothy S; Remmel, Rory P

2010-11-01

Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Evidence That GH115 α-Glucuronidase Activity, Which Is Required to Degrade Plant Biomass, Is Dependent on Conformational Flexibility*

PubMed Central

Rogowski, Artur; Baslé, Arnaud; Farinas, Cristiane S.; Solovyova, Alexandra; Mortimer, Jennifer C.; Dupree, Paul; Gilbert, Harry J.; Bolam, David N.

2014-01-01

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan. PMID:24214982

The Genetic Landscape of Renal Complications in Type 1 Diabetes

PubMed Central

Sandholm, Niina; Van Zuydam, Natalie; Ahlqvist, Emma; Juliusdottir, Thorhildur; Deshmukh, Harshal A.; Rayner, N. William; Di Camillo, Barbara; Forsblom, Carol; Fadista, Joao; Ziemek, Daniel; Salem, Rany M.; Hiraki, Linda T.; Pezzolesi, Marcus; Trégouët, David; Dahlström, Emma; Valo, Erkka; Oskolkov, Nikolay; Ladenvall, Claes; Marcovecchio, M. Loredana; Cooper, Jason; Sambo, Francesco; Malovini, Alberto; Manfrini, Marco; McKnight, Amy Jayne; Lajer, Maria; Harjutsalo, Valma; Gordin, Daniel; Parkkonen, Maija; Lyssenko, Valeriya; McKeigue, Paul M.; Rich, Stephen S.; Brosnan, Mary Julia; Fauman, Eric; Bellazzi, Riccardo; Rossing, Peter; Hadjadj, Samy; Krolewski, Andrzej; Paterson, Andrew D.; Hirschhorn, Joel N.; Maxwell, Alexander P.; Cobelli, Claudio; Colhoun, Helen M.; Groop, Leif; McCarthy, Mark I.

2017-01-01

Diabetes is the leading cause of ESRD. Despite evidence for a substantial heritability of diabetic kidney disease, efforts to identify genetic susceptibility variants have had limited success. We extended previous efforts in three dimensions, examining a more comprehensive set of genetic variants in larger numbers of subjects with type 1 diabetes characterized for a wider range of cross-sectional diabetic kidney disease phenotypes. In 2843 subjects, we estimated that the heritability of diabetic kidney disease was 35% (P=6.4×10−3). Genome-wide association analysis and replication in 12,540 individuals identified no single variants reaching stringent levels of significance and, despite excellent power, provided little independent confirmation of previously published associated variants. Whole-exome sequencing in 997 subjects failed to identify any large-effect coding alleles of lower frequency influencing the risk of diabetic kidney disease. However, sets of alleles increasing body mass index (P=2.2×10−5) and the risk of type 2 diabetes (P=6.1×10−4) associated with the risk of diabetic kidney disease. We also found genome-wide genetic correlation between diabetic kidney disease and failure at smoking cessation (P=1.1×10−4). Pathway analysis implicated ascorbate and aldarate metabolism (P=9.0×10−6), and pentose and glucuronate interconversions (P=3.0×10−6) in pathogenesis of diabetic kidney disease. These data provide further evidence for the role of genetic factors influencing diabetic kidney disease in those with type 1 diabetes and highlight some key pathways that may be responsible. Altogether these results reveal important biology behind the major cause of kidney disease. PMID:27647854

Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Medina-Rangel, Paulina Ximena; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-02-15

Altered glycosylation levels or distribution of sialic acids (SA) or hyaluronan in animal cells are indicators of pathological conditions like infection or malignancy. We applied fluorescently-labeled molecularly imprinted polymer (MIP) particles for bioimaging of fixed and living human keratinocytes, to localize hyaluronan and sialylation sites. MIPs were prepared with the templates D-glucuronic acid (GlcA), a substructure of hyaluronan, and N-acetylneuraminic acid (NANA), the most common member of SA. Both MIPs were found to be highly selective towards their target monosaccharides, as no cross-reactivity was observed with other sugars like N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucose and D-galactose, present on the cell surface. The dye rhodamine and two InP/ZnS quantum dots (QDs) emitting in the green and in the red regions were used as fluorescent probes. Rhodamine-MIPGlcA and rhodamine-MIPNANA were synthesized as monodispersed 400nm sized particles and were found to bind selectively their targets located in the extracellular region, as imaged by epifluorescence and confocal microscopy. In contrast, when MIP-GlcA and MIP-NANA particles with a smaller size (125nm) were used, the MIPs being synthesized as thin shells around green and red emitting QDs respectively, it was possible to stain the intracellular and pericellular regions as well. In addition, simultaneous dual-color imaging with the two different colored QDs-MIPs was demonstrated. Importantly, the MIPs were not cytotoxic and did not affect cell viability; neither was the cells morphology affected as demonstrated by live cell imaging. These synthetic receptors could offer a new and promising imaging tool to monitor disease progression. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of an unintended consequence of Nrf2-directed cytoprotection against a key tobacco carcinogen plus a counteracting chemopreventive intervention

PubMed Central

Paonessa, Joseph D.; Ding, Yi; Randall, Kristen L.; Munday, Rex; Argoti, Dayana; Vouros, Paul; Zhang, Yuesheng

2011-01-01

Nrf2 is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. While Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2+/+ mice than in Nrf2−/− mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. While glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, while higher liver UGT activity may protect the liver against ABP it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further demonstrate that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues, but does not appear to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2. PMID:21487034

Separation and characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by ammonium sulfate precipitation.

PubMed

Peng, Feng; Bian, Jing; Peng, Pai; Xiao, Huan; Ren, Jun-Li; Xu, Feng; Sun, Run-Cang

2012-04-25

Delignified Arundo donax was sequentially extracted with DMSO, saturated barium hydroxide, and 1.0 M aqueous NaOH solution. The yields of the soluble fractions were 10.2, 6.7, and 10.0% (w/w), respectively, of the dry Arundo donax materials. The DMSO-, Ba(OH)(2)- and NaOH-soluble hemicellulosic fractions were further fractionated into two subfractions by gradient 50% and 80% saturation ammonium sulfate precipitation, respectively. Monosaccharide, molecular weight, FT-IR, and 1D ((1)H and (13)C) and 2D (HSQC) NMR analysis revealed the differences in structural characteristics and physicochemical properties among the subfractions. The subfractions precipitated with 50% saturation ammonium sulfate had lower arabinose/xylose and glucuronic acid/xylose ratios but had higher molecular weight than those of the subfractions precipitated by 80% saturation ammonium sulfate. FT-IR and NMR analysis revealed that the highly acetylated DMSO-soluble hemicellulosic subfraction (H(D50)) could be precipitated with a relatively lower concentration of 50% saturated ammonium sulfate, and thus the gradient ammonium sulfate precipitation technique could discriminate acetyl and non-acetyl hemicelluloses. It was found that the DMSO-soluble subfraction H(D50) precipitated by 50% saturated ammonium sulfate mainly consisted of poorly substituted O-acetyl arabino-4-O-methylglucurono xylan with terminal units of arabinose linked on position 3 of xylose, 4-O-methylglucuronic acid residues linked on position 2 of the xylan bone, and the acetyl groups (degree of acetylation, 37%) linked on position 2 or 3. The DMSO-soluble subfraction H(D80) precipitated by 80% saturated ammonium sulfate was mainly composed of highly substituted arabino-4-O-methylglucurono xylan and β-d-glucan.

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates.

PubMed

Marín-Zamora, María Elisa; Rojas-Melgarejo, Francisco; García-Cánovas, Francisco; García-Ruiz, Pedro Antonio

2007-09-30

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.

Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

PubMed Central

Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

2012-01-01

Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

Roles of Chondroitin Sulfate and Dermatan Sulfate in the Formation of a Lesion Scar and Axonal Regeneration after Traumatic Injury of the Mouse Brain

PubMed Central

Li, Hong-Peng; Komuta, Yukari; Kimura-Kuroda, Junko; van Kuppevelt, Toin H.

2013-01-01

Abstract Dermatan sulfate (DS) is synthesized from chondroitin sulfate (CS) by epimerization of glucuronic acid of CS to yield iduronic acid. In the present study, the role of CS and DS was examined in mice that received transection of nigrostriatal dopaminergic pathway followed by injection of glycosaminoglycan degrading enzymes into the lesion site. Two weeks after injury, fibrotic and glial scars were formed around the lesion, and transected axons did not regenerate beyond the fibrotic scar. Injection of chondroitinase ABC (ChABC), which degrades both CS and DS, completely suppressed the fibrotic scar formation, reduced the glial scar, and promoted the regeneration of dopaminergic axons. Injection of the DS-degrading enzyme chondroitinase B (ChB) also yielded similar results. By contrast, injection of chondroitinase AC (ChAC), a CS-degrading enzyme, did not suppress the fibrotic and glial scar formation, but reduced CS immunoreactivity and promoted the axonal regeneration. Addition of transforming growth factor-β1 (TGF-β1) to a co-culture of meningeal fibroblasts and cerebral astrocytes induces a fibrotic scar-like cell cluster. The effect of TGF-β1 on cluster formation was suppressed by treatment with ChABC or ChB, but not by ChAC. TGF-β1-induced cell cluster repelled neurites of neonatal cerebellar neurons, but addition of ChABC or ChAC suppressed the inhibitory property of clusters on neurite outgrowth. The present study is the first to demonstrate that DS and CS play different functions after brain injury: DS is involved in the lesion scar formation, and CS inhibits axonal regeneration. PMID:23438307

SDN-1/Syndecan Acts in Parallel to the Transmembrane Molecule MIG-13 to Promote Anterior Neuroblast Migration.

PubMed

Sundararajan, Lakshmi; Norris, Megan L; Lundquist, Erik A

2015-05-28

The Q neuroblasts in Caenorhabditis elegans display left-right asymmetry in their migration, with QR and descendants on the right migrating anteriorly, and QL and descendants on the left migrating posteriorly. Initial QR and QL migration is controlled by the transmembrane receptors UNC-40/DCC, PTP-3/LAR, and the Fat-like cadherin CDH-4. After initial migration, QL responds to an EGL-20/Wnt signal that drives continued posterior migration by activating MAB-5/Hox activity in QL but not QR. QR expresses the transmembrane protein MIG-13, which is repressed by MAB-5 in QL and which drives anterior migration of QR descendants. A screen for new Q descendant AQR and PQR migration mutations identified mig-13 as well as hse-5, the gene encoding the glucuronyl C5-epimerase enzyme, which catalyzes epimerization of glucuronic acid to iduronic acid in the heparan sulfate side chains of heparan sulfate proteoglycans (HSPGs). Of five C. elegans HSPGs, we found that only SDN-1/Syndecan affected Q migrations. sdn-1 mutants showed QR descendant AQR anterior migration defects, and weaker QL descendant PQR migration defects. hse-5 affected initial Q migration, whereas sdn-1 did not. sdn-1 and hse-5 acted redundantly in AQR and PQR migration, but not initial Q migration, suggesting the involvement of other HSPGs in Q migration. Cell-specific expression studies indicated that SDN-1 can act in QR to promote anterior migration. Genetic interactions between sdn-1, mig-13, and mab-5 suggest that MIG-13 and SDN-1 act in parallel to promote anterior AQR migration and that SDN-1 also controls posterior migration. Together, our results indicate previously unappreciated complexity in the role of multiple signaling pathways and inherent left-right asymmetry in the control of Q neuroblast descendant migration. Copyright © 2015 Sundararajan et al.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

PubMed

Benini, Stefano; Toccafondi, Mirco; Rejzek, Martin; Musiani, Francesco; Wagstaff, Ben A; Wuerges, Jochen; Cianci, Michele; Field, Robert A

2017-11-01

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Sphingomonas pituitosa sp. nov., an exopolysaccharide-producing bacterium that secretes an unusual type of sphingan.

PubMed

Denner, E B; Paukner, S; Kämpfer, P; Moore, E R; Abraham, W R; Busse, H J; Wanner, G; Lubitz, W

2001-05-01

Strain EDIVT, an exopolysaccharide-producing bacterium, was subjected to polyphasic characterization. The bacterium produced copious amounts of an extracellular polysaccharide, forming slimy, viscous, intensely yellow-pigmented colonies on Czapek-Dox (CZD) agar. The culture fluids of the liquid version of CZD medium were highly viscous after cultivation for 5 d. Cells of strain EDIVT were Gram-negative, catalase-positive, oxidase-negative, nonspore-forming, rod-shaped and motile. Comparisons of 16S rDNA gene sequences demonstrated that EDIVT clusters phylogenetically with the species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (64.5 mol%), the presence of ubiquinone Q-10, the presence of 2-hydroxymyristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the detection of sym-homospermidine as the major component in the polyamine pattern, together with the presence of sphingoglycolipid, supported this delineation. 16S rDNA sequence analysis indicated that strain EDIVT is most closely related (99.4% similarity) to Sphingomonas trueperi LMG 2142T. DNA-DNA hybridization showed that the level of relatedness to S. trueperi is only 45.5%. Further differences were apparent in the cellular fatty acid profile, the polar lipid pattern, the Fourier-transform infrared spectrum and whole-cell proteins and in a number of biochemical characteristics. On the basis of the estimated phylogenetic position derived from 16S rDNA sequence data, DNA-DNA reassociation and phenotypic differences, strain EDIVT (= CIP 106154T = DSM 13101T) was recognized as a new species of Sphingomonas, for which the name Sphingomonas pituitosa sp. nov. is proposed. A component analysis of the exopolysaccharide (named PS-EDIV) suggested that it represents a novel type of sphingan composed of glucose, rhamnose and an unidentified sugar. Glucuronic acid, which is commonly found in sphingans, was absent. The mean molecular mass of PS-EDIV was approximately 3 x 10(6) Da.

Biotransformation of the novel inotropic agent toborinone (OPC-18790) in rats and dogs. Evidence for the formation of novel glutathione and two cysteine conjugates.

PubMed

Kitani, M; Miyamoto, G; Nagasawa, M; Yamada, T; Matsubara, J; Uchida, M; Odomi, M

1997-06-01

The metabolism of toborinone, (+/-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)-quin - olinone, a novel inotropic agent, was studied in rats and dogs after intravenous administration. Chemical structures of the 13 metabolites were characterized by direct-probe FAB/MS and field desorption/MS, LC/FAB/MS, and various NMR measurements. After intravenous dosing of 10 mg/kg [14C]toborinone, fecal and urinary recoveries of the 14C dose were approximately 70% and 26-30%, respectively, in both rats and dogs. The predominant component of radioactivity was the unchanged toborinone in every biological specimen in rats and dogs. Although unchanged toborinone was predominantly observed, toborinone underwent extensive conjugations with glucuronic acid, sulfate, and glutathione, either directly or following phase I reaction. Metabolites resulting from oxidative N-C cleavage were minor both in number and in quantity in every biological specimen in rats and dogs. In rats, toborinone underwent O-demethylation to form M-7 and successive phase it reaction to yield the glucuronide M-1 and the sulfoconjugate M-2, and deconjugation to yield M-7, which was a primary metabolite accounted for 35.67% of the radioactivity excreted in the feces by 48 hr. Conjugates M-1 and M-2 were the major metabolites in rat plasma. In dogs, toborinone was metabolized via mercapturic acid pathway to yield the primary metabolites, cysteine conjugates M-10 and M-11 that accounted for 19.10% and 6.70% of the radioactivity excreted in the feces by 48 hr and that were detected species specifically in dogs. The glutathione conjugate M-13, which was isolated from in vitro incubations using dog liver, led us to consider a possible mercapturic acid pathway from the parent compound to M-10. Metabolites in dog plasma and those in urine in both rats and dogs were minor in quantity. The metabolic pathways of toborinone in rats and dogs are proposed herein.

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

PubMed

Ismail, Sabariah; Hanapi, Nur Aziah; Ab Halim, Mohd Rohaimi; Uchaipichat, Verawan; Mackenzie, Peter I

2010-05-14

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

SDN-1/Syndecan Acts in Parallel to the Transmembrane Molecule MIG-13 to Promote Anterior Neuroblast Migration

PubMed Central

Sundararajan, Lakshmi; Norris, Megan L.; Lundquist, Erik A.

2015-01-01

The Q neuroblasts in Caenorhabditis elegans display left-right asymmetry in their migration, with QR and descendants on the right migrating anteriorly, and QL and descendants on the left migrating posteriorly. Initial QR and QL migration is controlled by the transmembrane receptors UNC-40/DCC, PTP-3/LAR, and the Fat-like cadherin CDH-4. After initial migration, QL responds to an EGL-20/Wnt signal that drives continued posterior migration by activating MAB-5/Hox activity in QL but not QR. QR expresses the transmembrane protein MIG-13, which is repressed by MAB-5 in QL and which drives anterior migration of QR descendants. A screen for new Q descendant AQR and PQR migration mutations identified mig-13 as well as hse-5, the gene encoding the glucuronyl C5-epimerase enzyme, which catalyzes epimerization of glucuronic acid to iduronic acid in the heparan sulfate side chains of heparan sulfate proteoglycans (HSPGs). Of five C. elegans HSPGs, we found that only SDN-1/Syndecan affected Q migrations. sdn-1 mutants showed QR descendant AQR anterior migration defects, and weaker QL descendant PQR migration defects. hse-5 affected initial Q migration, whereas sdn-1 did not. sdn-1 and hse-5 acted redundantly in AQR and PQR migration, but not initial Q migration, suggesting the involvement of other HSPGs in Q migration. Cell-specific expression studies indicated that SDN-1 can act in QR to promote anterior migration. Genetic interactions between sdn-1, mig-13, and mab-5 suggest that MIG-13 and SDN-1 act in parallel to promote anterior AQR migration and that SDN-1 also controls posterior migration. Together, our results indicate previously unappreciated complexity in the role of multiple signaling pathways and inherent left-right asymmetry in the control of Q neuroblast descendant migration. PMID:26022293

Back to the future with the AGP–Ca2+ flux capacitor

PubMed Central

Lamport, Derek T. A.; Varnai, Peter; Seal, Charlotte E.

2014-01-01

Background Arabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp–arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca2+ when paired with its adjacent sidechain. Scope AGPs bind Ca2+ (Kd ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H+-ATPase generates a low periplasmic pH that dissociates AGP–Ca2+ carboxylates (pka ∼3); the consequential large increase in free Ca2+ drives entry into the cytosol via Ca2+ channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca2+ waves. This differs markedly from animals, in which cytosolic Ca2+ originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca2+ storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca2+, hence an AGP–Ca2+ oscillator. Conclusions The novel concept of dynamic Ca2+ recycling by an AGP–Ca2+ oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca2+ signalling and auxin. This accounts for the involvement of AGPs in plant morphogenesis, including tropic and nastic movements. PMID:25139429

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

PubMed

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Detection of boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography-tandem mass spectrometry analyses.

PubMed

Chiesa, L; Nobile, M; Panseri, S; Sgoifo Rossi, C A; Pavlovic, R; Arioli, F

2014-12-10

The presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-boldenone conjugates, α-boldenone conjugates at concentrations higher than 2 ng mL(-1) and prednisolone above the cut-off level of 5 ng mL(-1) in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL(-1) for anabolic steroids, and 0.13 and 0.15 ng mL(-1) as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection and characterization of urinary metabolites of boldione by LC-MS/MS. Part I: Phase I metabolites excreted free, as glucuronide and sulfate conjugates, and released after alkaline treatment of the urine.

PubMed

Gómez, C; Pozo, O J; Fabregat, A; Marcos, J; Deventer, K; Van Eenoo, P; Segura, J; Ventura, R

2012-10-01

Boldione (1,4-androstadien-3,17-dione) is included in the list of prohibited substances, issued by the World Anti-Doping Agency (WADA). Endogenous production of low concentrations of boldione has also been reported. The objective of this study was to assess boldione metabolism in humans. Detection of boldione metabolites was accomplished by analysis by liquid chromatography coupled to tandem mass spectrometry of urine samples obtained after administration of the drug and subjected to different sample preparation procedures to analyze the different metabolic fractions (free, glucuronides, sulpfates and released in basic media). In addition to boldione, eight metabolites were detected in the free fraction. Four of them were identified by comparison with standards: 6β-hydroxy-boldenone (M3), androsta-1,4,6-triene-3,17-dione (M5), (5α)-1-androstenedione (M6) and (5α)-1-testosterone (M8). Metabolite M7 was identified as the 5β-isomer of 1-androstenedione, and metabolites M1, M2 and M4 were hydroxylated metabolites and tentative structures were proposed based on mass spectrometric data. After β-glucuronidase hydrolysis, five additional metabolites excreted only as conjugates with glucuronic acid were detected: boldenone, (5β)-1-testosterone (M9), and three metabolites resulting from reduction of the 3-keto group. Boldenone, epiboldenone, and hydroxylated metabolites of boldione, boldenone and 1-testosterone were detected as conjugates with sulfate. In addition, boldione and seven metabolites (boldenone, M2, M3, M4, M5, M7 and M9) increased their concentration in urine after treatment of the urine in alkaline conditions. In summary, 15 boldione metabolites were detected in all fractions. The longer detection time was observed for metabolite M4 after alkaline treatment of the urine, which was detected up to 5 days after boldione administration. Copyright © 2012 John Wiley & Sons, Ltd.

Determination of α- and β-boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis.

PubMed

Chiesa, Luca; Pavlovic, Radmila; Dusi, Guglielmo; Pasquale, Elisa; Casati, Alessio; Panseri, Sara; Arioli, Francesco

2015-01-01

The debate about the origins of boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-boldenone conjugate, is considered fully compliant up to 2 ng mL(-1). The conjugated form of β-boldenone must be absent. In recent years, the literature about boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β-boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β-boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-boldenone sulfate and glucuronide, α- and β-boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL(-1), the detection capabilities (CCβ) between 0.08 and 0.1 ng mL(-1). Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of boldenone in a trial on a significant number of animals. Copyright © 2014 Elsevier B.V. All rights reserved.

Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Increase in gap-junctional intercellular communications (GJIC) of normal human dermal fibroblasts (NHDF) on surfaces coated with high-molecular-weight hyaluronic acid (HMW HA).

PubMed

Park, Jeong Ung; Tsuchiya, Toshie

2002-06-15

Normal human dermal fibroblast (NHDF) cells were used to detect differences in gap-junctional intercellular communication (GJIC) by hyaluronic acid (HA), a linear polymer built from repeating disaccharide units that consist of N-acetyl-D-glucosamine (GlcNa) and D-glucuronic acid (GlcA) linked by a beta 1-4 glycosidic bond. The NHDF cells were cultured with different molecular weights (MW) of HA for 4 days. The rates of cell attachment in dishes coated with high-molecular-weight (HMW; 310 kDa or 800 kDa) HA at 2 mg/dish were significantly reduced at an early time point compared with low-molecular-weight (LMW; 4.8 kDa or 48 kDa) HA with the same coating amounts. HA-coated surfaces were observed by atomic force microscopy (AFM) under air and showed that HA molecules ran parallel in the dish coated with LMW HA and had an aggregated island structure in the dish coated with HMW HA surfaces. The cell functions of GJIC were assayed by a scrape-loading dye transfer (SLDT) method using a dye solution of Lucifer yellow. Promotion of the dye transfer was clearly obtained in the cell monolayer grown on the surface coated with HMW HA. These results suggest that HMW HA promotes the function of GJIC in NHDF cells. In contrast, when HMW HA was added to the monolayer of NHDF cells, the functions of GJIC clearly were lowered in comparison with the cells grown in the control dish or with those grown on the surface of HMW HA. Therefore it is concluded that the MW size of HA and its application method are important factors for generating biocompatible tissue-engineered products because of the manner in which the GJIC participates in cell differentiation and cell growth rate. Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 541-547, 2002

Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

PubMed

Zhao, Linlin; Krishnan, Sadagopan; Zhang, Yun; Schenkman, John B; Rusling, James F

2009-02-01

Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in causing liver carcinoma in humans, provided the glucuronidation pathway is active.

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

PubMed

De Santi, Concetta; Willassen, Nils Peder; Williamson, Adele

2016-01-01

The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate*

PubMed Central

Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-01-01

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. PMID:25122756

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

PubMed

Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

2014-10-03

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cotransformation of Trichoderma harzianum with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils†

PubMed Central

Bae, Yeoung-Seuk; Knudsen, Guy R.

2000-01-01

Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), β-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil. PMID:10653755

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum.

PubMed

Michikawa, Mari; Ichinose, Hitomi; Momma, Mitsuru; Biely, Peter; Jongkees, Seino; Yoshida, Makoto; Kotake, Toshihisa; Tsumuraya, Yoichi; Withers, Stephen G; Fujimoto, Zui; Kaneko, Satoshi

2012-04-20

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.

Enterolactone glucuronide and β-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment.

PubMed

Di, Yunyun; Ji, Shaoping; Wolf, Philipp; Krol, Ed S; Alcorn, Jane

2017-08-01

Evidence from preclinical and animal studies demonstrated an anticancer effect of flaxseed lignans, particularly enterolactone (ENL), against prostate cancer. However, extensive first-pass metabolism following oral lignan consumption results in their systemic availability primarily as glucuronic acid conjugates (ENL-Gluc) and their modest in vivo effects. To overcome the unfavorable pharmacokinetics and improve their effectiveness in prostate cancer, antibody-directed enzyme prodrug therapy (ADEPT) might offer a novel strategy to allow for restricted activation of ENL from circulating ENL-Gluc within the tumor environment. The anti-prostate-specific membrane antigen (PSMA) antibody D7 was fused with human β-glucuronidase (hβG) via a flexible linker. The binding property of the fusion construct, D7-hβG, against purified or cell surface PSMA was determined by flow cytometry and Octet Red 384 system, respectively, with a binding rate constant, K d, of 2.5 nM. The enzymatic activity of D7-hβG was first tested using the probe, 4-methylumbelliferone glucuronide. A 3.8-fold greater fluorescence intensity was observed at pH 4.5 at 2 h compared with pH 7.4. The ability of D7-hβG to activate ENL from ENL-Gluc was tested and detected using LC-MS/MS. Enhanced generation of ENL was observed with increasing ENL-Gluc concentrations and reached 3613.2 ng/mL following incubation with 100 μM ENL-Gluc at pH 4.5 for 0.5 h. D7-hβG also decreased docetaxel IC 50 value from 23 nM to 14.9 nM in C4-2 cells. These results confirmed the binding and activity of D7-hβG and additional in vitro investigation is needed to support the future possibility of introducing this ADEPT system to animal models.

Metabolic Responses to Low Temperature of Three Peach Fruit Cultivars Differently Sensitive to Cold Storage

PubMed Central

Brizzolara, Stefano; Hertog, Maarten; Tosetti, Roberta; Nicolai, Bart; Tonutti, Pietro

2018-01-01

Refrigerated storage is widely applied in order to maintain peach quality but it can also induce chilling injuries (CIs) such as flesh browning and bleeding, and mealiness. Peach fruit from three cultivars (‘Red Haven’, RH, ‘Regina di Londa’, RL, and ‘Flaminia’, FL) were stored for 4 weeks under low temperatures (0.5 and 5.5°C). GC-MS was employed to study changes in both metabolome and volatilome induced by cold storage in the mesocarp. CIs were assessed both at the end of each week of storage and after subsequent shelf-life (SL) at 20°C. Flesh browning and mealiness appeared to be more related to 5.5°C storage, while flesh bleeding revealed high incidence following 0.5°C storage. Compared to RL and FL, RH showed a marked lower incidence of CIs. Multivariate statistical analyses indicate that RH peaches indeed differ from RL and FL in particular when considering data from samples collected at the end of the cold storage. Common and divergent responses have been identified in terms of metabolic responses to the applied low temperatures. In all three cultivars raffinose, glucose-6P, fucose, xylose, sorbitol, GABA, epicatechin, catechin, and putrescine markedly increased during cold storage, while citramalic, glucuronic, mucic and shikimic acids decreased. Among volatile organic compounds (VOCs), aldehydes and alcohols generally accumulated more under low temperature conditions while esters and lactones evolved during subsequent SL. The main cultivar differences developed after cold storage during SL although some common responses (e.g., an increased production of ethyl acetate) were observed. The lower levels of flesh browning and bleeding displayed by RH peaches were related to compounds with antioxidant activity, or acting as osmotic protectants and membrane stabilizer. Indeed, RH showed higher levels of amino acids and urea, together with a marked increase in putrescine, sorbitol, maltitol, myoinositol and sucrose detected during storage and SL. PMID:29892309

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry.

PubMed

Lou, Yan; Zheng, Jinqi; Wang, Baohong; Zhang, Xingguo; Zhang, Xia; Zeng, Su

2014-01-01

Stellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. Twenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg(-1)), one intravenous injection group (5 mgkg(-1)), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and β-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. A total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. The present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites. © 2013 Elsevier Ireland Ltd. All rights reserved.

Electric field-assisted formation of organically modified hydroxyapatite (ormoHAP) spheres in carboxymethylated gelatin gels.

PubMed

Heinemann, C; Heinemann, S; Kruppke, B; Worch, H; Thomas, J; Wiesmann, H P; Hanke, T

2016-10-15

A biomimetic strategy was developed in order to prepare organically modified hydroxyapatite (ormoHAP) with spherical shape. The technical approach is based on electric field-assisted migration of calcium ions and phosphate ions into a hydrogel composed of carboxymethylated gelatin. The electric field as well as the carboxymethylation using glucuronic acid (GlcA) significantly accelerates the mineralization process, which makes the process feasible for lab scale production of ormoHAP spheres and probably beyond. A further process was developed for gentle separation of the ormoHAP spheres from the gelatin gel without compromising the morphology of the mineral. The term ormoHAP was chosen since morphological analyses using electron microscopy (SEM, TEM) and element analysis (EDX, FT-IR, XRD) confirmed that carboxymethylated gelatin molecules use to act as organic templates for the formation of nanocrystalline HAP. The hydroxyapatite (HAP) crystals self-organize to form hollow spheres with diameters ranging from 100 to 500nm. The combination of the biocompatible chemical composition and the unique structure of the nanocomposites is considered to be a useful basis for future applications in functionalized degradable biomaterials. A novel bioinspired mineralization process was developed based on electric field-assisted migration of calcium and phosphate ions into biochemically carboxymethylated gelatin acting as organic template. Advantages over conventional hydroxyapatite include particle size distribution and homogeneity as well as achievable mechanical properties of relevant composites. Moreover, specifically developed calcium ion or phosphate ion release during degradation can be useful to adjust the fate of bone cells in order to manipulate remodeling processes. The hollow structure of the spheres can be useful for embedding drugs in the core, encapsulated by the highly mineralized outer shell. In this way, controlled drug release could be achieved, which enables advanced strategies for threating bone-related diseases, e.g. osteoporosis and multiple myeloma. Copyright © 2016. Published by Elsevier Ltd.

Versatility of the Burkholderia cepacia Complex for the Biosynthesis of Exopolysaccharides: A Comparative Structural Investigation

PubMed Central

Silipo, Alba; Lanzetta, Rosa; Liut, Gianfranco; Rizzo, Roberto; Cescutti, Paola

2014-01-01

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable. PMID:24722641

Versatility of the Burkholderia cepacia complex for the biosynthesis of exopolysaccharides: a comparative structural investigation.

PubMed

Cuzzi, Bruno; Herasimenka, Yury; Silipo, Alba; Lanzetta, Rosa; Liut, Gianfranco; Rizzo, Roberto; Cescutti, Paola

2014-01-01

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.

PubMed

Rosa, Lorena; Ravanal, María Cristina; Mardones, Wladimir; Eyzaguirre, Jaime

2013-05-01

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91,725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100,000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5-5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introducing the "TCDD-inducible AhR-Nrf2 gene battery".

PubMed

Yeager, Ronnie L; Reisman, Scott A; Aleksunes, Lauren M; Klaassen, Curtis D

2009-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 mug/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid-synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine.

PubMed

Buechler, Jochen; Schwab, Matthias; Mikus, Gerd; Fischer, Beate; Hermle, Leo; Marx, Claudia; Grön, Georg; Spitzer, Manfred; Kovar, Karl Artur

2003-08-15

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.

Aldouronate utilization in Paenibacillus sp. strain JDR-2: Physiological and enzymatic evidence for coupling of extracellular depolymerization and intracellular metabolism.

PubMed

Nong, Guang; Rice, John D; Chow, Virginia; Preston, James F

2009-07-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 alpha-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/beta-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX(1)), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX(3)), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX(n)). The average rates of utilization of MeGAX(n), MeGAX(1), and MeGAX(3) were 149.8, 59.4, and 54.3 microg xylose equivalents.ml(-1).h(-1), respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX(1) and MeGAX(3), releasing 4-O-methyl-d-glucuronate alpha-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX(3), to xylose and xylobiose. The ability to utilize MeGAX(1) provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX(n) than that of MeGAX(3) indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

Metabolic Responses to Low Temperature of Three Peach Fruit Cultivars Differently Sensitive to Cold Storage.

PubMed

Brizzolara, Stefano; Hertog, Maarten; Tosetti, Roberta; Nicolai, Bart; Tonutti, Pietro

2018-01-01

Refrigerated storage is widely applied in order to maintain peach quality but it can also induce chilling injuries (CIs) such as flesh browning and bleeding, and mealiness. Peach fruit from three cultivars ('Red Haven', RH, 'Regina di Londa', RL, and 'Flaminia', FL) were stored for 4 weeks under low temperatures (0.5 and 5.5°C). GC-MS was employed to study changes in both metabolome and volatilome induced by cold storage in the mesocarp. CIs were assessed both at the end of each week of storage and after subsequent shelf-life (SL) at 20°C. Flesh browning and mealiness appeared to be more related to 5.5°C storage, while flesh bleeding revealed high incidence following 0.5°C storage. Compared to RL and FL, RH showed a marked lower incidence of CIs. Multivariate statistical analyses indicate that RH peaches indeed differ from RL and FL in particular when considering data from samples collected at the end of the cold storage. Common and divergent responses have been identified in terms of metabolic responses to the applied low temperatures. In all three cultivars raffinose, glucose-6P, fucose, xylose, sorbitol, GABA, epicatechin, catechin, and putrescine markedly increased during cold storage, while citramalic, glucuronic, mucic and shikimic acids decreased. Among volatile organic compounds (VOCs), aldehydes and alcohols generally accumulated more under low temperature conditions while esters and lactones evolved during subsequent SL. The main cultivar differences developed after cold storage during SL although some common responses (e.g., an increased production of ethyl acetate) were observed. The lower levels of flesh browning and bleeding displayed by RH peaches were related to compounds with antioxidant activity, or acting as osmotic protectants and membrane stabilizer. Indeed, RH showed higher levels of amino acids and urea, together with a marked increase in putrescine, sorbitol, maltitol, myoinositol and sucrose detected during storage and SL.

Monitoring OH-PCBs in PCB transport worker's urine as a non-invasive exposure assessment tool.

PubMed

Haga, Yuki; Suzuki, Motoharu; Matsumura, Chisato; Okuno, Toshihiro; Tsurukawa, Masahiro; Fujimori, Kazuo; Kannan, Narayanan; Weber, Roland; Nakano, Takeshi

2018-06-01

In this study, we analyzed hydroxylated polychlorinated biphenyls (OH-PCBs) in urine of both PCB transport workers and PCB researchers. A method to monitor OH-PCB in urine was developed. Urine was solid-phase extracted with 0.1% ammonia/ methanol (v/v) and glucuronic acid/sulfate conjugates and then decomposed using β-glucuronidase/arylsulfatase. After alkaline digestion/derivatization, the concentration of OH-PCBs was determined by HRGC/HRMS-SIM. In the first sampling campaign, the worker's OH-PCB levels increased several fold after the PCB waste transportation work, indicating exposure to PCBs. The concentration of OH-PCBs in PCB transport workers' urine (0.55~11 μg/g creatinine (Cre)) was higher than in PCB researchers' urine (< 0.20 μg/g Cre). However, also a slight increase of OH-PCBs was observed in the researchers doing the air sampling at PCB storage area. In the second sampling, after recommended PCB exposure reduction measures had been enacted, the worker's PCB levels did not increase during handling of PCB equipment. This suggests that applied safety measures improved the situation. Hydroxylated trichlorobiphenyls (OH-TrCBs) were identified as a major homolog of OH-PCBs in urine. Also, hydroxylated tetrachlorobiphenyls (OH-TeCBs) to hydroxylated hexachlorobiphenyls (OH-HxCBs) were detected. For the sum of ten selected major indicators, a strong correlation to total OH-PCBs were found and these can possibly be used as non-invasive biomarkers of PCB exposure in workers managing PCB capacitors and transformer oils. We suggest that monitoring of OH-PCBs in PCB management projects could be considered a non-invasive way to detect exposure. It could also be used as a tool to assess and improve PCB management. This is highly relevant considering the fact that in the next 10 years, approx. 14 million tons of PCB waste need to be managed. Also, the selected populations could be screened to assess whether exposure at work, school, or home has taken place.

Structural and Biochemical Characterization of Glycoside Hydrolase Family 79 β-Glucuronidase from Acidobacterium capsulatum

PubMed Central

Michikawa, Mari; Ichinose, Hitomi; Momma, Mitsuru; Biely, Peter; Jongkees, Seino; Yoshida, Makoto; Kotake, Toshihisa; Tsumuraya, Yoichi; Withers, Stephen G.; Fujimoto, Zui; Kaneko, Satoshi

2012-01-01

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-d-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)8-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu173 (acid base) and Glu287 (nucleophile), consistent with the retaining mechanism demonstrated by 1H NMR analysis. Glu45, Tyr243, Tyr292–Gly294, and Tyr334 form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln293 and Gly294 and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ∼200-fold lower (kcat/Km) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr334 in recognition of the C6 position of GlcA. The involvement of Glu45 in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (kcat/Km) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower. PMID:22367201

Inhibition of UDP-glucose dehydrogenase by 6-thiopurine and its oxidative metabolites: Possible mechanism for its interaction within the bilirubin excretion pathway and 6TP associated liver toxicity.

PubMed

Weeramange, Chamitha J; Binns, Cassie M; Chen, Chixiang; Rafferty, Ryan J

2018-03-20

6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a K i of 288 μM with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a K i of 7 μM. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with K i values of 54 and 14 μM, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities. Copyright © 2017 Elsevier B.V. All rights reserved.

Creosote bush (Larrea tridentata) resin increases water demands and reduces energy availability in desert woodrats (Neotoma lepida).

PubMed

Mangione, Antonio M; Dearing, M Denise; Karasov, William H

2004-07-01

Although many plant secondary compounds are known to have serious consequences for herbivores, the costs of processing them are generally unknown. Two potential costs of ingestion and detoxification of secondary compounds are elevation of the minimum drinking water requirement and excretion of energetically expensive metabolites (i.e., glucuronides) in the urine. To address these impacts, we studied the costs of ingestion of resin from creosote bush (Larrea tridentata) on desert woodrats (Neotoma lepida). The following hypotheses were tested: ingestion of creosote resin by woodrats (1) increases minimum water requirement and (2) reduces energy available by increasing fecal and urinary energy losses. We tested the first hypothesis, by measuring the minimum water requirement of woodrats fed a control diet with and without creosote resin. Drinking water was given in decreasing amounts until woodrats could no longer maintain constant body mass. In two separate experiments, the minimum drinking water requirement of woodrats fed resin was higher than that of controls by 18-30% (about 1-1.7 ml/d). We tested several potential mechanisms of increased water loss associated with the increase in water requirement. The rate of fecal water loss was higher in woodrats consuming resin. Neither urinary water nor evaporative water loss was affected by ingestion of resin. Hypothesis 2 was tested by measuring energy fluxes of woodrats consuming control vs. resin-treated diets. Woodrats on a resin diet had higher urinary energy losses and, thus, metabolized a lower proportion of the dietary energy than did woodrats on control diet. Fecal energy excretion was not affected by resin. The excretion of glucuronic acid represented almost half of the energy lost as a consequence of resin ingestion. The increased water requirement and energy losses of woodrats consuming a diet with resin could have notable ecological consequences.

Metabolomics in psoriatic disease: pilot study reveals metabolite differences in psoriasis and psoriatic arthritis

PubMed Central

Armstrong, April W.; Wu, Julie; Johnson, Mary Ann; Grapov, Dmitry; Azizi, Baktazh; Dhillon, Jaskaran; Fiehn, Oliver

2014-01-01

Importance: While “omics” studies have advanced our understanding of inflammatory skin diseases, metabolomics is mostly an unexplored field in dermatology. Objective: We sought to elucidate the pathogenesis of psoriatic diseases by determining the differences in metabolomic profiles among psoriasis patients with or without psoriatic arthritis and healthy controls. Design: We employed a global metabolomics approach to compare circulating metabolites from patients with psoriasis, psoriasis and psoriatic arthritis, and healthy controls. Setting: Study participants were recruited from the general community and from the Psoriasis Clinic at the University of California Davis in United States. Participants: We examined metabolomic profiles using blood serum samples from 30 patients age and gender matched into three groups: 10 patients with psoriasis, 10 patients with psoriasis and psoriatic arthritis and 10 control participants. Main outcome(s) and measures(s): Metabolite levels were measured calculating the mean peak intensities from gas chromatography time-of-flight mass spectrometry. Results: Multivariate analyses of metabolomics profiles revealed altered serum metabolites among the study population. Compared to control patients, psoriasis patients had a higher level of alpha ketoglutaric acid (Pso: 288 ± 88; Control: 209 ± 69; p=0.03), a lower level of asparagine (Pso: 5460 ± 980; Control: 7260 ± 2100; p=0.02), and a lower level of glutamine (Pso: 86000 ± 20000; Control: 111000 ± 27000; p=0.02). Compared to control patients, patients with psoriasis and psoriatic arthritis had increased levels of glucuronic acid (Pso + PsA: 638 ± 250; Control: 347 ± 61; p=0.001). Compared to patients with psoriasis alone, patients with both psoriasis and psoriatic arthritis had a decreased level of alpha ketoglutaric acid (Pso + PsA: 186 ± 80; Pso: 288 ± 88; p=0.02) and an increased level of lignoceric acid (Pso + PsA: 442 ± 280; Pso: 214 ± 64; p=0.02). Conclusions and relevance: The metabolite differences help elucidate the pathogenesis of psoriasis and psoriatic arthritis and they may provide insights for therapeutic development. PMID:25580230

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

PubMed Central

Puhka, Maija; Takatalo, Maarit; Nordberg, Maria-Elisa; Valkonen, Sami; Nandania, Jatin; Aatonen, Maria; Yliperttula, Marjo; Laitinen, Saara; Velagapudi, Vidya; Mirtti, Tuomas; Kallioniemi, Olli; Rannikko, Antti; Siljander, Pia R-M; af Hällström, Taija Maria

2017-01-01

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 1010 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials. PMID:29109780

Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics

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

Tocchetti, Guillermo Nicolás

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a groupmore » of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs. - Highlights: • Intestinal MRP2 (ABCC2) expression and activity can be regulated by xenobiotics. • PXR and CAR are major MRP2 modulators through a transcriptional mechanism. • Rifampicin, spironolactone and carbamazepine among others up-regulate MRP2 via PXR. • MRP2 activity influences the availability and efficacy of drugs administered orally.« less

Back to the future with the AGP-Ca2+ flux capacitor.

PubMed

Lamport, Derek T A; Varnai, Peter; Seal, Charlotte E

2014-10-01

Arabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp-arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca(2+) when paired with its adjacent sidechain. AGPs bind Ca(2+) (Kd ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H(+)-ATPase generates a low periplasmic pH that dissociates AGP-Ca(2+) carboxylates (pka ∼3); the consequential large increase in free Ca(2+) drives entry into the cytosol via Ca(2+) channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca(2+) waves. This differs markedly from animals, in which cytosolic Ca(2+) originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca(2+) storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca(2+), hence an AGP-Ca(2+) oscillator. The novel concept of dynamic Ca(2+) recycling by an AGP-Ca(2+) oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca(2+) signalling and auxin. This accounts for the involvement of AGPs in plant morphogenesis, including tropic and nastic movements. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A metabolomics strategy to explore urinary biomarkers and metabolic pathways for assessment of interaction between Danhong injection and low-dose aspirin during their synergistic treatment.

PubMed

Li, Jianping; Guo, Jianming; Shang, Erxin; Zhu, Zhenhua; Zhu, Kevin Yue; Li, Shujiao; Zhao, Buchang; Jia, Lifu; Zhao, Jing; Tang, Zhishu; Duan, Jinao

2016-07-15

The drug combination of Danhong injection (DHI) and low-dose aspirin (ASA) was frequently applied for the treatment of cardiovascular and cerebrovascular diseases. Due to the drug interactions, a lot of potential benefits and risks might exist side by side in the course of combination therapy. However, there had been no studies of interaction between DHI and ASA. Metabolomics was a powerful tool to explore endogenous biomarkers and metabolic pathways. In present study, metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis was performed to provide insight into understanding the interaction between DHI and low-dose ASA. Eleven potential biomarkers of three types were identified and seven metabolic pathways were constructed. The results showed that the interaction between DHI and low-dose ASA during synergistic treatment indeed affected some key endogenous biomarkers and metabolic pathways, which could not happen when DHI or low-dose ASA was used alone. The quality and quantity of endogenous metabolite were both influenced by interaction between DHI and low-dose ASA. In details, the amount of flavin mononucleotide, L-2, 4-diaminobutyric acid (DABA) and 4-aminohippuric acid were significantly increased. On the contrary, the amount of 3-methyluridine, 4, 6-dihydroxyquinoline, cortolone-3-glucuronide, and serotonin were significantly decreased. Furthermore, O-phosphotyrosine, 3-methyl-2-butenal, indoxyl sulfate and dolichyl diphosphate were disappeared in urine. As to metabolic pathways, riboflavin metabolism, pentose and glucuronate interconversions, and tryptophan metabolism were all significantly influenced. The emerging alterations of biomarkers and metabolic pathways were associated with a lot of drugs and diseases based on literature researches, which might influence the co-administration of other drugs or the treatments of relevant diseases. Our paper presented some hints to uncover the mechanism of interaction between DHI and low-dose ASA, which would provide some references for application of DHI and low-dose ASA combination. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: insights into the sugar sequences with bioactivities.

PubMed

Mizumoto, Shuji; Murakoshi, Saori; Kalayanamitra, Kittiwan; Deepa, Sarama Sathyaseelan; Fukui, Shigeyuki; Kongtawelert, Prachya; Yamada, Shuhei; Sugahara, Kazuyuki

2013-02-01

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

NASA Astrophysics Data System (ADS)

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

2007-12-01

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

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

PubMed Central

Dazzo, F B; Hubbell, D H

1975-01-01

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin. Images PMID:55100

Composition of Glycosaminoglycans in Elasmobranchs including Several Deep-Sea Sharks: Identification of Chondroitin/Dermatan Sulfate from the Dried Fins of Isurus oxyrinchus and Prionace glauca

PubMed Central

Higashi, Kyohei; Takeuchi, Yoshiki; Mukuno, Ann; Tomitori, Hideyuki; Miya, Masaki; Linhardt, Robert J.; Toida, Toshihiko

2015-01-01

Shark fin, used as a food, is a rich source of glycosaminoglyans (GAGs), acidic polysaccharides having important biological activities, suggesting their nutraceutical and pharmaceutical application. A comprehensive survey of GAGs derived from the fin was performed on 11 elasmobranchs, including several deep sea sharks. Chondroitin sulfate (CS) and hyaluronic acid (HA) were found in Isurus oxyrinchus, Prionace glauca, Scyliorhinus torazame, Deania calcea, Chlamydoselachus anguineus, Mitsukurina owatoni, Mustelus griseus and Dasyatis akajei, respectively. CS was only found from Chimaera phantasma, Dalatias licha, and Odontaspis ferox, respectively. Characteristic disaccharide units of most of the CS were comprised of C- and D-type units. Interestingly, substantial amount of CS/dermatan sulfate (DS) was found in the dried fin (without skin and cartilage) of Isurus oxyrinchus and Prionace glauca. 1H-NMR analysis showed that the composition of glucuronic acid (GlcA) and iduronic acid (IdoA) in shark CS/DS was 41.2% and 58.8% (Isurus oxyrinchus), 36.1% and 63.9% (Prionace glauca), respectively. Furthermore, a substantial proportion of this CS/DS consisted of E-, B- and D-type units. Shark CS/DS stimulated neurite outgrowth of hippocampal neurons at a similar level as DS derived from invertebrate species. Midkine and pleiotrophin interact strongly with CS/DS from Isurus oxyrinchus and Prionace glauca, affording Kd values of 1.07 nM, 6.25 nM and 1.70 nM, 1.88 nM, respectively. These results strongly suggest that the IdoA-rich domain of CS/DS is required for neurite outgrowth activity. A detailed examination of oligosaccharide residues, produced by chondroitinase ACII digestion, suggested that the IdoA and B-type units as well as A- and C-type units were found in clusters in shark CS/DS. In addition, it was discovered that the contents of B-type units in these IdoA-rich domain increased in a length dependent manner, while C- and D-type units were located particularly in the immediate vicinity of the IdoA-rich domain. PMID:25803296

The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles.

PubMed

Ramos, S; Kaback, H R

1977-03-08

In the previous paper [ramos, S., and Kaback, H.R. (1977), Biochemistry 16 (preceding paper in this issue)], it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces [i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)] were described. In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined. Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane. Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate). Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH. In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH. Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity. The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport.

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism ▿

PubMed Central

Nong, Guang; Rice, John D.; Chow, Virginia; Preston, James F.

2009-01-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 α-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/β-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX1), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX3), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAXn). The average rates of utilization of MeGAXn, MeGAX1, and MeGAX3 were 149.8, 59.4, and 54.3 μg xylose equivalents·ml−1·h−1, respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX1 and MeGAX3, releasing 4-O-methyl-d-glucuronate α-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX3, to xylose and xylobiose. The ability to utilize MeGAX1 provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAXn than that of MeGAX3 indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals. PMID:19395566

Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.

PubMed

Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

2004-10-01

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

Negative electrospray ionization mass spectrometry: a method for sequencing and determining linkage position in oligosaccharides from branched hemicelluloses.

PubMed

Quéméner, Bernard; Vigouroux, Jacqueline; Rathahao, Estelle; Tabet, Jean Claude; Dimitrijevic, Aleksandra; Lahaye, Marc

2015-01-01

Xyloglucans of apple, tomato, bilberry and tamarind were hydrolyzed by commercial endo β-1-4-D-endoglucanase. The xylo-gluco-oligosaccharides (XylGos) released were separated on CarboPac PA 200 column in less than 15 min, and, after purification, they were structurally characterized by negative electrospray ionization mass spectrometry using a quadrupole time-of-flight (ESI-Q-TOF), a hybrid linear ion trap (LTQ)/Orbitrap and a hybrid quadrupole Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. In order to corroborate the fragmentation routes observed on XylGos, some commercial galacto-manno-oligosaccharides (GalMOs) and glucurono-xylo-oligosaccharides were also studied. The fragmentation pathways of the ionized GalMos were similar to those of XylGos ones. The product ion spectra were mainly characterized by prominent double cleavage (D) ions corresponding to the entire inner side chains. The directed fragmentation from the reducing end to the other end was observed for the main glycosylated backbone but also for the side-chains, allowing their complete sequencing. Relevant cross-ring cleavage ions from (0,2)X(j)-type revealed to be diagnostic of the 1-2-linked- glycosyl units from XylGos together with the 1-2-linked glucuronic acid unit from glucuronoxylans. Resonant activation in the LTQ Orbitrap allowed not only determining the type of all linkages but also the O-acetyl group location on fucosylated side-chains. Moreover, the fragmentation of the different side chains using the MS(n) capabilities of the LTQ/Orbitrap analyzer also allowed differentiating terminal arabinosyl and xylosyl substituents inside S and U side-chains of XylGos, respectively. The CID spectra obtained were very informative for distinction of isomeric structures differing only in their substitution pattern. These features together makes the fragmentation in negative ionization mode a relevant and powerful technique useful to highlight the subtle structural changes generally observed during the development of plant organs such as during fruit ripening and for the screening of cell wall mutants with altered hemicellulose structure. Copyright © 2015 John Wiley & Sons, Ltd.

Transcriptome Analysis and Ultrastructure Observation Reveal that Hawthorn Fruit Softening Is due to Cellulose/Hemicellulose Degradation

PubMed Central

Xu, Jiayu; Zhao, Yuhui; Zhang, Xiao; Zhang, Lijie; Hou, Yali; Dong, Wenxuan

2016-01-01

Softening, a common phenomenon in many fruits, is a well coordinated and genetically determined process. However, the process of flesh softening during ripening has rarely been described in hawthorn. In this study, we found that ‘Ruanrou Shanlihong 3 Hao’ fruits became softer during ripening, whereas ‘Qiu JinXing’ fruits remained hard. At late developmental stages, the firmness of ‘Ruanrou Shanlihong 3 Hao’ fruits rapidly declined, and that of ‘Qiu JinXing’ fruits remained essentially unchanged. According to transmission electron microscopy, the middle lamella of ‘Qiu JinXing’ and ‘Ruanrou Shanlihong 3 Hao’ fruit flesh was largely degraded as the fruits matured. Microfilaments in ‘Qiu JinXing’ flesh were arranged close together and were deep in color, whereas those in ‘Ruanrou Shanlihong 3 Hao’ fruit flesh were arranged loosely, partially degraded and light in color. RNA-Seq analysis yielded approximately 46.72 Gb of clean data and 72,837 unigenes. Galactose metabolism and pentose and glucuronate interconversions are involved in cell wall metabolism, play an important role in hawthorn texture. We identified 85 unigenes related to the cell wall between hard- and soft-fleshed hawthorn fruits. Based on data analysis and real-time PCR, we suggest that β-GAL and PE4 have important functions in early fruit softening. The genes Ffase, Gns,α-GAL, PE63, XTH, and CWP, which are involved in cell wall degradation, are responsible for the different textures of hawthorn fruits. Thus, we hypothesize that the different textures of ‘Qiu JinXing’ and ‘Ruanrou Shanlihong 3 Hao’ fruits at maturity mainly result from cellulose/hemicelluloses degradation rather than from lamella degradation. Overall, we propose that different types of hydrolytic enzymes in cells interact to degrade the cell wall, resulting in ultramicroscopic Structure changes in the cell wall and, consequently, fruit softening. These results provide fundamental insight regarding the mechanisms by which hawthorn fruits acquire different textures and also lay a solid foundation for further research. PMID:27790234

Composition and metabolism of carbohydrates and lipids in Sparus aurata semen and its relation to viability expressed as sperm motility when activated.

PubMed

Lahnsteiner, Franz; Mansour, Nabil; Caberlotto, Stefano

2010-09-01

The present study investigated aspects of lipid and carbohydrate metabolism in Sparus aurata semen and tested the effect of lipids, carbohydrates and related metabolites on sperm viability using in vitro incubation experiments. Sparus aurata semen contained enzyme systems to metabolize sugars and lipids. Also key enzymes of the tricarboxylic acid cycle and enzymes involved in ATP metabolism were detected. When spermatozoa were incubated in sperm motility inhibiting saline solution for 48 h phospholipid levels decreased constantly and triglycerides levels during the first 24 h of incubation indicating that spermatozoa utilize lipids as energy resources. After 24 h triglycerides levels started to re-increase indicating a change in sperm metabolism, in particular the onset of triglycerides synthesis by the fatty acid synthase complex. In the incubation period from 0 to 24 h glucose levels were constant, and decreased thereafter. Glycogen levels did not change at all. Semen contained also considerable amounts of sialic acid, glucuronic acid and hexosamines, components of mucopolysaccharides. To find out whether lipids, carbohydrates, and related metabolites had a positive effect on sperm functionality semen was incubated together with the described compounds in sperm motility inhibiting saline solution and motility when activated was determined. In the control 37.2+/-10.1% of the spermatozoa were locally motile and 38.3+/-13.3% motile after 24 h, 36.4+/-5.2% were locally motile and 9.6+/-4.5% were motile after 48 h. The swimming velocity was 89.0+/-13.1 microm/s after 24 h and 61.3+/-12.6% after 48 h. Different types of lipids (arachidic acid, linoleic acid, and glycerol trimyristate) and metabolites acting as fuel for the tricarboxylic acid cycle (hydroxybutyrate, ketoglutarate, and pyruvate) had a positive effect on the sperm viability. Tested carbohydrates (fucose, galactose, glucosamine, glucose, glucoheptose, glycogen, and sialic acid) had no effect. Also lactate and fructose-6-phosphate had no effect on sperm viability while glucose-6-phosphate, oxalacetate, and phosphoglycerate had negative effects. 2010 Elsevier Inc. All rights reserved.

Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

PubMed Central

Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

2004-01-01

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed. PMID:15466046

Bauhinia championi (Benth.) Benth. polysaccharides upregulate Wnt/β-catenin signaling in chondrocytes.

PubMed

Li, Huiting; Li, Xihai; Liu, Guozhong; Chen, Jiashou; Weng, Xiaping; Liu, Fayuan; Xu, Huifeng; Liu, Xianxiang; Ye, Hongzhi

2013-12-01

Bauhinia championi (Benth.) Benth. polysaccharides (BCBPs), extracted from Bauhinia championi (Benth.) Benth., which has been used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA), are the bioactive constituents of Bauhinia championi (Benth.) rattan. However, the molecular mechanisms responsible for their effects on OA are poorly understood. The Wnt/β-catenin signaling pathway plays an important role in the proliferation of chondrocytes. In the present study, the effects of BCBPs on Wnt/β-catenin signaling in chondrocytes were investigated. BCBPs were obtained by hot-water extraction and identified by the modified high performance liquid chromatography (HPLC) method. Chondrocytes were isolated from the knees of Sprague‑Dawley rats and identified by type II collagen immunohistochemistry. The chondrocytes were treated with or without BCBPs for 48 h. Cell viability was evaluated by MTT assay. The mRNA and protein levels of Wnt-4, β-catenin, Frizzled-2, glycogen synthase kinase (GSK)-3β, cyclin D1 and collagen II were detected by western blot analysis and reverse transcription PCR (RT-PCR), respectively. We found that the BCBPs contained at least seven monosaccharides, including D-mannose, rhamnose, D-(+) glucuronic acid, D-(+) galacturonic acid, D-glucose, galactose and arabinose. The cell viability of the chondrocytes treated with 50, 100 and 200 µg/ml BCBPs was significantly higher than that of the chondroctyes in the control group (treated with 0 µg/ml BCBPs). Furthermore, compared with the control group, the mRNA and protein expression of Wnt-4, β-catenin, Frizzled-2 and cyclin D1 in the BCBP-treated groups markedly increased, whereas the mRNA and protein expression of GSK-3β significantly decreased. Of note, the dose of 100 µg/ml BCBPs was more effective than the dose of 50 µg/ml BCBPs and 200 µg/ml BCBPs. In addition, we found that treatment with BCBPs upregulated the protein levels of collagen II in the chondrocytes. These results indicate that BCBPs upregulate Wnt/β-catenin signaling, thus promoting chondrocyte proliferation.

FINAL REPORT

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

PETER, GARY F.

2014-07-16

Excellent progress was made in standardizing three complementary methods: Magnetic resonance imaging, x-ray micro CT, and MALDI imaging linear ion trap mass spectroscopy to image biomass and chemical, anatomical and functional changes that occur during pretreatment and hydrolysis. Magnetic resonance microscopy provides excellent images with as low as 5 uM resolution with hydrated biomass samples. We visualized dramatic changes in signal associated with the hydrolysis of the carbohydrates by strong acids. Quantitative diffusion approaches were used to probe more subtle structural changes in biomass. Diffusion tensor calculations reflect diffusion anisotropy and fractional anisotropy maps clearly show the longer range diffusionmore » within the vessels compared to within the fiber cells. The diffusion is increased along the cell walls of the vessels. Suggesting that further research with NMR imaging should be pursued. X-ray CT provides excellent images at as low as 3.5 uM resolution from dried biomass. Small increases in surface area, and decreases in local density have been quantified in with wood after mild pretreatments; these changes are expected to be underestimates of the hydrated wood, due to the ~12% shrinkage that occurs upon drying untreated wood. MALDI-MS spectra show high ion intensities at most mass to charge ratios in untreated and pretreated woody material. MALDI-MSn is required to improve specificity and reduce background for imaging. MALDI-TOF is not specific enough for carbohydrate identification. Using MALDI-LIT/MSn we can readily identify oligomeric glucans and xylans and their fragmentation patterns as well as those of the glucuronic acid side chains of birch 4-O-methyl glucuronxylan. Imaging of glucan and xylan oligomers show that many contain isobaric ions with different distributions, indicating again that MSn is needed for accurate imaging of lignocellulosic materials. We are now starting to integrate the three imaging methods by using the same set of biomass samples imaged with all three methods, and using common analytical software to quantify parameters from the three dimensional images. In addition to the proposed experiments, we conducted imaging studies with a novel TOF-SIMS instrument available through collaborations with the AMOLF goup led by Ron Heeren at the FOM Institute in Amersterdam, Netherlands. ToF-SIMS was used to image intact cross sections of Populus stems with high spatial resolution, chemically selectivity. ToF-SIMS images were correlated with fluorescence microscopy which allowed for more positive ion identification.« less

Toxicity and Detoxification Effects of Herbal Caowu via Ultra Performance Liquid Chromatography/Mass Spectrometry Metabolomics Analyzed using Pattern Recognition Method

PubMed Central

Yan, Yan; Zhang, Aihua; Dong, Hui; Yan, Guangli; Sun, Hui; Wu, Xiuhong; Han, Ying; Wang, Xijun

2017-01-01

Background: Caowu (Radix Aconiti kusnezoffii, CW), the root of Aconitum kusnezoffii Reichb., has widely used clinically in rheumatic arthritis, painful joints, and tumors for thousands of years. However, the toxicity of heart and central nervous system induced by CW still limited the application. Materials and Methods: Metabolomics was performed to identify the sensitive and reliable biomarkers and to characterize the phenotypically biochemical perturbations and potential mechanisms of CW-induced toxicity, and the detoxification by combinatorial intervention of CW with Gancao (Radix Glycyrrhizae) (CG), Baishao (Radix Paeoniae Alba) (CB), and Renshen (Radix Ginseng) (CR) was also analyzed by pattern recognition methods. Results: As a result, the metabolites were characterized and responsible for pentose and glucuronate interconversions, tryptophan metabolism, amino sugar and nucleotide sugar metabolism, taurine and hypotaurine metabolism, fructose and mannose metabolism, and starch and sucrose metabolism, six networks of which were the same to the metabolic pathways of Chuanwu (Radix Aconiti, CHW) group. The ascorbate and aldarate metabolism was also characterized by CW group. The urinary metabolomics also revealed CW-induced serious toxicity to heart and liver. Thirteen significant metabolites were identified and had validated as phenotypic toxicity biomarkers of CW, five biomarkers of which were commonly owned in Aconitum. The changes of toxicity metabolites obtained from combinatorial intervention of CG, CB, and CR also were analyzed to investigate the regulation degree of toxicity biomarkers adjusted by different combinatorial interventions at 6th month. Conclusion: Metabolomics analyses coupled with pattern recognition methods in the evaluation of drug toxicity and finding detoxification methods were highlighted in this work. SUMMARY Metabolomics was performed to characterize the biochemical potential mechanisms of Caowu toxicityThirteen significant metabolites were identified and validated as phenotypic toxicity biomarkers of CaowuMetabolite changes of toxicity obtained can be adjusted by different combinatorial interventions.Pattern recognition plot reflects the toxicity effects tendency of the urine metabolic fluctuations according to time after treatment of herbal Caowu. Abbreviations used: CW: Caowu (Radix Aconiti kusnezoffii); CHW: Chuanwu (Radix Aconiti); TCM: Traditional Chinese Medicine; CG: Caowu and Gancao; CB: Caowu and Baishao; CR: Caowu and Renshen; QC: Quality control; UPLC: Ultra performance liquid chromatography; MS: Mass spectrometry; PCA: Principal component analysis; PLS-DA: Partial least squares-discriminant analysis; OPLS: Orthogonal projection to latent structures analysis. PMID:29200734

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

Ogawa, E.; Suzuki, S.; Fukuda, R.

Pharmacological means to accelerate the elimination of Cs-137 introduced into the living organism are studied. Male dd mice and male Wistar rats are individually housed in metal metabolism cages, and provided with commercial solid diet and water. Radioactivity is determined in urine, and feces for 24 hours and 4 days after subcutaneous injection of a tracer dose of Cs/sup 137/Cl, and in various organs after sacrifices at the ends of these periods. Effects of various chemicals on these results are compared. Twenty five chemicals were investigated. They include inorganic Na salts such as Na bicarbonate, Na carbonate, Na suliate, Namore » thiosulfate, primary and secondary Na phosphates, and organic Na salts such as Na lactate, lactated Ringer, Na acetate, Na glucuronate, Na salt of thioctic acid, ATP Na, and Na pentobarbiturate. Na bicarbonate, Na phosphates, Na sulfate, and Na thiosulfate are found as eifective, especially Na bicarbonate, K bicarbonate shows scarcely any effect, nor do other K salts. It is therefore assumed that Cs will exchange with Na ion in the tubular cells. LiCl is found to accelerate the excretion of Cs-137 from mice and rats. This result is of interest with respect to the periodic law, since it is known that for the elimination of Sr-90, Ca salts are ineffective or slightly effective, whereas Mg salts are effective. Of the diuretics, chlorothiazide, which is considered to increase the excretion of K, does nor increase the elimination of Cs-137 in any dose. This result is different from that of Diamox, a diuretic of the same nature. Cardiac glycosides and xanthine derivatives are effective. Out of digitalis preparations, Digitamin (Shionogi), Digilanogen C (Fujisawa), Digosin (Chugai) are effective. Digitoxin and strospeside are ineffective, and after their application, retention of Cs-137 is observed in the heart muscle. G- strophanthin is ineffective in a smaller dose, but increases the elimination of Cs-137 in a larger dose. Caffeine and sodium benzoate are also effective, but theophylline and theobromine are not so effective. Out of osmotic diuretics, Na ferrocyanate alone increases fecal excretion of Cs-137, and decreases the retention in the body. NaI and KI have scarcely any effect. Phosphomolybdic acid inhibits the elimination of Cs-137 and increases its retention. The above mentioned effective chemicals are also investigated for effects on the elimination of Sr-90. (JAIF)« less

Acidicapsa borealis gen. nov., sp. nov. and Acidicapsa ligni sp. nov., subdivision 1 Acidobacteria from Sphagnum peat and decaying wood.

PubMed

Kulichevskaya, Irina S; Kostina, Lilia A; Valásková, Vendula; Rijpstra, W Irene C; Damsté, Jaap S Sinninghe; de Boer, Wietse; Dedysh, Svetlana N

2012-07-01

Two strains of subdivision 1 Acidobacteria, a pink-pigmented bacterium KA1(T) and a colourless isolate WH120(T), were obtained from acidic Sphagnum peat and wood under decay by the white-rot fungus Hyploma fasciculare, respectively. Cells of these isolates were Gram-negative-staining, non-motile, short rods, which were covered by large polysaccharide capsules and occurred singly, in pairs, or in short chains. Strains KA1(T) and WH120(T) were strictly aerobic mesophiles that grew between 10 and 33 °C, with an optimum at 22-28 °C. Both isolates developed under acidic conditions, but strain WH120(T) was more acidophilic (pH growth range 3.5-6.4; optimum, 4.0-4.5) than strain KA1(T) (pH growth range 3.5-7.3; optimum , 5.0-5.5). The preferred growth substrates were sugars. In addition, the wood-derived isolate WH120(T) grew on oxalate, lactate and xylan, while the peat-inhabiting acidobacterium strain KA1(T) utilized galacturonate, glucuronate and pectin. The major fatty acids were iso-C(15:0) and iso-C(17:1)ω8c; the cells also contained significant amounts of 13,16-dimethyl octacosanedioic acid. The quinone was MK-8. The DNA G+C contents of strains KA1(T) and WH120(T) were 54.1 and 51.7 mol%, respectively. Strains KA1(T) and WH120(T) displayed 97.8% 16S rRNA gene sequence similarity to each other. The closest recognized relatives were Acidobacterium capsulatum and Telmatobacter bradus (93.4-94.3% 16S rRNA gene sequence similarity). These species differed from strains KA1(T) and WH120(T) by their ability to grow under anoxic conditions, the absence of capsules, presence of cell motility and differing fatty acid composition. Based on these differences, the two new isolates are proposed as representing a novel genus, Acidicapsa gen. nov., and two novel species. Acidicapsa borealis gen. nov., sp. nov. is the type species for the new genus with strain KA1(T) (=DSM 23886(T)=LMG 25897(T)=VKM B-2678(T)) as the type strain. The name Acidicapsa ligni sp. nov. is proposed for strain WH120(T) (=LMG 26244(T)=VKM B-2677(T)=NCCB 100371(T)).

Alkaline/peracetic acid as a pretreatment of lignocellulosic biomass for ethanol fuel production

NASA Astrophysics Data System (ADS)

Teixeira, Lincoln Cambraia

Peracetic acid is a lignin oxidation pretreatment with low energy input by which biomass can be treated in a silo type system for improving enzymatic digestibility of lignocellulosic materials for ethanol production. Experimentally, ground hybrid poplar wood and sugar cane bagasse are placed in plastic bags and a peracetic acid solution is added to the biomass in different concentrations based on oven-dry biomass. The ratio of solution to biomass is 6:1; after initial mixing of the resulting paste, a seven-day storage period at about 20°C is used in this study. As a complementary method, a series of pre-pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide based on 4-methyl-glucuronic acid and acetyl content in the biomass is been performed before addition of peracetic acid. The alkaline solutions are added to the biomass in a ratio of 14:1 solution to biomass; the slurry is mixed for 24 hours at ambient temperature. The above procedures give high xylan content substrates. Consequently, xylanase/beta-glucosidase combinations are more effective than cellulase preparations in hydrolyzing these materials. The pretreatment effectiveness is evaluated using standard enzymatic hydrolysis and simultaneous saccharification and cofermentation (SSCF) procedures. Hybrid poplar wood pretreated with 15 and 21% peracetic acid based on oven-dry weight of wood gives glucan conversion yields of 76.5 and 98.3%, respectively. Sugar cane bagasse pretreated with the same loadings gives corresponding yields of 85.9 and 93.1%. Raw wood and raw bagasse give corresponding yields of 6.8 and 28.8%, respectively. The combined 6% NaOH/15% peracetic acid pretreatments increase the glucan conversion yields from 76.5 to 100.0% for hybrid poplar wood and from 85.9 to 97.6% for sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% are obtained from 6% NaOH/15% peracetic acid pretreated materials using recombinant Zymomonas mobilis CP4/pZB5. Peracetic acid pretreatment improves enzymatic digestibility of hybrid poplar wood and sugar cane bagasse. Based on reduction of acetyl groups in the two lignocellulosic materials, alkaline pre-pretreatments are helpful in reducing peracetic acid requirements in the pretreatment and consequently diminishing growth inhibition of the bacteria that was observed using higher peracetic acid loadings.

Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows.

PubMed

Schwartz-Zimmermann, Heidi E; Hametner, Christian; Nagl, Veronika; Fiby, Iris; Macheiner, Lukas; Winkler, Janine; Dänicke, Sven; Clark, Erica; Pestka, James J; Berthiller, Franz

2017-12-01

The Fusarium mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereal-based food and feed. Mammals metabolize DON by conjugation to glucuronic acid (GlcAc), the extent and regioselectivity of which is species-dependent. So far, only DON-3-glucuronide (DON-3-GlcAc) and DON-15-GlcAc have been unequivocally identified as mammalian DON glucuronides, and DON-7-GlcAc has been proposed as further DON metabolite. In the present work, qualitative HPLC-MS/MS analysis of urine samples of animals treated with DON (rats: 2 mg/kg bw, single bolus, gavage; mice: 1 mg/kg bw, single i.p. injection; pigs: 74 µg/kg bw, single bolus, gavage; cows: 5.2 mg DON/kg dry mass, oral for 13 weeks) revealed additional DON and deepoxy-DON (DOM) glucuronides. To elucidate their structures, DON and DOM were incubated with human (HLM) and rat liver microsomes (RLM). Besides the expected DON/DOM-3- and 15-GlcAc, minor amounts of four DON- and four DOM glucuronides were formed. Isolation and enzymatic hydrolysis of four of these compounds yielded iso-DON and iso-DOM, the identities of which were eventually confirmed by NMR. Incubation of iso-DON and iso-DOM with RLM and HLM yielded two main glucuronides for each parent compound, which were isolated and identified as iso-DON/DOM-3-GlcAc and iso-DON/DOM-8-GlcAc by NMR. Iso-DON-3-GlcAc, most likely misidentified as DON-7-GlcAc in the literature, proved to be a major DON metabolite in rats and a minor metabolite in pigs. In addition, iso-DON-8-GlcAc turned out to be one of the major DON metabolites in mice. DOM-3-GlcAc was the dominant DON metabolite in urine of cows and an important DON metabolite in rat urine. Iso-DOM-3-GlcAc was detected in urine of DON-treated rats and cows. Finally, DON-8,15-hemiketal-8-glucuronide, a previously described by-product of DON-3-GlcAc production by RLM, was identified in urine of DON-exposed mice and rats. The discovery of several novel DON-derived glucuronides in animal urine requires adaptation of the currently used methods for DON-biomarker analysis.

Phylogenetic analysis of pectin-related gene families in Physcomitrella patens and nine other plant species yields evolutionary insights into cell walls

PubMed Central

2014-01-01

Background Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families. Results Contrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families. Conclusions Several gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution. PMID:24666997

Effects and mechanisms of Shaofu-Zhuyu decoction and its major bioactive component for Cold - Stagnation and Blood - Stasis primary dysmenorrhea rats.

PubMed

Huang, Xiaochen; Su, Shulan; Duan, Jin-Ao; Sha, Xiuxiu; Zhu, Kavin Yue; Guo, Jianming; Yu, Li; Liu, Pei; Shang, Erxin; Qian, Dawei

2016-06-20

Traditional Chinese medicine (TCM) is used under the guidance of the theory of traditional Chinese medical sciences in clinical application. The Chinese herbal formula, Shaofu Zhuyu decoction (SFZYD), is considered as an effective prescription for treating Cold - Stagnation and Blood - Stasis (CSBS) primary dysmenorrhea. The previous studies showed the SFZYD exhibited significant anti-inflammation and analgesic effect. In this present study the metabolomics of CSBS primary dysmenorrhea diseased rats and the cytokine transcription in PHA stimulated-PBMC were investigated to explore the effects and mechanisms. Explore a valuable insight into the effects and mechanisms of SFZYD on Cold - Stagnation and Blood - Stasis primary dysmenorrhea rats. We established CSBS primary dysmenorrhea diseased rats according the clinical symptoms. A targeted tandem mass spectrometry (MS/MS)-based metabolomic platform was used to evaluate the metabolic profiling changes and the intervention effects by SFZYD. The PBMC cell was adopted to explore the mechanisms by analyzing the signaling pathway evaluated by expression of inflammatory cytokines, c-jun and c-fos and corresponding phosphorylation levels. Estradiol, oxytocin, progesterone, endothelin, β-endorphin and PGF2α were restored back to the normal level after the treatment of SFZYD. Total twenty-five metabolites (10 in plasma and 15 in urine), up-regulated or down-regulated, were identified. These identified biomarkers underpinning the metabolic pathway including pentose and glucuronate interconversions, steroid hormone biosynthesis, and glycerophospholipid metabolism are disturbed in model rats. Among these metabolites, twenty one potential biomarkers were regulated after SFZYD treated. The compound of paeoniflorin, a major bioactive compound in SFZYD, was proved to regulate the MAPK signaling pathway by inhibiting the expression of IL-1β, IL-2, IL-10, IL-12, TNFα, INFγ, c-jun and c-fos in PHA stimulated-PBMC. These findings indicated that SFZYD improved the metabolic profiling and biochemical indicators on CSBS primary dysmenorrhea rats. And the mechanisms were closely related with the regulation of the MAPK pathway by reduction in phosphorylated forms of the three MAPK (ERK1/2, p38 and JNK) and down regulation of c-jun and c-fos by paeoniflorin. The data could be provided the guidance for further research and new drug discovery. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[THE SYSTEM OF XENOBIOTICS BIOTRANSFORMATION OF HELMINTHS. RESEMBLANCE AND DIFFERENSES FROM SIMILAR HOST SYSTEMS (REWEW)].

PubMed

Smirnov, L P; Borvinskaya, E V; Suhovskaya, I V

2016-01-01

The three phases system xenobiotic biotransformation in cells as prokaryotes as eukaryotes was formed during the process of evolution. Clear and managed function of all three links of this system guarantee the survival of living organisms at alteration of chemical component of environment. Oxidation, reduction or hydrolysis of xenobiotics realize in phase I by insertion or opening reactive and hydrophilic groups in structure of drug molecule. In phase II xenobiotics or their metabolites from phase I conjugate with endogenic compounds, main of there are glutathione, glucuronic acid, amino acids and sulphates. Active transport of substrata, metabolites and conjugates through cell lipid membranes special transport proteins carry out (phase III). The system of xenobiotics biotransformation of helminths has essential differences from the same of vertebrate hosts. In particular, parasites do not reveal the activity of prime oxidases of phase I, such as CYP or FMO, in spite of the genes of these enzymes in DNA. As this phenomenon displays mainly in adult helminths, living in guts of vertebrates, then the hypothesis was formulated that this effect is related with adaptation to conditions of strong deficiency of oxygen, arise in a process of evolution (Kotze et al., 2006). Literature data testify the existence in helminths of unique forms of enzymes of phase II, the investigation of which present doubtless interest in relation with possible role in adaptation to parasitic mode of life. Notwithstanding that many of helminths GST in greater or lesser degree similar with enzymes of M, P, S and О classes of other organisms, nevertheless they have essential structural differences as compared with enzymes of hosts that makes perspective the search of specific anthelminthics vaccines. Transport of xenobiotics is now considered phase III of biotransformation. It was shown that proteins of this phase (ATP binding cassette transporters (ABC ) of parasites) play a key role in efflux of lipophilic xenobiotics, hydrophilic metabolites and conjugates and take part in forming of anthelminthics resistance. Some of these transporters, such as P-glycoprotein (Pgp), are important for drug resistance of helminths. In particular, a correlation between the level of expression of Pgp and resistance of S. mansoni and F. hepatica to widely used anthelminthics as praziquantel and triclabendazol exist.

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP.

PubMed

Järvinen, Erkka; Deng, Feng; Kidron, Heidi; Finel, Moshe

2018-04-01

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E 1 -G), estradiol-3- and 17-glucuronides (E 2 -3G and E 2 -17G), as well as estriol-3- and 16-glucuronides (E 3 -3G and E 3 -16G) are found in human plasma and urine. Unlike E 2 -17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E 1 -G, E 2 -3G, E 3 -3G, E 3 -16G and estrone-3-sulfate (E 1 -S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E 1 -S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E 1 -G and E 2 -3G, were still transported by BCRP at 10-fold higher rates than E 1 -S. BCRP also transported E 3 -16G at higher rates than the studied MRPs, while it transported E 3 -3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E 1 -G, E 2 -3G, E 3 -3G and E 3 -16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K m values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at K m values below 20 μM, but lower V max values than other transporters. In the case of E 3 -3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E 3 -16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Preliminary proteomics analysis of the total proteins of HL Type cytoplasmic male sterility rice anther].

PubMed

Wen, Li; Liu, Gai; Zhang, Zai-Jun; Tao, Jun; Wan, Cui-Xiang; Zhu, Ying-Guo

2006-03-01

The proteins of HL type cytoplasmic male sterility rice anther of YTA (CMS) and YTB (maintenance line) were separated by two-dimensional electrophoresis with immobilized ph (3-10 non-linear) gradients as the first dimension and SDS-PAGE as the second. The silver-stained proteins spots were analyzed using Image Master 2D software, there were about 1800 detectable spots on each 2D-gel, and about 85 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 9 protein spots out of 16 were identified. Among those proteins, there were Putative nucleic acid binding protein, glucose-1-phosphate adenylyltransferase (ADP-glucose pyrophosphorylase, AGPase) (EC: 2.7.7.27) large chain, UDP-glucuronic acid decarboxylase, putative calcium-binding protein annexin, putative acetyl-CoA synthetase and putative lipoamide dehydrogenase etc. They were closely associated with metabolism, protein biosynthesis, transcription, signal transduction and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins, i.e. AGPase, putative lipoamide dehydrogenase and putative acetyl-CoA synthetase were deeply discussed on the relationship to CMS. AGPase catalyzes a very important step in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants: synthesis of the activated glucosyl donor, ADP-glucose, from glucose-1-phosphate and ATP. The lack of the AGPase in male sterile line might directly result in the reduction of starch, and the synthesis of starch was the most important processes during the development of pollen. In present research, the descent or reduction of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase seemed involved in pollen sterility in rice. The degeneration and formation of various tissues during pollen development may impose high demands for energy and key biosynthetic intermediates. Under such conditions, the TCA cycle needs to operate fully, because the TCA cycle is an important source for many intermediates required for biosynthetic pathways, in addition to performing an oxidative, energy-producing role. Thus, it seemed reasonable to infer that the decrease of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase in anther might prevent the conversion of pyruvate into acetyl-CoA, and as a result, the TCA cycle could no longer operate at a sufficient rate to meet all requirements in anther cells, leading to pollen sterility. This study gave new insights into the mechanism of CMS in rice and demonstrated the power of the proteomic approach in plant biology studies.

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

PubMed

Price, R J; Surry, D; Renwick, A B; Meneses-Lorente, G; Lake, B G; Evans, D C

2000-08-01

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

Transcriptomic analysis reveals the gene expression profile that specifically responds to IBA during adventitious rooting in mung bean seedlings.

PubMed

Li, Shi-Weng; Shi, Rui-Fang; Leng, Yan; Zhou, Yuan

2016-01-12

Auxin plays a critical role in inducing adventitious rooting in many plants. Indole-3-butyric acid (IBA) is the most widely employed auxin for adventitious rooting. However, the molecular mechanisms by which auxin regulate the process of adventitious rooting are less well known. The RNA-Seq data analysis indicated that IBA treatment greatly increased the amount of clean reads and the amount of expressed unigenes by 24.29 % and 27.42 % and by 4.3 % and 5.04 % at two time points, respectively, and significantly increased the numbers of unigenes numbered with RPKM = 10-100 and RPKM = 500-1000 by 13.04 % and 3.12 % and by 24.66 % and 108.2 % at two time points, respectively. Gene Ontology (GO) enrichment analysis indicated that the enrichment of down-regulated GOs was 2.87-fold higher than that of up-regulated GOs at stage 1, suggesting that IBA significantly down-regulated gene expression at 6 h. The GO functional category indicated that IBA significantly up- or down-regulated processes associated with auxin signaling, ribosome assembly and protein synthesis, photosynthesis, oxidoreductase activity and extracellular region, secondary cell wall biogenesis, and the cell wall during the development process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated that ribosome biogenesis, plant hormone signal transduction, pentose and glucuronate interconversions, photosynthesis, phenylpropanoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, ribosome, cutin, flavonoid biosynthesis, and phenylalanine metabolism were the pathways most highly regulated by IBA. A total of 6369 differentially expressed (2-fold change > 2) unigenes (DEGs) with 3693 (58 %) that were up-regulated and 2676 (42 %) down-regulated, 5433 unigenes with 2208 (40.6 %) that were up-regulated and 3225 (59.4 %) down-regulated, and 7664 unigenes with 3187 (41.6 %) that were up-regulated and 4477 (58.4 %) down-regulated were detected at stage 1, stage 2, and between stage 1 and stage 2, respectively, suggesting that IBA treatment increased the number of DEGs. A total of 143 DEGs specifically involved in plant hormone signaling and 345 transcription factor (TF) genes were also regulated by IBA. qRT-PCR validation of the 36 genes with known functions indicated a strong correlation with the RNA-Seq data. The changes in GO functional categories, KEGG pathways, and global DEG profiling during adventitious rooting induced by IBA were analyzed. These results provide valuable information about the molecular traits of IBA regulation of adventitious rooting.

Polysaccharides from Epimedium koreanum Nakai with immunomodulatory activity and inhibitory effect on tumor growth in LLC-bearing mice.

PubMed

Wang, Chengcheng; Feng, Liang; Su, Jiayan; Cui, Li; Dan Liu; Yan, Jun; Ding, Chuanlin; Tan, Xiaobin; Jia, Xiaobin

2017-07-31

Epimedium koreanum Nakai is documented as tonic herbal in China for over a thousand years and has the potential to enhance the body's immunity according to the theory of traditional Chinese medicine. Polysaccharides are one of the most important effective compounds in Epimedium koreanum Nakai. Accumulating evidence indicated polysaccharides derived from traditional Chinese medicine have potent immune-enhancing properties and relatively nontoxic effects in cancer treatment. However, information about immunological regulation in tumor of Epimedium koreanum Nakai polysaccharides is limited and the reports of purification, characterization of polysaccharides have remained less. The purpose of our study was to further investigate the active polysaccharides from Epimedium koreanum Nakai by evaluating the immune-regulation activities in tumor-bearing mice and provide reasonable explanation for traditional application. We firstly purified Epimedium koreanum polysaccharide (EPS) from crude extracts and evaluated EPS in vitro using immunological experiments including maturation and Ag presentation function of DCs, CD4 T-cell differentiation and secretion of anti-cancer cytokines. In LLC-bearing mice model, we investigated its antitumor activities through evaluation of tumor cell proliferative activity, calculation of immune organ indexes and relative host immune system function tests. Results showed that EPS (180 × 10 4 Da) was composed of mannose (Man), rhamnose (Rha), glucuronic acid (GlcUA), galactosamine (GalN), glucose (Glc), galactose (Gal), arabinose (Ara) and fructose (Fuc). Chemical composition assay indicated EPS was a fraction with 28.20% uronic acid content. FT-IR suggested the presence of pyraoid ring in EPS and SEM displayed smooth surface embedded by several pores. Moreover, Our study suggested EPS could remarkably stimulate macrophages to secrete substantial anti-cancer cytokines and promote maturation as well as Ag presentation function of DCs. Strikingly, CD4 T-cell differentiation and increased INF-γ production stimulated by EPS-activated macrophages were observed in the research. Furthermore, EPS exhibited prominent antitumor activities through regulating host immune system function in LLC-bearing mice. Taken together, experimental findings suggested EPS could be regarded as a potential immune-stimulating modifier for cancer therapy. Our studies demonstrated the polysaccharide (180 × 10 4 Da) purified from Epimedium koreanum Nakai could promote maturation and Ag presentation function of DCs, increase the level of immunomodulatory cytokines and activate CD4 T-cell differentiation. Furthermore, it may inhibit the tumor growth in LLC-bearing mice through regulating host immune system function. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Complementary Sample Preparation Strategies for Analysis of Cereal β-Glucan Oxidation Products by UPLC-MS/MS.

PubMed

Boulos, Samy; Nyström, Laura

2017-01-01

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO • ) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO • -attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO • -attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be (1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and (2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan's health promoting and technological properties.

Complementary Sample Preparation Strategies for Analysis of Cereal β-Glucan Oxidation Products by UPLC-MS/MS

PubMed Central

Boulos, Samy; Nyström, Laura

2017-01-01

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO•) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO•-attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO•-attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be (1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and (2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan's health promoting and technological properties. PMID:29164106

The two Rasamsonia emersonii α-glucuronidases, ReGH67 and ReGH115, show a different mode-of-action towards glucuronoxylan and glucuronoxylo-oligosaccharides.

PubMed

Martínez, Patricia Murciano; Appeldoorn, Maaike M; Gruppen, Harry; Kabel, Mirjam A

2016-01-01

The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-)glucuronic acids (UAme). Hence, α-glucuronidases play an important role in the degradation of UAmexylan structures facilitating the complete utilisation of plant biomass. The characterisation of α-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UAmexylan structures. The mode-of-action of two α-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 °C. ReGH67 was able to release UAme from UAme-substituted xylo-oligosaccharides (UAmeXOS), but only the UAme linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UAmeXOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UAmeXOS (DP 3-4). On the contrary, ReGH115 was able to release UAme from both polymeric UAmexylan and UAmeXOS. ReGH115 cleaved UAme from both internal and non-reducing end xylosyl residues, with the exception of UAme attached to the non-reducing end of a xylotriose oligosaccharide. In this research, and for the first time, we define the mode-of-action of two α-glucuronidases from two different GH families both from the ascomycete R. emersonii. To date, only four α-glucuronidases classified in GH115 are characterised. ReGH67 showed limited substrate specificity towards only UAmeXOS, cleaving UAme only when attached to the non-reducing end xylosyl residue. ReGH115 was much less substrate specific compared to ReGH67, because UAme was released from both polymeric UAmexylan and UAmeXOS, from both internal and non-reducing end xylosyl residues. The characterisation of the mode-of-action of these two α-glucuronidases helps understand how R. emersonii attacks UAmexylan in plant biomass and the knowledge presented is valuable to improve enzyme cocktails for biorefinery applications.

Complementary sample preparation strategies for analysis of cereal β-glucan oxidation products by UPLC-MS/MS

NASA Astrophysics Data System (ADS)

Boulos, Samy; Nyström, Laura

2017-11-01

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO•) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO•-attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO•-attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be 1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and 2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan’s health promoting and technological properties.

All-Atom Internal Coordinate Mechanics (ICM) Force Field for Hexopyranoses and Glycoproteins.

PubMed

Arnautova, Yelena A; Abagyan, Ruben; Totrov, Maxim

2015-05-12

We present an extension of the all-atom internal-coordinate force field, ICMFF, that allows for simulation of heterogeneous systems including hexopyranose saccharides and glycan chains in addition to proteins. A library of standard glycan geometries containing α- and β-anomers of the most common hexapyranoses, i.e., d-galactose, d-glucose, d-mannose, d-xylose, l-fucose, N -acetylglucosamine, N -acetylgalactosamine, sialic, and glucuronic acids, is created based on the analysis of the saccharide structures reported in the Cambridge Structural Database. The new force field parameters include molecular electrostatic potential-derived partial atomic charges and the torsional parameters derived from quantum mechanical data for a collection of minimal molecular fragments and related molecules. The ϕ/ψ torsional parameters for different types of glycosidic linkages are developed using model compounds containing the key atoms in the full carbohydrates, i.e., glycosidic-linked tetrahydropyran-cyclohexane dimers. Target data for parameter optimization include two-dimensional energy surfaces corresponding to the ϕ/ψ glycosidic dihedral angles in the disaccharide analogues, as determined by quantum mechanical MP2/6-31G** single-point energies on HF/6-31G** optimized structures. To achieve better agreement with the observed geometries of glycosidic linkages, the bond angles at the O-linkage atoms are added to the internal variable set and the corresponding bond bending energy term is parametrized using quantum mechanical data. The resulting force field is validated on glycan chains of 1-12 residues from a set of high-resolution X-ray glycoprotein structures based on heavy atom root-mean-square deviations of the lowest-energy glycan conformations generated by the biased probability Monte Carlo (BPMC) molecular mechanics simulations from the native structures. The appropriate BPMC distributions for monosaccharide-monosaccharide and protein-glycan linkages are derived from the extensive analysis of conformational properties of glycoprotein structures reported in the Protein Data Bank. Use of the BPMC search leads to significant improvements in sampling efficiency for glycan simulations. Moreover, good agreement with the X-ray glycoprotein structures is achieved for all glycan chain lengths. Thus, average/median RMSDs are 0.81/0.68 Å for one-residue glycans and 1.32/1.47 Å for three-residue glycans. RMSD from the native structure for the lowest-energy conformation of the 12-residue glycan chain (PDB ID 3og2) is 1.53 Å. Additionally, results obtained for free short oligosaccharides using the new force field are in line with the available experimental data, i.e., the most populated conformations in solution are predicted to be the lowest energy ones. The newly developed parameters allow for the accurate modeling of linear and branched hexopyranose glycosides in heterogeneous systems.

Effects of Curcuma xanthorrhiza Extracts and Their Constituents on Phase II Drug-metabolizing Enzymes Activity.

PubMed

Salleh, Nurul Afifah Mohd; Ismail, Sabariah; Ab Halim, Mohd Rohaimi

2016-01-01

Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer. The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities. The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM. In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC 50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC 50 values ranging between 9.59-22.76 μg/mL and 110.71-526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC 50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC 50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition. These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes. Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions Abbreviations Used : BSA: Bovine serum albumin, CAM: Complementary and alternative medicine, cDNA: Complementary deoxyribonucleic acid, CDNB: 1-Chloro-2,4-dinitrobenzene, CuSO4.5H2O: Copper(II) sulfate pentahydrate, CXEE: Curcuma xanthorrhiza ethanol extract, CXAE: Curcuma xanthorrhiza aqueous extract, GC-MS: Gas chromatography-mass spectroscopy, GSH: Glutathione, GST: Glutathione S-transferase, KCl: Potassium chloride, min: Minutes, MgCl 2 : Magnesium chloride, mg/mL: Concentration (weight of test substance in milligrams per volume of test concentration), mM: Milimolar, Na 2 CO 3 : Sodium carbonate, NaOH: Sodium hydroxide, nmol: nanomol, NSAIDs: Non-steroidal antiinflammatory drug, p-NP: para-nitrophenol, RLU: Relative light unit, SEM: Standard error of mean, UDPGA: UDP-glucuronic acid, UGT: UDP-glucuronosyltransferase.

Final report on the safety assessment of benzaldehyde.

PubMed

Andersen, Alan

2006-01-01

Benzaldehyde is an aromatic aldehyde used in cosmetics as a denaturant, a flavoring agent, and as a fragrance. Currently used in only seven cosmetic products, its highest reported concentration of use was 0.5% in perfumes. Benzaldehyde is a generally regarded as safe (GRAS) food additive in the United States and is accepted as a flavoring substance in the European Union. Because Benzaldehyde rapidly metabolizes to Benzoic Acid in the skin, the available dermal irritation and sensitization data demonstrating no adverse reactions to Benzoic Acid were considered supportive of the safety of Benzaldehyde. Benzaldehyde is absorbed through skin and by the lungs, distributes to all well-perfused organs, but does not accumulate in any specific tissue type. After being metabolized to benzoic acid, conjugates are formed with glycine or glucuronic acid, and excreted in the urine. Little acute toxicity was seen. The oral LD(50) of Benzaldehyde in rats and mice ranged from 800 to 2850 mg/kg. The intraperitoneal LD(50) in white rats was 3265 mg/kg. In short-term oral studies, the no observed adverse effect level (NOAEL) was 400 mg/kg in rats and mice. In subchronic oral studies, the NOAEL was 400 mg/kg in rats and 600 mg/kg in mice. In a 16-week feeding study, rats given up to 10,000 ppm showed no signs of toxicity. Repeated inhalation of volatilized Benzaldehyde produced ocular and nasal irritation at 500 ppm and death in rabbits at 750 ppm. Undiluted Benzaldehyde was irritating to rabbit eyes, causing edema, erythema, and pain. Benzaldehyde was determined not to be a contact sensitizer, but did produce allergic reactions in a maximization test. Clinical reports of allergy to Benzaldehyde are rare. Benzoic Acid did not produce irritation or sensitization reactions in human clinical studies. Benzoic Acid also failed to produce reactions in phototoxicity and photosensitization tests. Neither Benzaldehyde, Benzoic Acid, nor Sodium Benzoate are reproductive or developmental toxicants at doses that are nontoxic to the mother. In a behavioral study, blood levels of 0.12 ng/ml Benzaldehyde produced a 44% reduction in motor activity in mice. Benzaldehyde did not produce mutations in bacterial assays, but did produce chromosomal abnormalities in Chinese hamster cells and increased mutations in a mouse lymphoma forward mutation assay. Benzaldehyde was evaluated by the National Toxicology Program, which found no evidence of carcinogenicity in rats, and some evidence of carcinogenicity in mice. Several studies have suggested that Benzaldehyde can have carcinostatic or antitumor properties. Overall, at the concentrations used in cosmetics, Benzaldehyde was not considered a carcinogenic risk to humans. Although there are limited irritation and sensitization data available for Benzaldehyde, the available dermal irritation and sensitization data and ultraviolet (UV) absorption and phototoxicity data demonstrating no adverse reactions to Benzoic Acid support the safety of Benzaldehyde as currently used in cosmetic products.

Metabolism of acetaminophen (paracetamol) in plants--two independent pathways result in the formation of a glutathione and a glucose conjugate.

PubMed

Huber, Christian; Bartha, Bernadett; Harpaintner, Rudolf; Schröder, Peter

2009-03-01

Pharmaceuticals and their metabolites are detected in the aquatic environment and our drinking water supplies. The need for high quality drinking water is one of the most challenging problems of our times, but still only little knowledge exists on the impact of these compounds on ecosystems, animals, and man. Biological waste water treatment in constructed wetlands is an effective and low-cost alternative, especially for the treatment of non-industrial, municipal waste water. In this situation, plants get in contact with pharmaceutical compounds and have to tackle their detoxification. The mechanisms for the detoxification of xenobiotics in plants are closely related to the mammalian system. An activation reaction (phase I) is followed by a conjugation (phase II) with hydrophilic molecules like glutathione or glucose. Phase III reactions can be summarized as storage, degradation, and transport of the xenobiotic conjugate. Until now, there is no information available on the fate of pharmaceuticals in plants. In this study, we want to investigate the fate and metabolism of N-acetyl-4-aminophenol (paracetamol) in plant tissues using the cell culture of Armoracia rusticana L. as a model system. A hairy root culture of A. rusticana was treated with acetaminophen in a liquid culture. The formation and identification of metabolites over time were analyzed using HPLC-DAD and LC-MSn techniques. With LC-MS technique, we were able to detect paracetamol and identify three of its metabolites in root cells of A. rusticana. Six hours after incubation with 1 mM of acetaminophen, the distribution of acetaminophen and related metabolites in the cells resulted in 18% paracetamol, 64% paracetamol-glucoside, 17% paracetamol glutathione, and 1% of the corresponding cysteine conjugate. The formation of two independently formed metabolites in plant root cells again revealed strong similarities between plant and mammalian detoxification systems. The detoxification mechanism of glucuronization in mammals is mirrored by glucosidation of xenobiotics in plants. Furthermore, in both systems, a glutathione conjugate is formed. Due to the existence of P450 enzymes in plants, the formation of the highly reactive NAPQI intermediate is possible. In this study, we introduce the hairy root cell culture of A. rusticana L. as a suitable model system to study the fate of acetaminophen in plant tissues. Our first results point to the direction of plants being able to take up and detoxify the model substrate paracetamol. These first findings underline the great potential of using plants for waste water treatments in constructed wetlands. This very first study on the detoxification of a widely used antipyretic agent in plant tissues again shows the flexibility of plant detoxification systems and their potential in waste water treatment facilities. This study covers only the very first steps of acetaminophen detoxification in plants; still, there is no data on long-term exposure as well as the possible impact of pharmaceuticals on the plant health and stress defense. Long-term experiments need to be performed to follow the fate of acetaminophen in root and leaf cells in a whole plant system, and to evaluate possible usage of plants for the remediation of acetaminophen from waste water.

Population pharmacokinetics of the photodynamic therapy agent 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a in cancer patients.

PubMed

Bellnier, David A; Greco, William R; Loewen, Gregory M; Nava, Hector; Oseroff, Allan R; Pandey, Ravindra K; Tsuchida, Takaaki; Dougherty, Thomas J

2003-04-15

Photodynamic therapy is an effective and often curative treatment for certain solid tumors. The porphyrin-based photosensitizer Photofrin, the only Food and Drug Administration-approved drug for this therapy, suffers from certain disadvantages: its complex chemical nature; retention by skin (leading to protracted cutaneous photosensitivity); and less than optimal photophysical properties. In this study, we examine the population pharmacokinetics and cutaneous phototoxicity of 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), a chlorin-type photosensitizer with more favorable photophysical properties. HPPH plasma concentration-time data were obtained in 25 patients enrolled in Phase I-II clinical trials for the treatment of partially obstructive esophageal carcinoma, high-grade dysplasia associated with Barrett's esophagus, carcinoma of the lung, or multiple basal cell carcinomas. Doses of 3, 4, 5, or 6 mg/m(2) were administered as 1-h i.v. infusions. The pharmacokinetic data for each patient were fitted with a standard two-compartment (biexponential) model with continuous infusion. The model fitting approach was iteratively reweighted nonlinear regression, with weights equal to the reciprocal of the square of the predicted HPPH plasma concentrations. The complete set of data for all 25 patients was then fitted simultaneously with nonlinear mixed effects modeling. Cutaneous phototoxicity responses were determined, as a function of time after HPPH infusion, following exposure to various doses of light from a solar simulator. The estimates of the population mean (variance) for each parameter were as follows: volume of distribution (V(C)), 2.40 liters/m(2) (0.259); steady-state volume (V(SS)), 9.58 liters/m(2) (11.6); systemic clearance (CL), 0.0296 liter/h/m(2) (0.000094); and distributional clearance (CL(D)), 0.144 liter/h/m(2) (0.00166). These parameters were independent of dose. Clearance increased with age. A relative error model was used for the difference in the raw and fitted data, and the overall coefficient of variation estimate across all of the data was 14.5%. The estimated mean population alpha and beta half-lives (95% confidence interval) were 7.77 h (3.46-17.6 h) and 596 h (120-2951 h), respectively. High-performance liquid chromatography analysis of serum showed no circulating HPPH metabolites, and in vitro incubation of HPPH with human liver microsomal preparations resulted in no metabolite or glucuronic acid-HPPH conjugate production. A minimal skin response to the solar simulator was observed, mostly in patients treated with the highest dose of HPPH, 6 mg/m(2). All of the HPPH pharmacokinetic parameters were consistent with a highly lipophilic agent that is concentrated in plasma and is nearly 100% bound to plasma proteins; this was verified by plasma protein binding studies. Whereas low concentrations of HPPH can be detected in plasma several months after a single infusion, no instances of cutaneous photosensitivity have been noted in these patients. In general, HPPH pharmacokinetic profiles are readily predictable from the global population model. This is the first comprehensive human population pharmacokinetic/pharmacodynamic study of a clinical anticancer photodynamic therapy agent.

Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure.

PubMed

Nigg, H N; Knaak, J B

2000-01-01

The organophosphorus pesticides of this review were discovered in 1936 during the search for a replacement for nicotine for cockroach control. The basic biochemical characteristics of RBC AChE and BChE were determined in the 1940s. The mechanism of inhibition of both enzymes and other serine esterases was known in the 1940s and, in general, defined in the 1950s. In 1949, the death of a parathion mixer-loader dictated blood enzyme monitoring to prevent acute illness from organophosphorus pesticide intoxication. However, many of the chemical and biochemical steps for serine enzyme inhibition by OP compounds remain unknown today. The possible mechanisms of this inhibition are presented kinetically beginning with simple (by comparison) Michaelis-Menten substrate enzyme interaction kinetics. As complicated as the inhibition kinetics appear here, PBPK model kinetics will be more complex. The determination of inter- and intraindividual variation in RBC ChE and BChE was recognized early as critical knowledge for a blood esterase monitoring program. Because of the relatively constant production of RBCs, variation in RBC AChE was determined by about 1970. The source of plasma (or serum) BChE was shown to be the liver in the 1960s with the change in BChE phenotype to the donor in liver transplant patients. BChE activity was more variable than RBC AChE, and only in the 1990s have BChE individual variation questions been answered. We have reviewed the chemistry, metabolism, and toxicity of organophosphorus insecticides along with their inhibitory action toward tissue acetyl- and butyrylcholinesterases. On the basis of the review, a monitoring program for individuals mixing-loading and applying OP pesticides for commercial applicators was recommended. Approximately 41 OPs are currently registered for use by USEPA in the United States. Under agricultural working conditions, OPs primarily are absorbed through the skin. Liver P-450 isozymes catalyze the desulfurization of phosphorothioates and phosphorodithioates (e.g., parathion and azinphosmethyl, respectively) to the more toxic oxons (P = O(S to O)). In some cases, P-450 isozymes catalyze the oxidative cleavage of P-O-aryl bonds (e.g., parathion, methyl parathion, fenitrothion, and diazinon) to form inactive water-soluble alkyl phosphates and aryl leaving groups that are readily conjugated with glucuronic or sulfuric acids and excreted. In addition to the P-450 isozymes, mammalian tissues contain ('A' and 'B') esterases capable of reacting with OPs to produce hydrolysis products or phosphorylated enzymes. 'A'-esterases hydrolyze OPs (i.e., oxons), while 'B'-esterases with serine at the active center are inhibited by OPs. OPs possessing carboxylesters, such as malathion and isofenphos, are hydrolyzed by the direct action of 'B'-esterases (i.e., carboxylesterase, CaE). Metabolic pathways shown for isofenphos, parathion, and malathion define the order in which these reactions occur, while Michaelis-Menten kinetics define reaction parameters (Vmax, K(m)) for the enzymes and substrates involved, and rates of inhibition of 'B'-esterases (kis, bimolecular rate constants) by OPs and their oxons. OPs exert their insecticidal action by their ability to inhibit AChE at the cholinergic synapse, resulting in the accumulation of acetylcholine. The extent to which AChE or other 'B'-esterases are inhibited in workers is dependent upon the rate the OP pesticide is activated (i.e., oxon formation), metabolized to nontoxic products by tissue enzymes, its affinity for AChE and other 'B'-esterases, and esterase concentrations in tissues. Rapid recovery of OP BChE inhibition may be related to reactivation of inhibited forms. AChE, BChE, and CaE appear to function in vivo as scavengers, protecting workers against the inhibition of AChE at synapses. Species sensitivity to OPs varies widely and results in part from binding affinities (Ka) and rates of phosphorylation (kp) rather than rates of activation and detoxif