Metabolomic Analysis of Blood Plasma after Oral Administration of N-acetyl-d-Glucosamine in Dogs

PubMed Central

Osaki, Tomohiro; Kurozumi, Seiji; Sato, Kimihiko; Terashi, Taro; Azuma, Kazuo; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Minami, Saburo; Okamoto, Yoshiharu

2015-01-01

N-acetyl-d-glucosamine (GlcNAc) is a monosaccharide that polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin. GlcNAc is a basic component of hyaluronic acid and keratin sulfate found on the cell surface. The aim of this study was to examine amino acid metabolism after oral GlcNAc administration in dogs. Results showed that plasma levels of ectoine were significantly higher after oral administration of GlcNAc than prior to administration (p < 0.001). To our knowledge, there have been no reports of increased ectoine concentrations in the plasma. The mechanism by which GlcNAc administration leads to increased ectoine plasma concentration remains unclear; future studies are required to clarify this mechanism. PMID:26262626

Hydrothermal conversion of N-acetyl-d-glucosamine to 5-hydroxymethylfurfural using ionic liquid as a recycled catalyst in a water-dimethyl sulfoxide mixture.

PubMed

Zang, Hongjun; Yu, Songbai; Yu, Pengfei; Ding, Hongying; Du, Yannan; Yang, Yuchan; Zhang, Yiwen

2017-04-10

Here, N-acetyl-d-glucosamine (GlcNAc), the monomer composing the second most abundant biopolymer, chitin, was efficiently converted into 5-hydroxymethylfurfural (5-HMF) using ionic liquid (IL) catalysts in a water/dimethyl sulfoxide (DMSO) mixture solvent. Various reaction parameters, including reaction temperature and time, DMSO/water mass ratios and catalyst dosage were optimized. A series of ILs with different structures were analyzed to explore their impact on GlcNAc conversion. The substrate scope was expanded from GlcNAc to d-glucosamine, chitin, chitosan and monosaccharides, although 5-HMF yields obtained from polymers and other monosaccharides were generally lower than those from GlcNAc. Moreover, the IL N-methylimidazolium hydrogen sulfate ([Hmim][HSO 4 ]) exhibited the best catalyst performance (64.6% yield) when GlcNAc was dehydrated in a DMSO/water mixture at 180 °C for 6 h without the addition of extra catalysts. To summarize, these results could provide knowledge essential to the production of valuable chemicals that are derived from renewable marine resources and benefit biofuel-related applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Attachment and internalization of a Chlamydia trachomatis lymphogranuloma venereum strain by McCoy cells: kinetics of infectivity and effect of lectins and carbohydrates.

PubMed Central

Söderlund, G; Kihlström, E

1983-01-01

The kinetics of attachment and ingestion of Chlamydia trachomatis serotype L1 by monolayers of McCoy cells were studied by using a method that discriminated between attachment and uptake. When about 1% of the McCoy cells was infected, the proteinase K-resistant chlamydial fraction, regarded as ingested chlamydiae, reached a constant value after about 3 h of incubation at 37 degrees C. Uptake of chlamydiae at 4 degrees C could not be demonstrated. The attached and ingested chlamydial fractions were constant over an eightfold increase in chlamydial inoculum. Chitobiose and chitotriose, the di- and trisaccharides of N-acetyl-D-glucosamine, reduced the association of C. trachomatis serotype L1 with McCoy cells. Higher concentrations of chitobiose also selectively inhibited ingestion of chlamydiae. A corresponding effect of chitobiose was also observed on the number of chlamydial inclusions. Wheat germ agglutinin, specific for N-acetyl-D-glucosamine residues, reduced the association of chlamydiae when incubated at 4 degrees C, but not at 37 degrees C. A small inhibiting effect of concanavalin A on association of chlamydiae, but no effect of the corresponding carbohydrates, indicates a nonspecific effect on chlamydial attachment of this lectin. These results suggest that beta 1 leads to 4-linked oligomers of N-acetyl-D-glucosamine are important in the specificity of attachment of C. trachomatis to McCoy cells. PMID:6642670

Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms

PubMed Central

Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

2013-01-01

The production of extracellular poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-d-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme. PMID:23357872

In vitro Studies of Sandfly Fever Viruses and Their Potential Significance for Vaccine Development.

DTIC Science & Technology

1980-02-01

more 14C-canavanine (DL-guanido-1LC-canavanine hydrochloride , Research Products International, Elk Grove, Illinois) than other viral proteins (data not...several laboratories have suggested that N-acetyl- glucosamine , glucose, and mannose residues are preassembled on dolichol phosphate (an isoprenoid...described below. Unlike the 0-glycosidic linkage between galactosamine and serine, the N-glycosidic linkage between N-acetyl glucosamine and asparagine

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria*

PubMed Central

Little, Dustin J.; Bamford, Natalie C.; Pokrovskaya, Varvara; Robinson, Howard; Nitz, Mark; Howell, P. Lynne

2014-01-01

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+, and Zn2+. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci. PMID:25359777

Glucosamine supplementation during late gestation alters placental development and increases litter size

USDA-ARS?s Scientific Manuscript database

Background: During late gestation the placental epithelial interface becomes highly folded, which involves changes in stromal hyaluronan. Hyaluronan is composed of glucoronate and N-acetyl-glucosamine. We hypothesized that supplementing gestating dams with glucosamine during this time would support ...

A novel analytical method for d-glucosamine quantification and its application in the analysis of chitosan degradation by a minimal enzyme cocktail.

PubMed

Mekasha, Sophanit; Toupalová, Hana; Linggadjaja, Eka; Tolani, Harish A; Anděra, Ladislav; Arntzen, Magnus Ø; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H; Agger, Jane W

2016-10-04

Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of fungal plant pathogens by synergistic action of chito-oligosaccharides and commercially available fungicides.

PubMed

Rahman, Md Hafizur; Shovan, Latifur Rahman; Hjeljord, Linda Gordon; Aam, Berit Bjugan; Eijsink, Vincent G H; Sørlie, Morten; Tronsmo, Arne

2014-01-01

Chitosan is a linear heteropolymer consisting of β 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DPn (average degree of polymerization) 206 and FA (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DPn in the range of 15-40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases.

Inhibition of Fungal Plant Pathogens by Synergistic Action of Chito-Oligosaccharides and Commercially Available Fungicides

PubMed Central

Rahman, Md. Hafizur; Shovan, Latifur Rahman; Hjeljord, Linda Gordon; Aam, Berit Bjugan; Eijsink, Vincent G. H.; Sørlie, Morten; Tronsmo, Arne

2014-01-01

Chitosan is a linear heteropolymer consisting of β 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DPn (average degree of polymerization) 206 and F A (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DPn in the range of 15–40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases. PMID:24770723

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

PubMed

Brockhausen, Inka; Nair, Dileep G; Chen, Min; Yang, Xiaojing; Allingham, John S; Szarek, Walter A; Anastassiades, Tassos

2016-04-01

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

PubMed

Balsanelli, Eduardo; Tuleski, Thalita Regina; de Baura, Valter Antonio; Yates, Marshall Geoffrey; Chubatsu, Leda Satie; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

2013-01-01

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides

PubMed Central

Balsanelli, Eduardo; Tuleski, Thalita Regina; de Baura, Valter Antonio; Yates, Marshall Geoffrey; Chubatsu, Leda Satie; de Oliveira Pedrosa, Fabio; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

2013-01-01

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization. PMID:24130823

Novel Task Functionalized Biopolymers for Enhanced Change Detection in Support of C-IED Operations

DTIC Science & Technology

2013-04-15

biopolymer was tested. All materials tested, PAM, chitin , the native biopolymer, and the MU conjugated biopolymer, were applied in small soil plots...phaseoli Strain 127 K36. Carbohydr. Res. 117, 141-156. EPA. 2001. Chitin ; Poly-N-acetyl-D-glucosamine (128991) Fact Sheet. USEPA Fact Sheet

Enzymatic production of N-acetyl-d-glucosamine from crayfish shell wastes pretreated via high pressure homogenization.

PubMed

Wei, Guoguang; Zhang, Alei; Chen, Kequan; Ouyang, Pingkai

2017-09-01

This study presents an efficient pretreatment of crayfish shell using high pressure homogenization that enables N-acetyl-d-glucosamine (GlcNAc) production by chitinase. Firstly, the chitinase from Serratia proteamaculans NJ303 was screened for its ability to degrade crayfish shell and produce GlcNAc as the sole product. Secondly, high pressure homogenization, which caused the crayfish shell to adopt a fluffy netted structure that was characterized by Scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD), was evaluated as the best pretreatment method. In addition, the optimal conditions of high pressure homogenization of crayfish shell were determined to be five cycles at a pressure of 400bar, which achieved a yield of 3.9g/L of GlcNAc from 25g/L of crayfish shell in a batch enzymatic reaction over 1.5h. The results showed high pressure homogenization might be an efficient method for direct utilization of crayfish shell for enzymatic production of GlcNAc. Copyright © 2017 Elsevier Ltd. All rights reserved.

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

PubMed

Balsanelli, Eduardo; Serrato, Rodrigo V; de Baura, Valter A; Sassaki, Guilherme; Yates, Marshall G; Rigo, Liu Un; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

2010-08-01

In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Global Analysis of Transcript and Protein Levels Across the Plasmodium falciparum Life Cycle

DTIC Science & Technology

2004-01-01

presence of 500 ng·mL1 pyrimethamine and gametocytogenesis was induced (Ifediba and Vanderberg 1981). N-acetyl-D- glucosamine (50 mM) was added to the...in 5 mM Tris(2-Carboxyethyl)phosphine hydrochloride (TCEP, Roche); (3) alkylated by 20 mM iodoacetamide (IAM); and (4) digested with proteinase K

Comparative opsonic and protective activities of Staphylococcus aureus conjugate vaccines containing native or deacetylated Staphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine.

PubMed

Maira-Litrán, Tomás; Kropec, Andrea; Goldmann, Donald A; Pier, Gerald B

2005-10-01

Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-beta-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an untypable strain.

Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex

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

Huet, Joëlle, E-mail: jhuet@ulb.ac.be; Azarkan, Mohamed; Looze, Yvan

2008-05-01

A chitinase isolated from the latex of the tropical species Carica papaya has been crystallized. The addition of N-acetyl-d-glucosamine to the crystallization solution has improved the diffraction quality resolution of the crystal to 1.8 Å resolution. A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 Å, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-d-glucosamine, the monomer resultingmore » from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 Å, β = 95.33° and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 Å. Structure refinement is currently in progress.« less

Antitumor activities of D-glucosamine and its derivatives*

PubMed Central

Zhang, Li; Liu, Wan-shun; Han, Bao-qin; Peng, Yan-fei; Wang, Dong-feng

2006-01-01

The growth inhibitory effects of D-glucosamine hydrochloride (GlcNH2·HCl), D-glucosamine (GlcNH2) and N-acetyl glucosamine (NAG) on human hepatoma SMMC-7721 cells in vitro were investigated. The results showed that GlcNH2·HCl and GlcNH2 resulted in a concentration-dependent reduction in hepatoma cell growth as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, human hepatoma SMMC-7721 cells treated with GlcNH2·HCl resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. NAG could not inhibit the proliferation of SMMC-7721 cells. GlcNH2·HCl exhibited antitumor activity against Sarcoma 180 in Kunming mice at dosage of 125~500 mg/kg, dose of 250 mg/kg being the best. GlcNH2·HCl at dose of 250 mg/kg could enhance significantly the thymus index, and spleen index and could promote T lymphocyte proliferation induced by ConA. The antitumor effect of GlcNH2·HCl is probably host-mediated and cytocidal. PMID:16845712

Oral Administration of N-Acetyl-D-Glucosamine Polymer Particles Down-Regulates Airway Allergic Responses

DTIC Science & Technology

2007-03-01

deoxygalactose and galactose, respectively. Relatively less mITLN-1 was eluted by these monosaccharides . The oligomeric Hu/Mo chimeric ITLN-1 had...Abeygunawardana, C., Bush, C. A. and Cisar, J. O. (1991) Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR...Hoogerhout, P. and van Boom, J. H. (1988) (1-5)-linked beta-D-galactofuranosides are immunodominant in extracellular polysaccharides of

Biologic properties and vaccine potential of the staphylococcal poly-N-acetyl glucosamine surface polysaccharide.

PubMed

Maira-Litran, Tomas; Kropec, Andrea; Goldmann, Donald; Pier, Gerald B

2004-02-17

Staphylococci have become the most common causes of nosocomial bacterial infections, and this fact, along with increasing problems associated with antimicrobial resistance, spurs the need for finding immunotherapeutic alternatives to prevent and possibly treat these infections. Most virulent, clinical isolates of both coagulase-negative staphylococci (CoNS) and Staphylococcus aureus carry the ica locus which encodes proteins that synthesize a polymer of beta-1-6 linked N-acetyl glucosamine residues (PNAG). Animal studies have shown purified PNAG can elicit protective immunity against both CoNS and S. aureus, suggesting its potential as a broadly protective vaccine for many clinically important strains of staphylococci.

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

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

de Miranda Santos, I.K.; Pereira, M.E.

1984-09-01

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeusmore » and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.« less

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

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

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

2006-01-01

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

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

PubMed

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

2004-01-01

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

Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

PubMed Central

Hildebrandt, K M; Anderson, J S

1990-01-01

Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

Oral Administration of N-acetyl-D Glucosamine Polymer Particles Down-Regulates Airway Allergic Responses

DTIC Science & Technology

2008-05-01

asthma in Cynomolgus monkeys. J Appl Physiol 96:1433-1444, 2003. Task 2. Shibata Y, A Nishiyama, H Ohata, J Gabbard , QN Myrvik, RA Henriksen...Proceeding of “International Symposium on Low-Dose Radiation Exposures and Bio-Defense System. Page 5, 2006. Task 2. Shibata Y, J Gabbard , M Yamashita...killed BCG. J Leukoc Biol 78:1281-1290. 4. Shibata, Y., J. Gabbard , M. Yamashita, S. Tsuji, M. Smith, A. Nishiyama, R. A. Henriksen, and Q. N. Myrvik

Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans

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

Olchowy, Jaroslaw; Jedrzejczak, Robert; Milewski, Slawomir

2005-11-01

The isomerase domain of glucosamine-6-phosphate synthase from C. albicans has been crystallized and X-ray diffraction data have been collected. Preliminary analysis of the data reveals the oligomeric structure of the eukaryotic synthase to be a ‘dimer’ of prokaryotic-like dimers. Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5′-diphospho-N-acetyl d-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346–712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to spacemore » group I4, with unit-cell parameters a = b = 149, c = 103 Å. Diffraction data were collected to 3.8 Å. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.« less

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

PubMed Central

Ficko-Blean, Elizabeth; Stuart, Christopher P.; Suits, Michael D.; Cid, Melissa; Tessier, Matthew; Woods, Robert J.; Boraston, Alisdair B.

2012-01-01

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract. PMID:22479408

Oral Administration of N-Acetyl-D Glucosamine Polymer Particles Down-Regulates Airway Allergic Responses

DTIC Science & Technology

2006-03-01

Cholesterol Depletion Enhances Chitin Phagocytosis-Induced Macrophage Activation. Abstract will be presented at AAI Meeting at Boston in May 2006...presented at AAI Meeting at Boston in May 2006. Task 2. Tsuji S, M Yamashita Tsuji, A Nishiyama, Y Shibata. Molecular structure of human and mouse...interlectin-1 and comparison of binding to a mycobacterial galactofuranosyl residue. Abstract will be presented at AAI Meeting at Boston in May 2006

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans.

PubMed

Pusztahelyi, T; Molnár, Z; Emri, T; Klement, E; Miskei, M; Kerékgyárto, J; Balla, J; Pócsi, I

2006-01-01

N-Acetyl-D-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-acetyl-beta-D-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress. The transcription of the chiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

Electrospun Microfiber Scaffolds with Anti-Inflammatory Tributanoylated N-Acetyl-d-Glucosamine Promote Cartilage Regeneration

PubMed Central

Kim, Chaekyu; Shores, Lucas; Guo, Qiongyu; Aly, Ahmed; Jeon, Ok Hee; Kim, Do Hun; Bernstein, Nicholas; Bhattacharya, Rahul; Chae, Jemin Jeremy; Yarema, Kevin J.

2016-01-01

Tissue-engineering strategies offer promising tools for repairing cartilage damage; however, these strategies suffer from limitations under pathological conditions. As a model disease for these types of nonideal systems, the inflammatory environment in an osteoarthritic (OA) joint limits the efficacy of engineered therapeutics by disrupting joint homeostasis and reducing its capacity for regeneration. In this work, we investigated a sugar-based drug candidate, a tributanoylated N-acetyl-d-glucosamine analogue, called 3,4,6-O-Bu3GlcNAc, that is known to reduce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in osteoarthritis. 3,4,6-O-Bu3GlcNAc not only inhibited NFκB signaling but also exerted chondrogenic and anti-inflammatory effects on chondrocytes isolated from patients with osteoarthritis. 3,4,6-O-Bu3GlcNAc also increased the expression of extracellular matrix proteins and induced cartilage tissue production in three-dimensional in vitro hydrogel culture systems. To translate these chondrogenic and anti-inflammatory properties to tissue regeneration in osteoarthritis, we implanted 3,4,6-O-Bu3GlcNAc-loaded poly(lactic-co-glycolic acid) microfiber scaffolds into rats. The drug-laden scaffolds were biocompatible, and when seeded with human OA chondrocytes, similarly promoted cartilage tissue formation. 3,4,6-O-Bu3GlcNAc combined with the appropriate structural environment could be a promising therapeutic approach for osteoarthritis. PMID:27019285

Fabrication of nonwoven fabrics consisting of gelatin nanofibers cross-linked by glutaraldehyde or N-acetyl-d-glucosamine by aqueous method.

PubMed

Furuike, Tetsuya; Chaochai, Thitirat; Okubo, Tsubasa; Mori, Takahiro; Tamura, Hiroshi

2016-12-01

Since gelatin (Gel) undergoes a sol-gel transition, a novel dry-spinning procedure for Gel was used. Here, nonwoven fabrics of Gel were electrospun by applying the principles of dry spinning. The diameter of the fibers and the viscosity and flow rate of the solution were directly dependent on the concentration of Gel. Nonwoven fabrics spun with a 25% (w/w) Gel concentration only exhibited a nanoscale fiber diameter. In order to improve the properties of the nonwoven fabrics, they were cross-linked with glutaraldehyde (GTA) vapor after spinning or by the addition of N-acetyl-d-glucosamine (GlcNAc) to the Gel solution prior to spinning followed by heating these fibers. The developed nonwoven fibers were characterized using SEM, rheometry, FTIR, TGA, and mechanical tensile testing. The nonwoven fabrics cross-linked by the GTA vapor exhibited improved mechanical properties compared to those without cross-linking or with GlcNAc cross-linking. The swelling and water uptake ability resulted in no morphological changes in the fibers with GTA cross-linking. The TGA thermogram confirmed no phase change in the composite structure. Further, in vitro cytocompatibility studies using human mesenchymal stem cells showed the compatible nature of the developed nonwoven fibers. Our studies showed that these nonwoven fibers could be useful in medical care. Copyright © 2016 Elsevier B.V. All rights reserved.

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers.

PubMed

Lu, Shih-Chin; Lin, Sung-Chyr

2012-01-05

Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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

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

2008-09-17

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

N-Acetylglucosamine Inhibits LuxR, LasR and CviR Based Quorum Sensing Regulated Gene Expression Levels

PubMed Central

Kimyon, Önder; Ulutürk, Zehra İ.; Nizalapur, Shashidhar; Lee, Matthew; Kutty, Samuel K.; Beckmann, Sabrina; Kumar, Naresh; Manefield, Mike

2016-01-01

N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50–80% reduction at low millimolar concentrations (0.25–5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities. PMID:27602027

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

In Vivo Anticancer Efficacy and Toxicity Studies of a Novel Polymer Conjugate N-Acetyl Glucosamine (NAG)-PEG-Doxorubicin for Targeted Cancer Therapy.

PubMed

Pawar, Smita; Mahajan, Ketan; Vavia, Pradeep

2017-11-01

A novel polymer-drug conjugate, polyethylene glycol-N-(acetyl)-glucosamine-doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer-drug conjugate comprised of polyethylene glycol-maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.

Differences in carbon source utilization of Salmonella Oranienburg and Saintpaul isolated from river water.

PubMed

Medrano-Félix, Andrés; Estrada-Acosta, Mitzi; Peraza-Garay, Felipe; Castro-Del Campo, Nohelia; Martínez-Urtaza, Jaime; Chaidez, Cristóbal

2017-08-01

Long-term exposure to river water by non-indigenous micro-organisms such as Salmonella may affect metabolic adaptation to carbon sources. This study was conducted to determine differences in carbon source utilization of Salmonella Oranienburg and Salmonella Saintpaul (isolated from tropical river water) as well as the control strain Salmonella Typhimurium exposed to laboratory, river water, and host cells (Hep-2 cell line) growth conditions. Results showed that Salmonella Oranienburg and Salmonella Saintpaul showed better ability for carbon source utilization under the three growth conditions evaluated; however, S. Oranienburg showed the fastest and highest utilization on different carbon sources, including D-Glucosaminic acid, N-acetyl-D-Glucosamine, Glucose-1-phosphate, and D-Galactonic acid, while Salmonella Saintpaul and S. Typhimurium showed a limited utilization of carbon sources. In conclusion, this study suggests that environmental Salmonella strains show better survival and preconditioning abilities to external environments than the control strain based on their plasticity on diverse carbon sources use.

The cuticular localization of integument peptides from particular routing categories.

PubMed

Locke, M; Kiss, A; Sass, M

1994-10-01

The distribution of integument peptides in relation to chitin and structural features has been studied in the surface epidermis of the caterpillar of Calpodes ethlius by immunoblotting and immunogold labelling using antibodies prepared to peptides isolated from lamellate endocuticle or from hemolymph. The intermoult cuticle consists of an epicuticle, an endocuticle of many chitin containing lamellae, and a chitin containing assembly zone directly above the apical epidermal microvilli and the perimicrovillar space. During the intermoult, the epidermis secretes peptides constitutively, that is, secretory vesicles containing peptides exocytose without accumulating, traverse the perimicrovillar space and form lamellae in the assembly zone. At moulting, the epidermis deposits ecdysial droplets in addition. These interrupt the last few lamellae which later go on to become the perforated ecdysial membrane. The integument is involved with four routing classes of peptide. Secretion is apical into the cuticle (C), basal into the hemolymph (H), bidirectional (BD), or transported to the cuticle across the epidermis from the hemolymph (T). Some peptides change their routing at moulting. There are several patterns of localization. (1) C and BD cuticular peptides occur mainly in chitin containing lamellate cuticle. (2) Some are also present in epicuticle, and are therefore not obligatorily linked to chitin or matrix between chitin fibers. Cuticular peptides that also occur in the hemolymph are glycosylated, whereas most that are only secreted apically into the cuticle are not. All BD but few C peptides carry alpha-D-glucose/alpha-D-mannose. Some C and BD peptides carry N-acetyl glucosamine. (3) C36 extracted from cuticle has most N-acetyl glucosamine and colocalizes with chitin rather than the protein matrix. It is therefore probably the main link between chitin fibers and the matrix. (4) H235 is barely detectable at the apical cell surface during the intermoult but is abundant at moulting around and below the ecdysial droplets. (5) T66 occurs in intermoult lamellate cuticle. At moulting, alone among the peptides examined, it is in ecdysial droplets. Intermoult C and BD peptides are not in ecdysial droplets but continue to be present in the ecdysial membrane, suggesting that constitutive secretion is independent from the exocytosis of transported moult peptides. T66 differs from most hemolymph peptides in that it does not carry N-acetyl glucosamine or alpha-D-glucose/alpha-D-mannose. (6) Weakly reacting BD peptides (and some H peptides barely detectable in cuticle) localize near the apical surface. Their distribution therefore favours apical secretion and retrieval as a mechanism for basal secretion.

Identification of the glycoproteins of lymphocystis disease virus (LDV) of fish.

PubMed

Robin, J; Laperrière, A; Berthiaume, L

1986-01-01

Analysis of highly purified fish Lymphocystis Disease Virus (LDV), strain Leetown NFH, by three different methods, namely periodic Acid Schiff reaction, radiolabelling with tritiated fucose and N-acetyl-D-glucosamine and staining with three lectins, indicated that ten glycoproteins were associated with the virus structure. Six of them were detected by all of the three methods, three by both radiolabelling and lectin staining but only one by the lectin technique. Localization of these glycoproteins at the surface or inside the virion is discussed.

Synthesis and characterization of sugar based low molecular weight gelators and the preparation of chiral sulfinamides

NASA Astrophysics Data System (ADS)

Mangunuru, Hari Prasad Reddy

Low molecular weight gelators (LMWGs) have received considerable attention in the field of chemistry from last few decades. These compounds form self-assembled fibrous networks like micelles, cylindrical, sheets, fibers, layers and so on. The fibrous network entraps the solvent and forms gel, because of the self-assembly phenomenon and their demonstrated potential uses in a variety of areas, ranging from environmental to medicinal applications. Sugars are good starting materials to synthesize the new class of LMWG's, because these are different from some expensive materials, these are natural products. We have synthesized and characterized the LMGS's based on D-glucose and D-glucosamine. D-glucosamine is the versatile starting material to make different peptoids and triazoles. Several series of compounds were synthesized using compounds 1-3 as starting material and studied the gelation behavior all the compounds. We have studied the self-assembling properties of a new class of tripeptoids, synthesized by one-pot Ugi reaction from simple starting materials. Among the focused library of tripeptoids synthesized, we found that several efficient low molecular weight organogelators were obtained for aqueous DMSO and ethanol mixtures. We have also synthesized and characterized a series of monosaccharide triazole derivatives. These compounds were synthesized from N-acetyl glucosamine and D-glucose via a Cu(I) catalyzed azide/alkyne cycloaddition reaction (CuAAc). The compounds have been screened for their gelation properties and several efficient low molecular weight organo/hydro gelators were obtained, among these compounds, five per-acetyl glucosamine derivatives and one peracetyl glucose derivative were able to form gels in water. These new molecules are expected to be useful in drug delivery and tissue engineering.*. Asymmetric synthesis of chiral amines is a challenging in synthetic organic chemistry. The development of new catalysts for asymmetric organic transformations is a very important research goal in modern synthetic organic chemistry. We have synthesized a new class of chiral oxathiozinone from chiral amino phenol. From this synthesized chiral sulfinamides, ketimines followed by reducing the ketimines synthesized the highly hindered chiral amines. *Please refer to dissertation for diagrams.

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

PubMed Central

Jagtap, Pravin Kumar Ankush; Soni, Vijay; Vithani, Neha; Jhingan, Gagan Deep; Bais, Vaibhav Singh; Nandicoori, Vinay Kumar; Prakash, Balaji

2012-01-01

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU. PMID:22969087

A 4-deoxy analogue of N-acetyl-D-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

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

Wijk, Xander M.R. van; Oosterhof, Arie; Broek, Sebastiaan A.M.W. van den

2010-09-10

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -{beta}1-4GlcA-{alpha}1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-D-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect.more » 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.« less

G₂/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora.

PubMed

Rouf, Razina; Stephens, Alexandre S; Spaan, Lina; Arndt, Nadia X; Day, Christopher J; May, Tom W; Tiralongo, Evelin; Tiralongo, Joe

2014-01-01

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6-10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

VARIATION IN THE GROUP-SPECIFIC CARBOHYDRATE OF GROUP A STREPTOCOCCI

PubMed Central

McCarty, Maclyn

1956-01-01

Soil organisms have been isolated which elaborate induced enzymes capable of attacking group A and variant (V) streptococcal carbohydrates. The V enzyme hydrolyzes V carbohydrate extensively to dialyzable split products with resultant total loss of precipitating activity with homologous antisera. The split products inhibit the reaction between intact V carbohydrate and its antiserum: evidence is presented which indicates that rhamnose oligosaccharides are responsible for the inhibitory effect. The serological specificity of the V carbohydrate thus appears to be primarily dependent on a rhamnose-rhamnose linkage. The effect of the A enzyme on A carbohydrate is characterized by the removal of 50 to 70 per cent of the total glucosamine in the form of free N-acetyl-glucosamine. As a result of this treatment, the residual carbohydrate loses its reactivity with specific group A antisera and at the same time develops markedly increased cross-reactivity with V antisera. This cross-reactivity is in turn eliminated by treatment with V enzyme. The evidence suggests that the specificity of group A carbohydrate is determined to a large extent by side chains of N-acetyl-glucosamine which also serve to mask underlying rhamnose-rhamnose linkages with V specificity. PMID:13367334

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

PubMed

Eisenbeis, Simone; Lohmiller, Stefanie; Valdebenito, Marianne; Leicht, Stefan; Braun, Volkmar

2008-08-01

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound 'high-mannose' oligosaccharides.

PubMed Central

Bailey, D S; Burke, J; Sinclair, R; Mukherjee, B B

1981-01-01

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis. PMID:7306042

Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity.

PubMed

Kidibule, Peter Elias; Santos-Moriano, Paloma; Jiménez-Ortega, Elena; Ramírez-Escudero, Mercedes; Limón, M Carmen; Remacha, Miguel; Plou, Francisco José; Sanz-Aparicio, Julia; Fernández-Lobato, María

2018-03-22

Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5-6.5 and 30-40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the k cat /K m ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (D-glucosamine) 1-8 -GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.

Anti-streptococcal, tubulin, and dopamine receptor 2 antibodies in children with PANDAS and Tourette syndrome: single-point and longitudinal assessments.

PubMed

Morris-Berry, C M; Pollard, M; Gao, S; Thompson, C; Singer, H S

2013-11-15

Single-point-in-time ELISA optical densities for three putative antibodies identified in Sydenham's chorea, the streptococcal group A carbohydrate antigen, N-acetyl-beta-d-glucosamine, tubulin, and the dopamine 2 receptor, showed no differences in children with PANDAS (n=44) or Tourette syndrome (n=40) as compared to controls (n=24). Anti-tubulin and D2 receptor antibodies assessed in serial samples from 12 PANDAS subjects obtained prior to a documented exacerbation, during the exacerbation (with or without a temporally associated streptococcal infection), and following the exacerbation, showed no evidence of antibody levels correlating with a clinical exacerbation. These data do not support hypotheses suggesting an autoimmune hypothesis in either TS or PANDAS. © 2013.

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars.

PubMed

Popowska, Magdalena; Osińska, Magdalena; Rzeczkowska, Magdalena

2012-04-01

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

Identification of rat serum alkaline phosphatase isoenzyme by means of wheat germ agglutinin.

PubMed

Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

1997-01-01

Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-D-glucosamine (GlcNAc)-Sepharose 6E column chromatography. It was concluded that this method is useful for clinical examination in the rat.

The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate.

PubMed

Paszkiewicz-Gadek, A; Porowska, H; Gałasiński, W

1992-01-01

UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

PubMed Central

Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

Mining marine shellfish wastes for bioactive molecules: chitin and chitosan--Part A: extraction methods.

PubMed

Hayes, Maria; Carney, Brian; Slater, John; Brück, Wolfram

2008-07-01

Legal restrictions, high costs and environmental problems regarding the disposal of marine processing wastes have led to amplified interest in biotechnology research concerning the identification and extraction of additional high grade, low-volume by-products produced from shellfish waste treatments. Shellfish waste consisting of crustacean exoskeletons is currently the main source of biomass for chitin production. Chitin is a polysaccharide composed of N-acetyl-D-glucosamine units and the multidimensional utilization of chitin derivatives including chitosan, a deacetylated derivative of chitin, is due to a number of characteristics including: their polyelectrolyte and cationic nature, the presence of reactive groups, high adsorption capacities, bacteriostatic and fungistatic influences, making them very versatile biomolecules. Part A of this review aims to consolidate useful information concerning the methods used to extract and characterize chitin, chitosan and glucosamine obtained through industrial, microbial and enzymatic hydrolysis of shellfish waste.

Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates.

PubMed

Cardozo, Flávio Augusto; Gonzalez, Juan Miguel; Feitosa, Valker Araujo; Pessoa, Adalberto; Rivera, Irma Nelly Gutierrez

2017-10-27

N-Acetyl-D-glucosamine (GlcNAc) is a monosaccharide with great application potential in the food, cosmetic, pharmaceutical, and biomaterial areas. GlcNAc is currently produced by chemical hydrolysis of chitin, but the current processes are environmentally unfriendly, have low yield and high cost. This study demonstrates the potential to produce GlcNAc from α-chitin using chitinases of ten marine-derived Aeromonas isolates as a sustainable alternative to the current chemical process. The isolates were characterized as Aeromonas caviae by multilocus sequence analysis (MLSA) using six housekeeping genes (gltA, groL, gyrB, metG, ppsA, and recA), not presented the virulence genes verified (alt, act, ast, ahh1, aer, aerA, hlyA, ascV and ascFG), but showed hemolytic activity on blood agar. GlcNAc was produced at 37 °C, pH 5.0, 2% (w/v) colloidal chitin and crude chitinase extracts (0.5 U mL -1 ) by all the isolates with yields from 14 to 85% at 6 h, 17-89% at 12 h and 19-93% after 24 h. The highest yield of GlcNAc was observed by A. caviae CH129 (93%). This study demonstrates one of the most efficient chitin enzymatic hydrolysis procedures and A. caviae isolates with great potential for chitinases expression and GlcNAc production.

Metabolism-independent sugar sensing in central orexin neurons.

PubMed

González, J Antonio; Jensen, Lise T; Fugger, Lars; Burdakov, Denis

2008-10-01

Glucose sensing by specialized neurons of the hypothalamus is vital for normal energy balance. In many glucose-activated neurons, glucose metabolism is considered a critical step in glucose sensing, but whether glucose-inhibited neurons follow the same strategy is unclear. Orexin/hypocretin neurons of the lateral hypothalamus are widely projecting glucose-inhibited cells essential for normal cognitive arousal and feeding behavior. Here, we used different sugars, energy metabolites, and pharmacological tools to explore the glucose-sensing strategy of orexin cells. We carried out patch-clamp recordings of the electrical activity of individual orexin neurons unambiguously identified by transgenic expression of green fluorescent protein in mouse brain slices. RESULTS- We show that 1) 2-deoxyglucose, a nonmetabolizable glucose analog, mimics the effects of glucose; 2) increasing intracellular energy fuel production with lactate does not reproduce glucose responses; 3) orexin cell glucose sensing is unaffected by glucokinase inhibitors alloxan, d-glucosamine, and N-acetyl-d-glucosamine; and 4) orexin glucosensors detect mannose, d-glucose, and 2-deoxyglucose but not galactose, l-glucose, alpha-methyl-d-glucoside, or fructose. Our new data suggest that behaviorally critical neurocircuits of the lateral hypothalamus contain glucose detectors that exhibit novel sugar selectivity and can operate independently of glucose metabolism.

Modulation of cytokine-induced prostaglandin E₂ production in cultures of articular chondrocytes obtained from carpal joints of camels (Camelus dromedarius).

PubMed

Frondoza, Carmelita G; Heinecke, Lowella F; Grzanna, Mark W; Au, Angela Y; Ownby, Stacy L

2011-01-01

To determine whether camel articular chondrocytes can be maintained in tissue culture without phenotype loss and whether the response to cytokine stimulation can be modulated. Cartilage from 4 carpal joints of healthy adult dromedary camels (Camelus dromedarius). Chondrocytes were evaluated for type II collagen and aggrecan production They were incubated with control media or with 2 test mixtures (alone and then in combination) that have anti-inflammatory activity (avocado-soybean unsaponifiables, glucosamine, and chondroitin sulfate [ie, ASU + GLU + CS] and pentosan polysulfate and N-acetyl glucosamine [ie, PPS + NG]). Cells were then stimulated with interleukin-1β and tumor necrosis factor-α to determine prostaglandin (PG) E₂ production and nuclear factor (NF)-κB activation. Chondrocytes proliferated in media used for propagating equine chondrocytes; they produced type II collagen and aggrecan. Cytokine stimulation induced PGE₂ production and translocation of NF-κB. Incubation with each test mixture significantly inhibited PGE₂ production. The combination of ASU + GLU + CS and PPS + NG significantly potentiated PGE₂ inhibition and disrupted NF-κB translocation, compared with effects for either mixture alone. Chondrocytes proliferated without loss of the cartilage phenotype. Responses to cytokines were significantly inhibited by the mixtures of ASU + GLU + CS and PPS + NG, which indicated that this response can be modulated. This culture technique can be used to study the functional properties of camel chondrocytes and identify agents that may potentially be used to treat and manage joint inflammation.

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

PubMed

Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F

2018-05-17

O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

THE PRESENCE OF A GROUP A VARIANT-LIKE ANTIGEN IN STREPTOCOCCI OF OTHER GROUPS WITH SPECIAL REFERENCE TO GROUP N

PubMed Central

Elliott, S. D.; Hayward, John; Liu, T. Y.

1971-01-01

A Group A variant-like antigen has been detected in streptococci belonging to Groups D, E, G, M, and N. In Groups D and N the variant-like antigen was located in the streptococcal cell walls. In two strains of Group N streptococci (C559 and B209) the cell walls were chemically different and serologically distinct. In strain C559 N-acetylgalactosamine, and in strain B209, N-acetylglucosamine were the major determinants of serological specificity. The cell walls of strain C559 contained at least three serologically reactive components: a rhamnose-containing fraction that precipitated with an antiserum to Group A-variant carbohydrate; a strain-specific polysaccharide composed of galactosamine and glucosamine, both in the N-acetylated form and probably polymerized with an unidentified phosphorylated substance; and a component of unknown composition serologically related to a Group D streptococcus strain C3 (S. durans). An analogy is drawn between the cell wall structure in streptococcus and Salmonella. PMID:5111438

Inhibitors of acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 1: Hit to lead evaluation of a novel arylsulfonamide series.

PubMed

Green, Oluyinka M; McKenzie, Andrew R; Shapiro, Adam B; Otterbein, Ludovic; Ni, Haihong; Patten, Arthur; Stokes, Suzanne; Albert, Robert; Kawatkar, Sameer; Breed, Jason

2012-02-15

A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of the glycosylation-pattern of the middle ear mucosa in guinea pigs.

PubMed

Engleder, Elisabeth; Demmerer, Elisabeth; Wang, Xueyan; Honeder, Clemens; Zhu, Chengjing; Studenik, Christian; Wirth, Michael; Arnoldner, Christoph; Gabor, Franz

2015-04-30

In the present study the glycosylation pattern of the middle ear mucosa (MEM) of guinea pigs, an approved model for middle ear research, was characterized with the purpose to identify bioadhesive ligands which might prolong the contact time of drug delivery systems with the middle ear mucosa (MEM). To assess the utility of five fluorescein labeled plant lectins with different carbohydrate specificities as bioadhesive ligands, viable MEM specimens were incubated at 4°C and the lectin binding capacities were calculated from the MEM-associated relative fluorescence intensities. Among all lectins under investigation, fluorescein-labeled wheat germ agglutinin (F-WGA) emerged as the highest bioadhesive lectin. In general, the accessibility of carbohydrate moieties of the MEM followed the order: sialic acid and N-acetyl-d-glucosamine (WGA)>mannose and galactosamine (Lensculinaris agglutinin)>N-acetyl-d-glucosamine (Solanumtuberosum agglutinin)>fucose (Ulexeuropaeus isoagglutinin I)>terminal mannose α-(1,3)-mannose (Galanthusnivalis agglutinin). Competitive inhibition studies with the corresponding carbohydrate revealed that F-WGA-binding was inhibited up to 90% confirming specificity of the F-WGA-MEM interaction. The cilia of the MEM were identified as F-WGA binding sites by fluorescence imaging as well as a z-stack of overlays of transmission, F-WGA- and nuclei-stained images of the MEM. Additionally, co-localisation experiments revealed that F-WGA bound to acidic mucopolysaccharides of the MEM. All in all, lectin-mediated bioadhesion to the MEM is proposed as a new concept for drug delivery to prolong the residence time of the drug in the tympanic cavity especially for successful therapy for difficult-to-treat diseases such as otitis media. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Potential of chitosan (chemically-modified chitin) for extraction of lead-arsenate contaminated soils

USDA-ARS?s Scientific Manuscript database

Arsenic (As), phosphorous (P), and lead (Pb) contamination in soils represents a health risk to humans and the environment. Chitosan (poly-N-acetyl glucosamine) is a non-toxic and inexpensive food industry byproduct derived from chitin that has been used as an adsorbent of heavy metals. The object...

In Vitro Studies of Interaction of Rickettsia and Macrophages: Effect of Ultraviolet Light on Coxiella burnetii Inactivation and Macrophage Enzymes

DTIC Science & Technology

1980-03-01

aminoethyl ether)-N,N-tetraacetic acid and 3 mM im- data). These results suggest that exposure to UV idazole hydrochloride buffer (pH 7.5) with a syringe...acetyl-f- glucosamin - 40- idase, no detectable quantities of these macro- - Kphage marker enzyme activities were found in 20- either the phase I or the

A Lectin-Like Receptor is Involved in Invasion of Erythrocytes by Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Jungery, M.; Pasvol, G.; Newbold, C. I.; Weatherall, D. J.

1983-02-01

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

Uptake of chitosan-derived D-glucosamine oligosaccharides in Streptomyces coelicolor A3(2).

PubMed

Viens, Pascal; Dubeau, Marie-Pierre; Kimura, Akane; Desaki, Yoshitake; Shinya, Tomonori; Shibuya, Naoto; Saito, Akihiro; Brzezinski, Ryszard

2015-05-01

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Post-polymerization modification of poly(L-glutamic acid) with D-(+)-glucosamine.

PubMed

Perdih, Peter; Cebašek, Sašo; Možir, Alenka; Zagar, Ema

2014-11-27

Carboxyl functional groups of poly(L-glutamic acid) (PGlu) were modified with a D-(+)-glucosamine (GlcN) by amidation using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling reagent. The coupling reaction was performed in aqueous medium without protection of hydroxyl functional groups of D-(+)-glucosamine. Poly(L-glutamic acid) and GlcN functionalized polyglutamates (P(Glu-GlcN)) were thoroughly characterized by 1D and 2D NMR spectroscopy and SEC-MALS to gain detailed information on their structure, composition and molar mass characteristics. The results reveal successful functionalization with GlcN through the amide bond and also to a minor extent through ester bond formation in position 1 of GlcN. In addition, a ratio between the α- and β-form of glucosamine substituent coupled to polyglutamate repeating units as well as the content of residual dimethoxy triazinyl active ester moiety in the samples were evaluated.

Application of a Granular Mineral-Based Hemostatic Agent (QuikClot) to Reduce Blood Loss After Grade V Liver Injury in Swine

DTIC Science & Technology

2004-09-01

tion of tiletamine hydrochloride (HCl) and zolazepam HCl (Telazol, Fort Dodge Laboratories, Fort Dodge, IA), anesthe- sia was induced by mask using...Demceva M, Vournakis J, Finkielsztein S, Connolly RJ. Comparison of poly-N-acetyl glucosamine (P-GlcNAc) with absorbable collagen (Actifoam) and fibrin

Isothermal Titration Calorimetry and Macromolecular Visualization for the Interaction of Lysozyme and Its Inhibitors

ERIC Educational Resources Information Center

Wei, Chin-Chuan; Jensen, Drake; Boyle, Tiffany; O'Brien, Leah C.; De Meo, Cristina; Shabestary, Nahid; Eder, Douglas J.

2015-01-01

To provide a research-like experience to upper-division undergraduate students in a biochemistry teaching laboratory, isothermal titration calorimetry (ITC) is employed to determine the binding constants of lysozyme and its inhibitors, N-acetyl glucosamine trimer (NAG[subscript 3]) and monomer (NAG). The extremely weak binding of lysozyme/NAG is…

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

PubMed Central

Pérez, M. M.; Prenafeta, A.; Valle, J.; Penadés, J.; Rota, C.; Solano, C.; Marco, J.; Grilló, M.J.; Lasa, I.; Irache, J.M.; Maira-Litran, T.; Jiménez-Barbero, J.; Costa, L.; Pier, G.B.; de Andrés, D.; Amorena, B.

2010-01-01

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-β-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis. PMID:19428854

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit of Branchiostoma belcheri

NASA Astrophysics Data System (ADS)

Fang, Y. Q.; Welsch, U.

1997-03-01

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins ( Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Concanavalin A (Con A), Ulex europaeus agglutinin I (UEA I) and Ricinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.

Comparative study of hemagglutination and lectin activity in Australian medicinal mushrooms (higher Basidiomycetes).

PubMed

Rouf, Razina; Tiralongo, Evelin; Krahl, Anja; Maes, Karen; Spaan, Lina; Wolf, Stefan; May, Tom W; Tiralongo, Joe

2011-01-01

Fifteen Australian mushroom species (higher Basidiomycetes) were assessed for hemagglutination and lectin activity. Hemagglutination activity was evaluated using both neuraminidase treated and untreated rabbit and human A, B, and O erythrocytes. Lectin activity was determined by the ability of various mono- and oligosaccharides to inhibit hemagglutination activity. Of the mushrooms evaluated, seven contained lectin activity. However, five (Agaricus bitorquis, Chlorophyllum brunneum, Coprinus comatus, Cortinarius sp. TWM 1710, and Omphalotus nidiformis) expressed lectin activity in only one of two collections tested. The two remaining lectin active mushroom species (Phlebopus marginatus and Psathyrella asperospora) possessed lectin activity with the same sugar specificity in both collections. Although lectins were identified with diverse specificity, lactose-specific lectin activity was most frequently identified, being present in Agaricus bitorquis, Copronus comatus, Omphalotus nidiformis, and Phlebopus marginatus. In contrast, Psathyrella asperospora, Cortinarius sp. TWM 1710, and Chlorophyllum brunneum were found to possess lectin activity specific for N-acetyl-D-glucosamine, galactose, and N-acetyl-neurammic acid, respectively. Significantly, the galactose-specific lectin activity identified in Cortinarius sp. TWM 1710 and the lactose-specific lectin activity in Phlebopus marginatus have not been previously reported.

Structural analysis of a type 1 ribosome inactivating protein reveals multiple L-asparagine-N-acetyl-D-glucosamine monosaccharide modifications: Implications for cytotoxicity

PubMed Central

HOGG, TANIS; MENDEL, JAMESON T.; LAVEZO, JONATHAN L.

2015-01-01

Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome-inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin-ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X-ray crystal structure of PAP-S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP-S1aci shares ~95% sequence identity with PAP-S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP-S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP-S1aci, which has no effect on catalytic Glu175 positioning or overall active-site topology, yet appears to come at the expense of strained main-chain geometry at the pre-proline residue Val173. Notably, a rare type of N-glycosylation was detected consisting of N-acetyl-D-glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP-S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N-glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA-binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N-glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor-mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition. PMID:26238506

Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

PubMed Central

Leatham, Gary F.

1985-01-01

Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.

PubMed Central

Holroyde, M J; Chesher, J M; Trayer, I P; Walker, D G

1976-01-01

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand. PMID:1275893

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

PubMed

Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

2003-02-11

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

PubMed Central

Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

2003-01-01

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE. PMID:12694627

Long range molecular dynamics study of interactions of the eukaryotic glucosamine-6-phosphate synthase with fructose-6-phosphate and UDP-GlcNAc.

PubMed

Miszkiel, Aleksandra; Wojciechowski, Marek

2017-11-01

Glucosamine-6-phosphate synthase (EC 2.6.1.16) is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5' diphospho N-acetyl-d-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes of it a potential target for anti-fungal, anti-bacterial and anti-diabetic therapy. The crystal structure of isomerase domain from human pathogenic fungus Candida albicans has been solved recently but it doesn't reveal the molecular mechanism details of inhibition taking place under UDP-GlcNAc influence, the unique feature of eukaryotic enzyme. The following study is a continuation of the previous research based on comparative molecular dynamics simulations of the structures with and without the enzyme's physiological inhibitor (UDP-GlcNAc) bound. The models used for this study included fructose-6-phosphate, one of the enzyme's substrates in its binding pocket. The simulation results studies demonstrated differences in mobility of the compared structures. Some amino acid residues were determined, for which flexibility is evidently different between the models. Importantly, it has been confirmed that the most fixed residues are related to the inhibitor binding process and to the catalysis reaction. The obtained results constitute an important step towards understanding of the inhibition that GlcN-6-P synthase is subjected by UDP-GlcNAc molecule. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane.

PubMed

Wang, Yan; Andole Pannuri, Archana; Ni, Dongchun; Zhou, Haizhen; Cao, Xiou; Lu, Xiaomei; Romeo, Tony; Huang, Yihua

2016-05-06

The partially de-N-acetylated poly-β-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane β-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the β-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose.

PubMed

Jorge, João M P; Nguyen, Anh Q D; Pérez-García, Fernando; Kind, Stefanie; Wendisch, Volker F

2017-04-01

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L -1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L -1  h -1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Glycosylated polyacrylate nanoparticles by emulsion polymerization

PubMed Central

Abeylath, Sampath C.; Turos, Edward

2007-01-01

A selection of glycosylated polyacrylate nanoparticles has been prepared by radical-initiated emulsion polymerization in aqueous media. Using ethyl acrylate as a co-monomer, carbohydrate acrylates were incorporated into the poly(ethyl acrylate) framework to give stable emulsions of glyconanoparticles with an average particle size of around 40 nm. Using this technique a variety of glyconanoparticles were prepared from 3-O-acryloyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 1-O-acryloyl-2,3:5,6-di-O-isopropylidene-α-D-mannofuranose, 6-O-acryloyl-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose, 2-N-acryloyl-1,3,4,6-tetra-O-acetyl-β-D-glucosamine, 5-O-acryloyl-2,3-isopropylidene-1-methoxy-β-D-ribofuranose and 4-N-acetyl-5’-O-acryloyl-2’,3’-O-isopropylidene cytidine. Scanning electron microscopy, dynamic light scattering and proton NMR analysis of the emulsions indicated essentially 100% incorporation of the carbohydrate acrylate monomer into the polymer with the exception of O-benzyl- and O-benzoyl-protected carbohydrate acrylates, which gave incomplete incorporation. Formation of larger glyconanoparticles of ~80nm with (unprotected) 3-O-acryloyl-D-glucose and 5-O-acryloyl-1-methoxy-β-D-ribofuranose revealed the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. This methodology allows for a new, simple route to the synthesis of polymeric glyconanoparticles with potential applications in targeted drug delivery and materials development. PMID:18677404

Characterizing the glycocalyx of poultry spermatozoa: III. Semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates.

PubMed

Peláez, J; Bongalhardo, D C; Long, J A

2011-02-01

The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and to contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, and N-acetyl-lactosamine. Our objective here was to evaluate the effects of 3 different cryopreservation methods on the sperm glycocalyx. Semen from roosters was pooled, diluted, cooled to 5°C, and aliquoted for cryopreservation using 6% dimethylacetamide (DMA), 11% dimethylsulfoxide (DMSO), or 11% glycerol (GOH). For the DMA method, semen was equilibrated for 1 min with cryoprotectant and rapidly frozen by dropping 25-µL aliquots into liquid nitrogen. For the other methods, semen was equilibrated for either 1 min (DMSO) or 20 min (GOH), loaded into straws, and frozen with a programmable freezer. Thawing rates mimicked the freezing rates (e.g., rapid for DMA; moderate for DMSO and GOH). Aliquots of thawed and fresh, unfrozen semen were incubated with 1 of 12 fluorescein isothiocyanate-conjugated lectins and counterstained with propidium iodide, and mean fluorescence intensity (MFI) was assessed by flow cytometry. For each lectin, the MFI of propidium iodide-negative (viable sperm) was compared among the fresh and frozen-thawed treatments (n = 5). For sperm frozen with GOH and DMA, the MFI of most lectins was similar (P > 0.05) to that of fresh sperm, whereas only 5 of 12 lectins were similar between fresh and DMSO-frozen sperm. Sperm from all 3 methods had higher (P < 0.05) MFI for lectins specific for N-acetyl-glucosamine and β-galactose than did fresh sperm. Fewer sperm were damaged (P < 0.001) with GOH than with DMA or DMSO, and membrane integrity was correlated with MFI for 9 of 12 lectins (P < 0.05). These data indicate that surface carbohydrates are altered during cryopreservation, and that cryoprotectant type and freezing-thawing rates affect the degree of modification. Although the glycoconjugates have not yet been identified, it is likely that these cryopreservation-induced changes contribute to the reduced fertility of frozen-thawed chicken semen.

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

PubMed Central

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

2014-01-01

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

Utilization of carbon sources by clinical isolates of Aeromonas.

PubMed

Prediger, Karoline C; Surek, Monica; Dallagassa, Cibelle B; Assis, Flávia E A; Piantavini, Mario S; Souza, Emanuel M; Pedrosa, Fábio O; Farah, Sônia M S S; Alberton, Dayane; Fadel-Picheth, Cyntia M T

2017-04-01

Bacteria in the genus Aeromonas are primarily aquatic organisms; however, some species can cause diseases in humans, ranging from wound infections to septicemia, of which diarrhea is the most common condition. The ability to use a variety of carbon substrates is advantageous for pathogenic bacteria. Therefore, we used Biolog GN2 microplates to analyze the ability of 103 clinical, predominantly diarrheal, isolates of Aeromonas to use various carbon sources, and we verified whether, among the substrates metabolized by these strains, there were some endogenous to the human intestine. The results indicate that Aeromonas present great diversity in the utilization of carbon sources, and that they preferentially use carbohydrates and amino acids as carbon sources. Among the carbon sources metabolized by Aeromonas in vitro, some were found to be components of intestinal mucin, including aspartic acid, glutamic acid, l-serine, galactose, N-acetyl-glucosamine, and glucose, which were used by all strains tested. Additionally, mannose, d-serine, proline, threonine, and N-acetyl-galactosamine were used by several strains. The potential to metabolize substrates endogenous to the intestine may contribute to Aeromonas' capacity to grow in and colonize the intestine. We speculate that this may help explain the ability of Aeromonas to cause diarrhea.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells*

PubMed Central

Hwang, Ji-Sun; Kwon, Mi-Youn; Kim, Kyung-Hong; Lee, Yunkyoung; Lyoo, In Kyoon; Kim, Jieun E.; Oh, Eok-Soo; Han, Inn-Oc

2017-01-01

We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess. PMID:27927986

A Second β-Hexosaminidase Encoded in the Streptococcus pneumoniae Genome Provides an Expanded Biochemical Ability to Degrade Host Glycans*

PubMed Central

Robb, Melissa; Robb, Craig S.; Higgins, Melanie A.; Hobbs, Joanne K.; Paton, James C.; Boraston, Alisdair B.

2015-01-01

An important facet of the interaction between the pathogen Streptococcus pneumoniae (pneumococcus) and its human host is the ability of this bacterium to process host glycans. To achieve cleavage of the glycosidic bonds in host glycans, S. pneumoniae deploys a wide array of glycoside hydrolases. Here, we identify and characterize a new family 20 glycoside hydrolase, GH20C, from S. pneumoniae. Recombinant GH20C possessed the ability to hydrolyze the β-linkages joining either N-acetylglucosamine or N-acetylgalactosamine to a wide variety of aglycon residues, thus revealing this enzyme to be a generalist N-acetylhexosaminidase in vitro. X-ray crystal structures were determined for GH20C in a ligand-free form, in complex with the N-acetylglucosamine and N-acetylgalactosamine products of catalysis and in complex with both gluco- and galacto-configured inhibitors O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc), O-(2-acetamido-2-deoxy-d-galactopyranosylidene)amino N-phenyl carbamate (GalPUGNAc), N-acetyl-d-glucosamine-thiazoline (NGT), and N-acetyl-d-galactosamine-thiazoline (GalNGT) at resolutions from 1.84 to 2.7 Å. These structures showed N-acetylglucosamine and N-acetylgalactosamine to be recognized via identical sets of molecular interactions. Although the same sets of interaction were maintained with the gluco- and galacto-configured inhibitors, the inhibition constants suggested preferred recognition of the axial O4 when an aglycon moiety was present (Ki for PUGNAc > GalPUGNAc) but preferred recognition of an equatorial O4 when the aglycon was absent (Ki for GalNGT > NGT). Overall, this study reveals GH20C to be another tool that is unique in the arsenal of S. pneumoniae and that it may implement the effort of the bacterium to utilize and/or destroy the wide array of host glycans that it may encounter. PMID:26491009

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.

The Cation-Responsive Protein NhaR of Escherichia coli Activates pgaABCD Transcription, Required for Production of the Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine▿

PubMed Central

Goller, Carlos; Wang, Xin; Itoh, Yoshikane; Romeo, Tony

2006-01-01

The pgaABCD operon of Escherichia coli is required for production of the biofilm adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA). We establish here that NhaR, a DNA-binding protein of the LysR family of transcriptional regulators, activates transcription of this operon. Disruption of the nhaR gene decreased biofilm formation without affecting planktonic growth. PGA production was undetectable in an nhaR mutant strain. Expression of a pgaA′-′lacZ translational fusion was induced by NaCl and alkaline pH, but not by CaCl2 or sucrose, in an nhaR-dependent fashion. Primer extension and quantitative real-time reverse transcription-PCR analyses further revealed that NhaR affects the steady-state level of pga mRNA. A purified recombinant NhaR protein bound specifically and with high affinity within the pgaABCD promoter region; one apparent binding site overlaps the −35 element, and a second site lies immediately upstream of the first. This protein was necessary and sufficient for activation of in vitro transcription from the pgaA promoter. These results define a novel mechanism for regulation of biofilm formation in response to environmental conditions and suggest an expanded role for NhaR in promoting bacterial survival. PMID:16997959

Chitin Adsorbents for Toxic Metals: A Review

PubMed Central

Anastopoulos, Ioannis; Bhatnagar, Amit; Bikiaris, Dimitrios N.; Kyzas, George Z.

2017-01-01

Wastewater treatment is still a critical issue all over the world. Among examined methods for the decontamination of wastewaters, adsorption is a promising, cheap, environmentally friendly and efficient procedure. There are various types of adsorbents that have been used to remove different pollutants such as agricultural waste, compost, nanomaterials, algae, etc., Chitin (poly-β-(1,4)-N-acetyl-d-glucosamine) is the second most abundant natural biopolymer and it has attracted scientific attention as an inexpensive adsorbent for toxic metals. This review article provides information about the use of chitin as an adsorbent. A list of chitin adsorbents with maximum adsorption capacity and the best isotherm and kinetic fitting models are provided. Moreover, thermodynamic studies, regeneration studies, the mechanism of adsorption and the experimental conditions are also discussed in depth. PMID:28067848

Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

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

Fuentes-Silva, D.; Mendoza-Hernández, G.; Stojanoff, V.

2007-09-01

Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoformsmore » were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.« less

Rare sugar D-allose suppresses gibberellin signaling through hexokinase-dependent pathway in Oryza sativa L.

PubMed

Fukumoto, Takeshi; Kano, Akihito; Ohtani, Kouhei; Yamasaki-Kokudo, Yumiko; Kim, Bong-Gyu; Hosotani, Kouji; Saito, Miu; Shirakawa, Chikage; Tajima, Shigeyuki; Izumori, Ken; Ohara, Toshiaki; Shigematsu, Yoshio; Tanaka, Keiji; Ishida, Yutaka; Nishizawa, Yoko; Tada, Yasuomi; Ichimura, Kazuya; Gomi, Kenji; Akimitsu, Kazuya

2011-12-01

One of the rare sugars, D-allose, which is the epimer of D-glucose at C3, has an inhibitory effect on rice growth, but the molecular mechanisms of the growth inhibition by D-allose were unknown. The growth inhibition caused by D-allose was prevented by treatment with hexokinase inhibitors, D-mannoheptulose and N-acetyl-D-glucosamine. Furthermore, the Arabidopsis glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1, showed a D-allose-insensitive phenotype. D-Allose strongly inhibited the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half rice seeds. The growth of the slender rice1 (slr1) mutant, which exhibits a constitutive gibberellin-responsive phenotype, was also inhibited by D-allose, and the growth inhibition of the slr1 mutant by D-allose was also prevented by D-mannoheptulose treatment. The expressions of gibberellin-responsive genes were down-regulated by D-allose treatment, and the down-regulations of gibberellin-responsive genes were also prevented by D-mannoheptulose treatment. These findings reveal that D-allose inhibits the gibberellin-signaling through a hexokinase-dependent pathway.

New structures and composition of cell wall teichoic acids from Nocardiopsis synnemataformans, Nocardiopsis halotolerans, Nocardiopsis composta and Nocardiopsis metallicus: a chemotaxonomic value.

PubMed

Tul'skaya, Elena M; Shashkov, Alexander S; Streshinskaya, Galina M; Potekhina, Natalia V; Evtushenko, Ludmila I

2014-12-01

The structures of the cell wall teichoic acids (TA) from some species of the genus Nocardiopsis were established by chemical and NMR spectroscopic methods. The cell walls of Nocardiopsis synnemataformans VKM Ac-2518(T) and Nocardiopsis halotolerans VKM Ac-2519(T) both contain two TA with unique structures-poly(polyol phosphate-glycosylpolyol phosphate)-belonging to the type IV TA. In both organisms, the minor TA have identical structures: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-4 of the amino sugar. This structure is found for the first time. The major TA of N. halotolerans has a hitherto unknown structure: poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate), the N-acetyl-β-galactosamine being acetalated with pyruvic acid at positions 4 and 6. The major TA of N. synnemataformans is a poly(glycerol phosphate-N-acetyl-β-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-3 of the amino sugar. The cell walls of Nocardiopsis composta VKM Ac-2520 and N. composta VKM Ac-2521(T) contain only one TA, namely 1,3-poly(glycerol phosphate) partially substituted with N-acetyl-α-glucosamine. The cell wall of Nocardiopsis metallicus VKM Ac-2522(T) contains two TA. The major TA is 1,5-poly(ribitol phosphate), each ribitol unit carrying a pyruvate ketal group at positions 2 and 4. The structure of the minor TA is the same as that of N. composta. The results presented correlate well with the phylogenetic grouping of strains and confirm the species and strain specific features of cell wall TA in members of the genus Nocardiopsis.

N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

PubMed Central

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L.

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species. PMID:21655093

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

PubMed

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

The versatile biopolymer chitosan: potential sources, evaluation of extraction methods and applications.

PubMed

Kaur, Surinder; Dhillon, Gurpreet Singh

2014-05-01

Among the biopolymers, chitin and its derivative chitosan (CTS) have been receiving increasing attention. Both are composed of randomly distributed β-(1-4)-linked d-glucosamine and N-acetyl glucosamine units. On commercial scale, CTS is mainly obtained from the crustacean shells. The chemical methods employed for extraction of CTS from crustacean shells are laden with many disadvantages. Waste fungal biomass represents a potential biological source of CTS, in fact with superior physico-chemical properties, such as high degree of deacetylation, low molecular weight, devoid of protein contamination and high bioactivity. Researchers around the globe are attempting to commercialize CTS production and extraction from fungal sources. Fungi are promising and environmentally benign source of CTS and they have the potential to completely replace crustacean-derived CTS. Waste fungal biomass resulting from various pharmaceutical and biotechnological industries is grown on inexpensive agro-industrial wastes and its by-products are a rich and inexpensive source of CTS. CTS is emerging as an important natural polymer having broad range of applications in different fields. In this context, the present review discusses the potential sources of CTS and their advantages and disadvantages. This review also deals with potential applications of CTS in different fields. Finally, the various attributes of CTS sought in different applications are discussed.

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

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

Thoden, James B.; Holden, Hazel M.

2010-09-08

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

An Overview of the Protective Effects of Chitosan and Acetylated Chitosan Oligosaccharides against Neuronal Disorders.

PubMed

Hao, Cui; Wang, Wei; Wang, Shuyao; Zhang, Lijuan; Guo, Yunliang

2017-03-23

Chitin is the second most abundant biopolymer on Earth and is mainly comprised of a marine invertebrate, consisting of repeating β-1,4 linked N-acetylated glucosamine units, whereas its N-deacetylated product, chitosan, has broad medical applications. Interestingly, chitosan oligosaccharides have therapeutic effects on different types of neuronal disorders, including, but not limited to, Alzheimer's disease, Parkinson's disease, and nerve crush injury. A common link among neuronal disorders is observed at a sub-cellular level, such as atypical protein assemblies and induced neuronal death. Chronic activation of innate immune responses that lead to neuronal injury is also common in these diseases. Thus, the common mechanisms of neuronal disorders might explain the general therapeutic effects of chitosan oligosaccharides and their derivatives in these diseases. This review provides an update on the pathogenesis and therapy for neuronal disorders and will be mainly focused on the recent progress made towards the neuroprotective properties of chitosan and acetylated chitosan oligosaccharides. Their structural features and the underlying molecular mechanisms will also be discussed.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3)

PubMed Central

van Wijk, Xander M.; Lawrence, Roger; Thijssen, Victor L.; van den Broek, Sebastiaan A.; Troost, Ran; van Scherpenzeel, Monique; Naidu, Natasha; Oosterhof, Arie; Griffioen, Arjan W.; Lefeber, Dirk J.; van Delft, Floris L.; van Kuppevelt, Toin H.

2015-01-01

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 μM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50–60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5′-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.—Van Wijk, X. M., Lawrence, R., Thijssen, V. L., van den Broek, S. A., Troost, R., van Scherpenzeel, M., Naidu, N., Oosterhof, A., Griffioen, A. W., Lefeber, D. J., van Delft, F. L., van Kuppevelt, T. H. A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3). PMID:25868729

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation.

PubMed

Hrynets, Yuliya; Ndagijimana, Maurice; Betti, Mirko

2015-01-01

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation

PubMed Central

2015-01-01

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure. PMID:26406447

A histochemical study of the distribution of lectin binding sites in the developing branchial area of the trout Salmo trutta.

PubMed Central

Rojo, M C; Blánquez, M J; González, M E

1996-01-01

A histochemical study of the branchial area of brown trout embryos from 35 to 71 d of incubation is reported. A battery of 6 different horseradish peroxidase-labelled lectins, the PAS reaction and Alcian blue staining were used to study the distribution of carbohydrate residues in glycoconjugates along the pharyngeal and branchial epithelia. Con A and WGA reacted at every site of the branchial region thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. WGA, DBA and SBA were good markers for the hatching gland cells (HGCs) and mucous cells. Other lectins, such as PNA and UEA I, reacted only for a short time at some sites during the considered period of incubation. From 35 d until posthatching stages, a manifest strong reaction was noted both in the dorsal epithelium of branchial arches and the HGCs as shown by SBA reactivity. This may be significant with regard to the controversial origin of HGCs, which is thought to be endodermal. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8982837

Use of an Enzyme-Linked Lectinsorbent Assay To Monitor the Shift in Polysaccharide Composition in Bacterial Biofilms

PubMed Central

Leriche, V.; Sibille, P.; Carpentier, B.

2000-01-01

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: d-glucose or d-mannose for ConA and N-acetyl-d-glucosamine or N-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix. PMID:10788349

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

PubMed Central

Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

2015-01-01

Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

PubMed Central

Berck, S.; Perret, X.; Quesada-Vincens, D.; Promé, J.-C.; Broughton, W. J.; Jabbouri, S.

1999-01-01

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not. PMID:9922261

Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae.

PubMed

Mishra, C; Semino, C E; McCreath, K J; de la Vega, H; Jones, B J; Specht, C A; Robbins, P W

1997-03-30

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

The Antemortem Detection and Conformational Switches of Prion Proteins

DTIC Science & Technology

2006-07-01

distribution of di-, mono-, and unglycosylated PrPC molecules (Fig. 7B, lane 2). Subsequent elution of the column with N-acetyl glucosamine yielded...pellet was washed in 50 mM Tris, pH 8.0, 150 mM NaCl, and then solubilized in 6 M guanidinium hydrochloride , 50 mM Tris pH 8.0, 150 mM NaCl. The...and 150mM sodium chloride (TCl) containing 6M guanidinium hydrochloride . The supernatant was centrifuged for 30min at 18,000g. The protein was

A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification.

PubMed

Chuanxiang, Wu; Lian, Xia; Lijie, Liu; Fengli, Qu; Zhiwei, Sun; Xianen, Zhao; Jinmao, You

2016-02-01

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12μM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Degradation and mineralization of chitin in an estuary

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

Boyer, J.

1987-01-01

A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described. /sup 14/C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D-(/sup 14/C)-glucosamine, allowing for its incorporation into the exoskeleton. Rates of chitin degradation and mineralization in estuarine water and sediments were determined as functions of temperature, inoculum source, and oxygen condition. Significant differences in rates between temperature treatments were evident. Q/sub 10/ values ranged from 1.2 to 2.5 for water and sediment, respectively. Increased incubation temperature also resulted in decreased lag times before onset of chitinoclastic bacterial growth and chitin degradation.more » The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other bacterial types. Actively growing chitinoclastic bacterial isolates produced primarily acetate, hydrogen, and carbon dioxide in broth culture.« less

Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

PubMed

Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

2015-10-01

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

Chitin, Chitosan, and Its Derivatives for Wound Healing: Old and New Materials

PubMed Central

Azuma, Kazuo; Izumi, Ryotaro; Osaki, Tomohiro; Ifuku, Shinsuke; Morimoto, Minoru; Saimoto, Hiroyuki; Minami, Saburo; Okamoto, Yoshiharu

2015-01-01

Chitin (β-(1-4)-poly-N-acetyl-d-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. It is often converted to its more deacetylated derivative, chitosan. Previously, many reports have indicated the accelerating effects of chitin, chitosan, and its derivatives on wound healing. More recently, chemically modified or nano-fibrous chitin and chitosan have been developed, and their effects on wound healing have been evaluated. In this review, the studies on the wound-healing effects of chitin, chitosan, and its derivatives are summarized. Moreover, the development of adhesive-based chitin and chitosan are also described. The evidence indicates that chitin, chitosan, and its derivatives are beneficial for the wound healing process. More recently, it is also indicate that some nano-based materials from chitin and chitosan are beneficial than chitin and chitosan for wound healing. Clinical applications of nano-based chitin and chitosan are also expected. PMID:25780874

Chemical Analysis for Chitin as a Measure of Fungal Infiltration of Cellulosic Materials.

DTIC Science & Technology

1976-12-01

the addition of 50 n’.illiliters of I 2N hydrochloric acid). Store at .~lO0 C. (11) Bushnell-Haas medium (12) Glucosamine hydrochloride (I milliliter...Infiltration Cellu!.jsic Materials Fungus-Induced Deterioration Glucosamine A TRACT (Cl~ i~s ,.v.ra. ia. ~V ~~~~~a . y d Sd.niIl ~ By block ni b.,) A chemical...EXPERIMENTAL PROCEDURE 3. Approach to the Problem. Carry out laboratory experiments to investigate variables as: shelf life of stock glucosamine , digestion

A Computational Framework for High-Throughput Isotopic Natural Abundance Correction of Omics-Level Ultra-High Resolution FT-MS Datasets

PubMed Central

Carreer, William J.; Flight, Robert M.; Moseley, Hunter N. B.

2013-01-01

New metabolomics applications of ultra-high resolution and accuracy mass spectrometry can provide thousands of detectable isotopologues, with the number of potentially detectable isotopologues increasing exponentially with the number of stable isotopes used in newer isotope tracing methods like stable isotope-resolved metabolomics (SIRM) experiments. This huge increase in usable data requires software capable of correcting the large number of isotopologue peaks resulting from SIRM experiments in a timely manner. We describe the design of a new algorithm and software system capable of handling these high volumes of data, while including quality control methods for maintaining data quality. We validate this new algorithm against a previous single isotope correction algorithm in a two-step cross-validation. Next, we demonstrate the algorithm and correct for the effects of natural abundance for both 13C and 15N isotopes on a set of raw isotopologue intensities of UDP-N-acetyl-D-glucosamine derived from a 13C/15N-tracing experiment. Finally, we demonstrate the algorithm on a full omics-level dataset. PMID:24404440

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

Genomic, proteomic and biochemical analysis of the chitinolytic machinery of Serratia marcescens BJL200.

PubMed

Tuveng, Tina R; Hagen, Live Heldal; Mekasha, Sophanit; Frank, Jeremy; Arntzen, Magnus Øverlie; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

2017-04-01

The chitinolytic machinery of Serratia marcescens BJL200 has been studied in detail over the last couple of decades, however, the proteome secreted by this Gram-negative bacterium during growth on chitin has not been studied in depth. In addition, the genome of this most studied chitinolytic Serratia strain has until now, not been sequenced. We report a draft genome sequence for S. marcescens BJL200. Using label-free quantification (LFQ) proteomics and a recently developed plate-method for assessing secretomes during growth on solid substrates, we find that, as expected, the chitin-active enzymes (ChiA, B, C, and CBP21) are produced in high amounts when the bacterium grows on chitin. Other proteins produced in high amounts after bacterial growth on chitin provide interesting targets for further exploration of the proteins involved in degradation of chitin-rich biomasses. The genome encodes a fourth chitinase (ChiD), which is produced in low amounts during growth on chitin. Studies of chitin degradation with mixtures of recombinantly produced chitin-degrading enzymes showed that ChiD does not contribute to the overall efficiency of the process. ChiD is capable of converting N,N'-diacetyl chitobiose to N-acetyl glucosamine, but is less efficient than another enzyme produced for this purpose, the Chitobiase. Thus, the role of ChiD in chitin degradation, if any, remains unclear. Copyright © 2017 Elsevier B.V. All rights reserved.

Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

PubMed

Klinkspoor, J H; Yoshida, T; Lee, S P

1998-05-15

1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with beta-cyclodextrin (2.5-15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (>100 microM Triton X-100, >0.8 mM Tween-20), caused increased LDH release. Incubation with beta-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.

In vitro synthesis of intermediates involved in the assembly of enterobacterial common antigen (ECA)

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

Barr, K.; Wolski, S.; Kroto, J.

1986-05-01

ECA is a cell surface antigen found in all bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of trisaccharide repeat units; the component sugars are N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-D-fucose (Fuc4NAc). In vivo studies have suggested that GlcNAc-pyrophosphorylundecaprenol (GlcNAc-PP-lipid) is an intermediate in ECA synthesis. More recently, they have demonstrated UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase activity in cell envelope preparations of E. coli. Radioactivity from UDP-(/sup 3/H)Glc-NAc was incorporated into endogenous lipid acceptor, and the labeled product was characterized as GlcNAc-PP-lipid (lipid I). Transferase activity was inhibited by tunicamycin andmore » UMP, but it was unaffected by UDP. The reaction was reversible, and the synthesis of UDP-(/sup 3/H)GlcNAc from UMP and (/sup 3/H)GlcNAc-PP-lipid was also sensitive to tunicamycin. The simultaneous addition of UDP-(/sup 14/C)ManNAcUA and UDP-(/sup 3/H)GlcNAc to cell envelope preparations resulted in the synthesis of a more polar lipid (lipid II) that contained both labeled sugars in equimolar amounts. Synthesis of lipid II was dependent on prior synthesis of lipid I. Accordingly, (/sup 3/H)GlcNAc-PP-lipid that had been synthesized in vivo served as an acceptor in vitro of ManNAcUA residues from UDP-ManNAcUA. Lipid II has been tentatively identified as ManNAcUA-GlcNAc-pyrophosphorylundecaprenol.« less

Nanostructured Lipid Carrier for Topical Application of N-Acetyl Glucosamine.

PubMed

Aliasgharlou, Lavin; Ghanbarzadeh, Saeed; Azimi, Hamideh; Zarrintan, Mohammad Hossein; Hamishehkar, Hamed

2016-12-01

Purpose: Hyperpigmentation occurs when melanin is overproduced in certain spots on the skin and is one of the most challenging skin conditions to treat. Although it is usually harmless, for cosmetic reasons, it is dreadfully bothersome to those who undergo it. It was reported that N-acetyl-glucosamine (NAGA) prevents melanin synthesis and alters the expression of numerous genes related to pigmentation. In spite of these advantages, NAGA cannot be employed in topical formulations due to its extremely polar characteristics. Nanoparticles, especially lipid-based ones, have been introduced as an efficient carrier for dermal drug delivery. Methods: The aim of the present study was to load adequate hydrophilic NAGA to the lipophilic nanostructured lipid carriers (NLCs) for potential dermal application. NAGA-loaded NLCs were formulated, using hot homogenization technique, and the characteristics of the optimized formulation were analyzed by laser light scattering, X-ray diffraction, and scanning electron microscopy methods. Loading capacity percentage and in vitro release study were carried out by applying a validated HPLC method. The optimum formulation was utilized for the in vivo skin lightening evaluations in healthy volunteers. Results: NAGA-loaded NLCs demonstrated promising results (the size of 190 nm, narrow size distribution, loading capacity of 9%, and appropriate NAGA release profile) suitable for dermal delivery. XRD results exhibited a dramatic reduction in the crystalline structure of encapsulated NAGA. Dermoscopy images indicated a considerable decline in melanin distribution pattern in the majority of the cases treated with NAGA-loaded NLCs. Conclusion: Thus, this study has opened new horizons for the potential use of lipid based nanoparticles in the managing of hyperpigmentation.

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.

Characterization of Two Campylobacter jejuni Strains for Use in Volunteer Experimental-Infection Studies▿ †

PubMed Central

Poly, Frédéric; Read, Timothy D.; Chen, Yu-Han; Monteiro, Mario A.; Serichantalergs, Oralak; Pootong, Piyarat; Bodhidatta, Ladaporn; Mason, Carl J.; Rockabrand, David; Baqar, Shahida; Porter, Chad K.; Tribble, David; Darsley, Michael; Guerry, Patricia

2008-01-01

The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, d-glycero-d-ido-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-altro-heptose, N-acetyl-glucosamine, and minor amounts of 6-deoxy-3-O-Me-altro-heptose. Both CPSs were shown to carry O-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011. PMID:18809665

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

USGS Publications Warehouse

Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

2004-01-01

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

O-linked N-acetyl-glucosamine deposition in placental proteins varies according to maternal glycemic levels.

PubMed

Dela Justina, Vanessa; Dos Passos Junior, Rinaldo R; Bressan, Alecsander F; Tostes, Rita C; Carneiro, Fernando S; Soares, Thaigra S; Volpato, Gustavo T; Lima, Victor Vitorino; Martin, Sebastian San; Giachini, Fernanda R

2018-05-07

Hyperglycemia increases glycosylation with O-linked N‑acetyl‑glucosamine (O-GlcNAc) contributing to placental dysfunction and fetal growth impairment. Our aim was to determine how O-GlcNAc levels are affected by hyperglycemia and the O-GlcNAc distribution in different placental regions. Female Wistar rats were divided into the following groups: severe hyperglycemia (>300 mg/dL; n = 5); mild hyperglycemia (>140 mg/dL, at least than two time points during oral glucose tolerance test; n = 7) or normoglycemia (<120 mg/dL; n = 6). At 21 days of pregnancy, placental tissue was collected and processed for morphometry and immunohistochemistry analyses, or properly stored at -80 °C for protein quantification by western blot. Placental index was increased only in severe hyperglycemic rats. Morphometric analysis showed increased junctional zone and decreased labyrinth region in placentas exclusively from the severe hyperglycemic group. Proteins targeted by O-GlcNAc were detected in all regions, with increased O-GlcNAc levels in the hyperglycemic group compared to control and mild hyperglycemic rats. Proteins in endothelial and trophoblast cells were the main target for O-GlcNAc. Whereas no changes in O-GlcNAc transferase (OGT) expression were detected, O-GlcNAcase (OGA) expression was reduced in placentas from the severe hyperglycemic group and augmented in placentas from the mild hyperglycemic group, compared with their respective control groups. Placental O-GlcNAc overexpression may contribute to placental dysfunction, as indicated by the placental index. Additionally, morphometric alterations, occurring simultaneously with increased O-GlcNAc accumulation in the placental tissue may contribute to placental dysfunction during hyperglycemia. Copyright © 2017. Published by Elsevier Inc.

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

PubMed

Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

2016-11-18

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Effects of Plant Growth Hormones on Mucor indicus Growth and Chitosan and Ethanol Production.

PubMed

Safaei, Zahra; Karimi, Keikhosro; Golkar, Poorandokht; Zamani, Akram

2015-07-22

The objective of this study was to investigate the effects of indole-3-acetic acid (IAA) and kinetin (KIN) on Mucor indicus growth, cell wall composition, and ethanol production. A semi-synthetic medium, supplemented with 0-5 mg/L hormones, was used for the cultivations (at 32 °C for 48 h). By addition of 1 mg/L of each hormone, the biomass and ethanol yields were increased and decreased, respectively. At higher levels, however, an inverse trend was observed. The glucosamine fraction of the cell wall, as a representative for chitosan, followed similar but sharper changes, compared to the biomass. The highest level was 221% higher than that obtained without hormones. The sum of glucosamine and N-acetyl glucosamine (chitin and chitosan) was noticeably enhanced in the presence of the hormones. Increase of chitosan was accompanied by a decrease in the phosphate content, with the lowest phosphate (0.01 g/g cell wall) being obtained when the chitosan was at the maximum (0.45 g/g cell wall). In conclusion, IAA and KIN significantly enhanced the M. indicus growth and chitosan production, while at the same time decreasing the ethanol yield to some extent. This study shows that plant growth hormones have a high potential for the improvement of fungal chitosan production by M. indicus.

O-Linked N-Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter▿

PubMed Central

Jochmann, Ramona; Thurau, Mathias; Jung, Susan; Hofmann, Christian; Naschberger, Elisabeth; Kremmer, Elisabeth; Harrer, Thomas; Miller, Matthew; Schaft, Niels; Stürzl, Michael

2009-01-01

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5′ long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5′ LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-d-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4+ T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS. PMID:19193796

Is there any scientific evidence for the use of glucosamine in the management of human osteoarthritis?

PubMed Central

2012-01-01

Glucosamine in its acetylated form is a natural constituent of some glycosaminoglycans (for example, hyaluronic acid and keratan sulfate) in the proteoglycans found in articular cartilage, intervertebral disc and synovial fluid. Glucosamine can be extracted and stabilized by chemical modification and used as a drug or a nutraceutical. It has been approved for the treatment of osteoarthritis (OA) in Europe to promote cartilage and joint health and is sold over the counter as a dietary supplement in the United States. Various formulations of glucosamine have been tested, including glucosamine sulfate and glucosamine hydrochloride. In vitro and in vivo studies have uncovered glucosamine's mechanisms of action on articular tissues (cartilage, synovial membrane and subchondral bone) and justified its efficacy by demonstrating structure-modifying and anti-inflammatory effects at high concentrations. However, results from clinical trials have raised many concerns. Pharmacokinetic studies have shown that glucosamine is easily absorbed, but the current treatment doses (for example, 1,500 mg/day) barely reach the required therapeutic concentration in plasma and tissue. The symptomatic effect size of glucosamine varies greatly depending on the formulation used and the quality of clinical trials. Importantly, the effect size reduces when evidence is accumulated chronologically and evidence for the structure-modifying effects of glucosamine are sparse. Hence, glucosamine was at first recommended by EULAR and OARSI for the management of knee pain and structure improvement in OA patients, but not in the most recent NICE guidelines. Consequently, the published recommendations for the management of OA require revision. Glucosamine is generally safe and although there are concerns about potential allergic and salt-related side effects of some formulations, no major adverse events have been reported so far. This paper examines all the in vitro and in vivo evidence for the mechanism of action of glucosamine as well as reviews the results of clinical trials. The pharmacokinetics, side effects and differences observed with different formulations of glucosamine and combination therapies are also considered. Finally, the importance of study design and criteria of evaluation are highlighted as new compounds represent new interesting options for the management of OA. PMID:22293240

Is there any scientific evidence for the use of glucosamine in the management of human osteoarthritis?

PubMed

Henrotin, Yves; Mobasheri, Ali; Marty, Marc

2012-01-30

Glucosamine in its acetylated form is a natural constituent of some glycosaminoglycans (for example, hyaluronic acid and keratan sulfate) in the proteoglycans found in articular cartilage, intervertebral disc and synovial fluid. Glucosamine can be extracted and stabilized by chemical modification and used as a drug or a nutraceutical. It has been approved for the treatment of osteoarthritis (OA) in Europe to promote cartilage and joint health and is sold over the counter as a dietary supplement in the United States. Various formulations of glucosamine have been tested, including glucosamine sulfate and glucosamine hydrochloride. In vitro and in vivo studies have uncovered glucosamine's mechanisms of action on articular tissues (cartilage, synovial membrane and subchondral bone) and justified its efficacy by demonstrating structure-modifying and anti-inflammatory effects at high concentrations. However, results from clinical trials have raised many concerns. Pharmacokinetic studies have shown that glucosamine is easily absorbed, but the current treatment doses (for example, 1,500 mg/day) barely reach the required therapeutic concentration in plasma and tissue. The symptomatic effect size of glucosamine varies greatly depending on the formulation used and the quality of clinical trials. Importantly, the effect size reduces when evidence is accumulated chronologically and evidence for the structure-modifying effects of glucosamine are sparse. Hence, glucosamine was at first recommended by EULAR and OARSI for the management of knee pain and structure improvement in OA patients, but not in the most recent NICE guidelines. Consequently, the published recommendations for the management of OA require revision. Glucosamine is generally safe and although there are concerns about potential allergic and salt-related side effects of some formulations, no major adverse events have been reported so far. This paper examines all the in vitro and in vivo evidence for the mechanism of action of glucosamine as well as reviews the results of clinical trials. The pharmacokinetics, side effects and differences observed with different formulations of glucosamine and combination therapies are also considered. Finally, the importance of study design and criteria of evaluation are highlighted as new compounds represent new interesting options for the management of OA.

Biodiversity of Exopolysaccharides Produced by Streptococcus thermophilus Strains Is Reflected in Their Production and Their Molecular and Functional Characteristics

PubMed Central

Vaningelgem, Frederik; Zamfir, Medana; Mozzi, Fernanda; Adriany, Tom; Vancanneyt, Marc; Swings, Jean; De Vuyst, Luc

2004-01-01

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures. PMID:14766570

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

PubMed Central

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass—namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production. PMID:29381705

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

PubMed

Nag, Ambarish; St John, Peter C; Crowley, Michael F; Bomble, Yannick J

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

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

PubMed

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

2018-04-01

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

Microbial Community-Level Physiological Profiles (CLPP) and herbicide mineralization potential in groundwater affected by agricultural land use

NASA Astrophysics Data System (ADS)

Janniche, Gry Sander; Spliid, Henrik; Albrechtsen, Hans-Jørgen

2012-10-01

Diffuse groundwater pollution from agricultural land use may impact the microbial groundwater community, which was investigated as Community-Level Physiological Profiles (CLPP) using EcoPlate™. Water was sampled from seven piezometers and a spring in a small agricultural catchment with diffuse herbicide and nitrate pollution. Based on the Shannon-Wiener and Simpson's diversity indices the diversity in the microbial communities was high. The response from the EcoPlates™ showed which substrates support groundwater bacteria, and all 31 carbon sources were utilized by organisms from at least one water sample. However, only nine carbon sources were utilized by all water samples: D-Mannitol, N-acetyl-D-glucosamine, putrescine, D-galacturonic acid, itaconic acid, 4-hydroxy benzoic acid, tween 40, tween 80, and L-asparagine. In all water samples the microorganisms preferred D-mannitol, D-galacturonic acid, tween 40, and 4-hydroxy benzoic acid as substrates, whereas none preferred 2-hydroxy benzoic acid, α-D-lactose, D,L-α-glycerol phosphate, α-ketobutyric acid, L-threonine and glycyl-L-glutamic acid. Principal Component Analysis of the CLPP's clustered the most agriculturally affected groundwater samples, indicating that the agricultural land use affects the groundwater microbial communities. Furthermore, the ability to mineralize atrazine and isoproturon, which have been used in the catchment, was also associated with this cluster.

Structural analysis and biological activity of a highly regular glycosaminoglycan from Achatina fulica.

PubMed

Liu, Jie; Zhou, Lutan; He, Zhicheng; Gao, Na; Shang, Feineng; Xu, Jianping; Li, Zi; Yang, Zengming; Wu, Mingyi; Zhao, Jinhua

2018-02-01

Edible snails have been widely used as a health food and medicine in many countries. A unique glycosaminoglycan (AF-GAG) was purified from Achatina fulica. Its structure was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, analysis of monosaccharide composition, and 1D/2D nuclear magnetic resonance spectroscopy. Chemical composition analysis indicated that AF-GAG is composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) and its average molecular weight is 118kDa. Structural analysis clarified that the uronic acid unit in glycosaminoglycan (GAG) is the fully epimerized and the sequence of AF-GAG is →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. Although its structure with a uniform repeating disaccharide is similar to those of heparin and heparan sulfate, this GAG is structurally highly regular and homogeneous. Anticoagulant activity assays indicated that AF-GAG exhibits no anticoagulant activities, but considering its structural characteristic, other bioactivities such as heparanase inhibition may be worthy of further study. Copyright © 2017 Elsevier Ltd. All rights reserved.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

PubMed

Ismail, Sadek; Dubois-Vedrenne, Ingrid; Laval, Marie; Tikhonova, Irina G; D'Angelo, Romina; Sanchez, Claire; Clerc, Pascal; Gherardi, Marie-Julie; Gigoux, Véronique; Magnan, Remi; Fourmy, Daniel

2015-10-15

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide. Copyright © 2015. Published by Elsevier Ireland Ltd.

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

PubMed Central

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

2014-01-01

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

Identification of sex-specific urinary biomarkers for major depressive disorder by combined application of NMR- and GC-MS-based metabonomics.

PubMed

Zheng, P; Chen, J-J; Zhou, C-J; Zeng, L; Li, K-W; Sun, L; Liu, M-L; Zhu, D; Liang, Z-H; Xie, P

2016-11-15

Women are more vulnerable to major depressive disorder (MDD) than men. However, molecular biomarkers of sex differences are limited. Here we combined gas chromatography-mass spectrometry (GC-MS)- and nuclear magnetic resonance (NMR)-based metabonomics to investigate sex differences of urinary metabolite markers in MDD, and further explore their potential of diagnosing MDD. Consequently, the metabolite signatures of women and men MDD subjects were significantly different from of that in their respective healthy controls (HCs). Twenty seven women and 36 men related differentially expressed metabolites were identified in MDD. Fourteen metabolites were changed in both women and men MDD subjects. Significantly, the women-specific (m-Hydroxyphenylacetate, malonate, glycolate, hypoxanthine, isobutyrate and azelaic acid) and men-specific (tyrosine, N-acetyl-d-glucosamine, N-methylnicotinamide, indoxyl sulfate, citrate and succinate) marker panels were further identified, which could differentiate men and women MDD patients from their respective HCs with higher accuracy than previously reported sex-nonspecific marker panels. Our findings demonstrate that men and women MDD patients have distinct metabonomic signatures and sex-specific biomarkers have promising values in diagnosing MDD.

Myxospore Coat Synthesis in Myxococcus xanthus: In Vivo Incorporation of Acetate and Glycine

PubMed Central

Filer, D.; White, D.; Kindler, S. H.; Rosenberg, E.

1977-01-01

Myxospore coat synthesis in Myxococcus xanthus was studied by incorporation of [14C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27°C, the radioactivity was shown to be located primarily in N-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of N-acetylglucosamine 6-phosphate and uridine 5′-diphosphate-N-acetylgalactosamine (UDPGalNAc) plus uridine 5′-diphosphate-N-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was N-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor d-cycloserine. In contrast, bacitracin inhibited the incorporation of [14C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [14C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [14C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [14C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [14C]glycine was resistant to penicillin but sensitive to bacitracin. PMID:408325

Glucosamine Modulates T Cell Differentiation through Down-regulating N-Linked Glycosylation of CD25*

PubMed Central

Chien, Ming-Wei; Lin, Ming-Hong; Huang, Shing-Hwa; Fu, Shin-Huei; Hsu, Chao-Yuan; Yen, B. Lin-Ju; Chen, Jiann-Torng; Chang, Deh-Ming; Sytwu, Huey-Kang

2015-01-01

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis. PMID:26468284

Offline and online capillary electrophoresis enzyme assays of β-N-acetylhexosaminidase.

PubMed

Křížek, Tomáš; Doubnerová, Veronika; Ryšlavá, Helena; Coufal, Pavel; Bosáková, Zuzana

2013-03-01

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N',N″-triacetylchitotriose, N,N'-diacetylchitobiose, and N-acetyl-D-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N',N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N'-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N'-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N',N″-triacetylchitotriose as a substrate was also performed.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

PubMed Central

Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F.; Vörös, Andrea; Hegedűs, Zoltán; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

2015-01-01

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.

PubMed

Ferrari, Alessandro R; Lee, Misun; Fraaije, Marco W

2015-06-01

Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature. © 2015 Wiley Periodicals, Inc.

Effect of farnesol on structure and composition of Staphylococcus epidermidis biofilm matrix.

PubMed

Gomes, Fernanda; Teixeira, Pilar; Cerca, Nuno; Azeredo, Joana; Oliveira, Rosário

2011-10-01

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections in which biofilm formation is considered to be one of the main virulence mechanisms. Moreover, their increased resistance to conventional antibiotic therapy enhances the need to develop new therapeutical agents. Farnesol, a natural sesquiterpenoid present in many essential oils, has been described as impairing bacterial growth. The aim of this study was to evaluate the effect of farnesol on the structure and composition of biofilm matrix of S. epidermidis. Biofilms formed in the presence of farnesol (300 μM) contained less biomass, and displayed notable changes in the composition of the biofilm matrix. Changes in the spacial structure were also verified by confocal scanning laser microscopy (CSLM). The results obtained by the quantification of extracellular polymers and by wheat germ agglutinin (WGA) fluorescent detection of glycoproteins containing β(1→4)-N-acetyl-D: -glucosamine support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content.

[Separation of osteoclasts by lectin affinity chromatography].

PubMed

Itokazu, M; Tan, A; Tanaka, S

1991-09-01

Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

PubMed

Ilic, M Z; Martinac, B; Samiric, T; Handley, C J

2008-12-01

To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

Seasonal Alterations in Organic Phosphorus Metabolism Drive the Phosphorus Economy of Annual Growth in F. sylvatica Trees on P-Impoverished Soil.

PubMed

Netzer, Florian; Herschbach, Cornelia; Oikawa, Akira; Okazaki, Yozo; Dubbert, David; Saito, Kazuki; Rennenberg, Heinz

2018-01-01

Phosphorus (P) is one of the most important macronutrients limiting plant growth and development, particularly in forest ecosystems such as temperate beech ( Fagus sylvatica ) forests in Central Europe. Efficient tree internal P cycling during annual growth is an important strategy of beech trees to adapt to low soil-P. Organic P (P org ) is thought to play a decisive role in P cycling, but the significance of individual compounds and processes has not been elucidated. To identify processes and metabolites involved in P cycling of beech trees, polar-metabolome and lipidome profiling was performed during annual growth with twig tissues from a sufficient (Conventwald, Con) and a low-soil-P (Tuttlingen, Tut) forest. Autumnal phospholipid degradation in leaves and P export from senescent leaves, accumulation of phospholipids and glucosamine-6-phosphate (GlcN6P) in the bark, storage of N-acetyl-D-glucosamine-6-phosphate (GlcNAc6P) in the wood, and establishing of a phospholipid "start-up capital" in buds constitute main processes involved in P cycling that were enhanced in beech trees on low-P soil of the Tut forest. In spring, mobilization of P from storage pools in the bark contributed to an effective P cycling. Due to the higher phospholipid "start-up capital" in buds of Tut beeches, the P metabolite profile in developing leaves in spring was similar in beech trees of both forests. During summer, leaves of Tut beeches meet their phosphate (P i ) needs by replacing phospholipids by galacto- and sulfolipids. Thus, several processes contribute to adequate P i supply on P impoverished soil thereby mediating similar growth of beech at low and sufficient soil-P availability.

Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity.

PubMed

Warda, Alicja K; Siezen, Roland J; Boekhorst, Jos; Wells-Bennik, Marjon H J; de Jong, Anne; Kuipers, Oscar P; Nierop Groot, Masja N; Abee, Tjakko

2016-01-01

We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.

Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity

PubMed Central

Warda, Alicja K.; Siezen, Roland J.; Boekhorst, Jos; Wells-Bennik, Marjon H. J.; de Jong, Anne; Kuipers, Oscar P.; Nierop Groot, Masja N.; Abee, Tjakko

2016-01-01

We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed. PMID:27272929

40 CFR 180.1072 - Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... of the biological plant growth regulator poly-D-glucosamine when used as a seed treatment in or on... established for residues of the biological plant growth regulator poly-D-glucosamine when used as a pesticide...

40 CFR 180.1072 - Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... of the biological plant growth regulator poly-D-glucosamine when used as a seed treatment in or on... established for residues of the biological plant growth regulator poly-D-glucosamine when used as a pesticide...

40 CFR 180.1072 - Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... of the biological plant growth regulator poly-D-glucosamine when used as a seed treatment in or on... established for residues of the biological plant growth regulator poly-D-glucosamine when used as a pesticide...

40 CFR 180.1072 - Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... of the biological plant growth regulator poly-D-glucosamine when used as a seed treatment in or on... established for residues of the biological plant growth regulator poly-D-glucosamine when used as a pesticide...

Ultrastructure and Glycoconjugate Pattern of the Foot Epithelium of the Abalone Haliotis tuberculata (Linnaeus, 1758) (Gastropoda, Haliotidae)

PubMed Central

Bravo Portela, I.; Martinez-Zorzano, V. S.; Molist- Perez, I.; Molist García, P.

2012-01-01

The foot epithelium of the gastropod Haliotis tuberculata is studied by light and electron microscopy in order to contribute to the understanding of the anatomy and functional morphology of the mollusks integument. Study of the external surface by scanning electron microscopy reveals that the side foot epithelium is characterized by a microvillus border with a very scant presence of small ciliary tufts, but the sole foot epithelium bears a dense field of long cilia. Ultrastructural examination by transmission electron microscopy of the side epithelial cells shows deeply pigmented cells with high electron-dense granular content which are not observed in the epithelial sole cells. Along the pedal epithelium, seven types of secretory cells are present; furthermore, two types of subepithelial glands are located just in the sole foot. The presence and composition of glycoconjugates in the secretory cells and subepithelial glands are analyzed by conventional and lectin histochemistry. Subepithelial glands contain mainly N-glycoproteins rich in fucose and mannose whereas secretory cells present mostly acidic sulphated glycoconjugates such as glycosaminoglycans and mucins, which are rich in galactose, N-acetyl-galactosamine, and N-acetyl-glucosamine. No sialic acid is present in the foot epithelium. PMID:22645482

Purification and partial characterization of a new mannose/glucose-specific lectin from Dialium guineense Willd seeds that exhibits toxic effect.

PubMed

Bari, Alfa U; Silva, Helton C; Silva, Mayara T L; Pereira Júnior, Francisco N; Cajazeiras, João B; Sampaio, Alexandre H; Leal, Rodrigo B; Teixeira, Edson H; Rocha, Bruno A M; Nascimento, Kyria S; Nagano, Celso S; Cavada, Benildo S

2013-08-01

A new mannose/glucose-specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b-Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d-mannose, d-glucose, and derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28-30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate-binding site. Copyright © 2013 John Wiley & Sons, Ltd.

Poly (Acetyl, Arginyl) Glucosamine as a Biofilm-reducing Water Line Treatment

USDA-ARS?s Scientific Manuscript database

Bacteria can attach and form biofilms on a surface hindering removal by common disinfectants. Some bacteria are better than others at forming this biofilm but once it is formed many pathogens can reside in the matrix. Salmonella spp. have been shown to have some biofilm forming capabilities but will...

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene.

PubMed

Hanin, M; Jabbouri, S; Quesada-Vincens, D; Freiberg, C; Perret, X; Promé, J C; Broughton, W J; Fellay, R

1997-06-01

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N-acetyl-D-glucosamine of LCOs is substituted at C6 with 2-O-methyl-L-fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeE, which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRdelta noeE) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRdelta noeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2-O-methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

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

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

DOE PAGES

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.; ...

2018-01-30

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

cap alpha. -D-Mannopyranosylmethyl-P-nitrophenyltriazene effects on the degradation and biosynthesis of N-linked oligosaccharide chains on. cap alpha. /sub 1/-acid glycoprotein by liver cells

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

Docherty, P.A.; Aronson, N.N. Jr.

1986-05-01

The effects of ..cap alpha..-D-mannopyranosylmethyl-p-nitrophenyltriazene (..cap alpha..-ManMNT) on the degradation and processing of oligosaccharide chains on ..cap alpha../sub 1/-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver blocked the complete degradation of endocytosed N-acetyl (/sup 14/C)glucosamine-labeled asialo-AGP and caused the accumulation of Man/sub 2/GlcNAc/sub 1/ fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally-derived (/sup 14/C)GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully-glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the ..cap alpha..-ManMNT-treated hepatocytes remained sensitive to digestionmore » with endoglycosidase H, were resistant to neuraminidase, and consisted of Man/sub 9-7/GlcNAc/sub 2/ structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since ManMNT is likely to irreversibly inactivate ..cap alpha..-D-mannosidases, the return of AGP to secretory forms with complex chains after 24 h probably resulted from synthesis of new processing enzymes.« less

Effects of Agaricus lilaceps fairy rings on soil aggregation and microbial community structure in relation to growth stimulation of western wheatgrass (Pascopyrum smithii) in Eastern Montana rangeland.

PubMed

Caesar-Tonthat, The Can; Espeland, Erin; Caesar, Anthony J; Sainju, Upendra M; Lartey, Robert T; Gaskin, John F

2013-07-01

Stimulation of plant productivity caused by Agaricus fairy rings has been reported, but little is known about the effects of these fungi on soil aggregation and the microbial community structure, particularly the communities that can bind soil particles. We studied three concentric zones of Agaricus lilaceps fairy rings in Eastern Montana that stimulate western wheatgrass (Pascopyrum smithii): outside the ring (OUT), inside the ring (IN), and stimulated zone adjacent to the fungal fruiting bodies (SZ) to determine (1) soil aggregate proportion and stability, (2) the microbial community composition and the N-acetyl-β-D-glucosaminidase activity associated with bulk soil at 0-15 cm depth, (3) the predominant culturable bacterial communities that can bind to soil adhering to wheatgrass roots, and (4) the stimulation of wheatgrass production. In bulk soil, macroaggregates (4.75-2.00 and 2.00-0.25 mm) and aggregate stability increased in SZ compared to IN and OUT. The high ratio of fungal to bacteria (fatty acid methyl ester) and N-acetyl-β-D-glucosaminidase activity in SZ compared to IN and OUT suggest high fungal biomass. A soil sedimentation assay performed on the predominant isolates from root-adhering soil indicated more soil-binding bacteria in SZ than IN and OUT; Pseudomonas fluorescens and Stenotrophomonas maltophilia isolates predominated in SZ, whereas Bacillus spp. isolates predominated in IN and OUT. This study suggests that growth stimulation of wheatgrass in A. lilaceps fairy rings may be attributed to the activity of the fungus by enhancing soil aggregation of bulk soil at 0-15 cm depth and influencing the amount and functionality of specific predominant microbial communities in the wheatgrass root-adhering soil.

40 CFR 180.1072 - Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Poly-D-glucosamine (chitosan); exemption from the requirement of a tolerance. 180.1072 Section 180.1072 Protection of Environment... RESIDUES IN FOOD Exemptions From Tolerances § 180.1072 Poly-D-glucosamine (chitosan); exemption from the...

Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat.

PubMed

Tabandeh, Mohammad Reza; Oryan, Ahmad; Mohammadalipour, Adel

2014-04-01

Polysaccharides are the main macromolecules of Aloe vera gel but no data about their effect on extracellular matrix (ECM) elements are available. Here, mannose rich Aloe vera polysaccharides (AVP) with molecular weight between 50 and 250 kDa were isolated and characterized. Open cutaneous wounds on the back of 45 rats (control and treated) were daily treated with 25mg (n=15) and 50 mg (n=15) AVP for 30 days. The levels of MMP-3 and TIMP-2 gene expression were analyzed using real time PCR. The levels of n-acetyl glucosamine (NAGA), n-acetyl galactosamine (NAGLA) and collagen contents were also measured using standard biochemical methods. Faster wound closure was observed at day 15 post wounding in AVP treated animals in comparison with untreated group. At day 10 post wounding, AVP inhibited MMP-3 gene expression, while afterwards MMP-3 gene expression was upregulated. AVP enhanced TIMP-2 gene expression, collagen, NAGLA and NAGA synthesis in relation to untreated wounds. Our results suggest that AVP has positive effects on the regulation of ECM factor synthesis, which open up new perspectives for the wound repair activity of Aloe vera polysaccharide at molecular level. Copyright © 2014 Elsevier B.V. All rights reserved.

An evaluation of D-glucosamine as a gratuitous catabolite repressor of Saccharomyces carlsbergensis.

PubMed

Furst, A; Michels, C A

1977-10-24

Glucose represses mitochondrial biogenesis and the fermentation of maltose, galactose and sucrose in yeast. We have analyzed the effect of D-glucosamine on these functions in order to determine if it can produce a similar repression. It was found that glucosamine represses the respiration rate (QO2) but more rapidly than glucose and to a final level slightly higher than in glucose-treated cells. Derepression of the respiration rate following either glucose or glucosamine repression was similar. A two hour lag was followed by a linear increase in QO2 to the derepressed level. Both glucose and glucosamine repressed the level of cytochrome oxidase to the same level. Glucosamine was also found to repress maltose and galactose fermentation but not sucrose fermentation. The derepression of maltase synthesis was inhibited by glucosamine. The constitutive synthesis of maltase was repressed by the addition of glucosamine. Glucosamine was judged to produce a repressed state similar to glucose repression in many respects.

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

EPA Science Inventory

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

Comparative analysis of pharmacological treatments with N-acetyl-DL-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

PubMed

Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

2015-12-15

Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. Copyright © 2015 Elsevier B.V. All rights reserved.

The Effects of Glucosamine Sulfate on Intervertebral Disc Annulus Fibrosus Cells in Vitro

PubMed Central

Sowa, Gwendolyn; Coelho, J. Paulo; Jacobs, Lloydine; Komperda, Kasey; Sherry, Nora; Vo, Nam; Preuss, Harry; Balk, Judith; Kang, Jame

2014-01-01

Background context Glucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection. Purpose The goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading. Study Design Controlled in vitro laboratory setting Methods Intervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1beta and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM and the authors have no conflicts of interest. Results Under conditions of inflammatory stimulation alone, glucosamine demonstrated a dose dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, MMP-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested. Conclusions These results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation, but not under conditions where mechanical loading is present or in which matrix synthesis is needed. PMID:24361347

Sugar-Annulated Oxazoline Ligands: A Novel Pd(II) Complex and Its Application in Allylic Substitution.

PubMed

Kraft, Jochen; Mill, Katharina; Ziegler, Thomas

2016-12-10

Two novel carbohydrate-derived pyridyl (PYOX)- and cyclopropyl (CYBOX)-substituted oxazoline ligands were prepared from d-glucosamine hydrochloride and 1,3,4,6-tetra- O -acetyl-2-amino-2-deoxy-β-d-glucopyranose hydrochloride in two steps, respectively. The sugar-annulated PYOX ligand formed a stable metal complex with Pd(II), which was fully characterized by NMR spectroscopy and X-ray crystallography. NMR and X-ray analysis revealed a change of the conformation in the sugar moiety upon complexation with the palladium(II) species. Both glycosylated ligands resulted in high asymmetric induction (up to 98% ee ) upon application as chiral ligands in the Pd-catalyzed allylic alkylation of rac -1,3-diphenylallyl acetate with dimethyl malonate (Tsuji-Trost reaction). Both ligands provided mainly the ( R )-enantiomer of the alkylation product.

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis.

PubMed

Becker, Sidnei; Soares, Cíntia; Porto, Luismar Marques

2009-07-01

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.

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

PubMed

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

2002-04-16

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

Oligosaccharide composition is similar in drusen and dense deposits in membranoproliferative glomerulonephritis type II.

PubMed

D'souza, Yvonne B; Jones, Carolyn J P; Short, Colin D; Roberts, Ian S D; Bonshek, Richard E

2009-04-01

Drusen are a feature of age-related macular degeneration (AMD). Lesions similar in appearance to drusen are also found in the fundi of patients with membranoproliferative glomerulonephritis type II (dense deposit disease, DDD). The lamina densa of the glomerular basement membrane, in DDD, is transformed into an electron-dense structure by deposition of microscopically homogeneous material. Our study sought to compare the saccharide composition of drusen and dense deposits in the formalin-fixed, paraffin-embedded tissue from the eye and kidney. Six eye specimens were obtained from patients diagnosed with AMD but another eye was obtained from a patient with partial lipodystrophy, who died after renal failure presumably because of DDD. The kidney specimens were from three biopsy-proven cases of DDD. Glycosylation patterns were measured by the binding of 19 biotinylated lectins before and after neuraminidase pre-treatment. High mannose, bi/tri-antennary non-bisected and bisected complex N-glycan, N-acetyl glucosamine, galactose, and sialic acid residues were found in both drusen and dense deposits. Treatment with neuraminidase exposed subterminal galactose in both sites and sparse N-acetyl galactosamine residues in drusen alone. Our study found similar pathologic oligosaccharide structures in the eye and kidney, suggesting that drusen may be a common end result of retinal and glomerular disease.

Search for the antiprostatic principle of stinging nettle (Urtica dioica) roots.

PubMed

Wagner, H; Willer, F; Samtleben, R; Boos, G

1994-12-01

While searching for the antiprostatic active principle of the roots of Urtica dioica we ethanol-precipitated a polysaccharide mixture from an aqueous root extract and obtained chemically defined acidic polysaccharides with molecular masses of 15-210kDa. The chemical structures of these compounds have been determined. Some polysaccharides stimulated T lymphocytes in vitro while others influenced the complement system or triggered the release of TNF-α. The crude polysaccharide extract showed a prolonged antiinflammatory activity in the rat paw edema test for 22 hr, which is comparable to the pharmacological efficacy of indometacin. We have reisolated the isolectin mixture (UDA) originally detected in Urtica roots by Peumans et al. (1984). This mixture displayed immunomodulatory effects on T lymphocytes in a dose-dependent manner. In addition, UDA also directly inhibited cell proliferation and blocked binding of epidermal growth factor to its receptor on a tumor cell line, as determined by a [(125)I]-EGF binding assay. These investigations suggest that Urtica polysaccharides and also the N-acetyl-glucosamine specific lectin UDA play a major role in the antiprostatic activity of the drug and phytopreparations containing it. Copyright © 1994 Gustav Fischer Verlag, Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

PubMed

Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

2016-10-01

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

PubMed Central

Tsuchido, T; Hiraoka, T; Takano, M; Shibasaki, I

1985-01-01

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids. PMID:2858469

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

PubMed

Engelmann, Péter; Hayashi, Yuya; Bodó, Kornélia; Ernszt, Dávid; Somogyi, Ildikó; Steib, Anita; Orbán, József; Pollák, Edit; Nyitrai, Miklós; Németh, Péter; Molnár, László

2016-12-01

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets. Copyright © 2016 Elsevier Ltd. All rights reserved.

EXCI-CEST: Exploiting pharmaceutical excipients as MRI-CEST contrast agents for tumor imaging.

PubMed

Longo, Dario Livio; Moustaghfir, Fatima Zzahra; Zerbo, Alexandre; Consolino, Lorena; Anemone, Annasofia; Bracesco, Martina; Aime, Silvio

2017-06-15

Chemical Exchange Saturation Transfer (CEST) approach is a novel tool within magnetic resonance imaging (MRI) that allows visualization of molecules possessing exchangeable protons with water. Many molecules, employed as excipients for the formulation of finished drug products, are endowed with hydroxyl, amine or amide protons, thus can be exploitable as MRI-CEST contrast agents. Their high safety profiles allow them to be injected at very high doses. Here we investigated the MRI-CEST properties of several excipients (ascorbic acid, sucrose, N-acetyl-d-glucosamine, meglumine and 2-pyrrolidone) and tested them as tumor-detecting agents in two different murine tumor models (breast and melanoma cancers). All the investigated molecules showed remarkable CEST contrast upon i.v. administration in the range 1-3ppm according to the type of mobile proton groups. A marked increase of CEST contrast was observed in tumor regions up to 30min post injection. The combination of marked tumor contrast enhancement and lack of toxicity make these molecules potential candidates for the diagnosis of tumors within the MRI-CEST approach. Copyright © 2017 Elsevier B.V. All rights reserved.

Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

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

Cummings, J.; Fedorov, A; Xu, C

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} =more » 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.« less

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

PubMed Central

Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu; Carr, Heather S.; Zuo, Yan; Frost, Jeffrey A.

2015-01-01

ABSTRACT Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization. PMID:25588829

D-Glucosamine as a novel chiral auxiliary for the stereoselective synthesis of P-stereogenic phosphine oxides.

PubMed

D'Onofrio, A; Copey, L; Jean-Gérard, L; Goux-Henry, C; Pilet, G; Andrioletti, B; Framery, E

2015-09-14

D-Glucosamine was successfully employed as a chiral auxiliary for the enantioselective synthesis of phosphine oxides. The influence of the anomeric position was also investigated and revealed the excellent ability of the α-anomer to perform this transformation in a highly selective fashion. The methodology employed consisted of three steps: diastereoselective formation of the oxazaphospholidine followed by subsequent selective cleavage of P-N and P-O bonds by reaction with two Grignard reagents. P-epimers oxazaphospholidines were prepared switching from a P(v) to a P(III) precursor, thus allowing for the synthesis of enantiomeric phosphine oxides. In addition, the chiral auxiliary could be recovered and efficiently recycled.

Study on feasibility of determination of glucosamine content of fermentation process using a micro NIR spectrometer.

PubMed

Sun, Zhongyu; Li, Can; Li, Lian; Nie, Lei; Dong, Qin; Li, Danyang; Gao, Lingling; Zang, Hengchang

2018-08-05

N-acetyl-d-glucosamine (GlcNAc) is a microbial fermentation product, and NIR spectroscopy is an effective process analytical technology (PAT) tool in detecting the key quality attribute: the GlcNAc content. Meanwhile, the design of NIR spectrometers is under the trend of miniaturization, portability and low-cost nowadays. The aim of this study was to explore a portable micro NIR spectrometer with the fermentation process. First, FT-NIR spectrometer and Micro-NIR 1700 spectrometer were compared with simulated fermentation process solutions. The R c 2 , R p 2 , RMSECV and RMSEP of the optimal FT-NIR and Micro-NIR 1700 models were 0.999, 0.999, 3.226 g/L, 1.388 g/L and 0.999, 0.999, 1.821 g/L, 0.967 g/L. Passing-Bablok regression method and paired t-test results showed there were no significant differences between the two instruments. Then the Micro-NIR 1700 was selected for the practical fermentation process, 135 samples from 10 batches were collected. Spectral pretreatment methods and variables selection methods (BiPLS, FiPLS, MWPLS and CARS-PLS) for PLS modeling were discussed. The R c 2 , R p 2 , RMSECV and RMSEP of the optimal GlcNAc content PLS model of the practical fermentation process were 0.994, 0.995, 2.792 g/L and 1.946 g/L. The results have a positive reference for application of the Micro-NIR spectrometer. To some extent, it could provide theoretical supports in guiding the microbial fermentation or the further assessment of bioprocess. Copyright © 2018. Published by Elsevier B.V.

N-Acetyl-L-Leucine Accelerates Vestibular Compensation after Unilateral Labyrinthectomy by Action in the Cerebellum and Thalamus

PubMed Central

Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

2015-01-01

An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus. PMID:25803613

N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.

PubMed

Günther, Lisa; Beck, Roswitha; Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

2015-01-01

An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus.

a Combined Molecular Dynamics and NMR Spectroscopic Protocol for the Conformational Analysis of Oligosaccharides.

NASA Astrophysics Data System (ADS)

Varma, Vikram

A combined experimental and theoretical protocol for the conformational analysis of oligosaccharides is presented. Three disaccharides, methyl alpha - scD-mannopyranosyl-(1 to 3)-alpha- scD-mannopyranoside, methyl beta- scD-galactopyranosyl-(1 to 4)-beta- scD-glucopyranoside, and propyl beta- scD-2-acetamido -2-deoxy glucopyranosyl-(1 to 3)- alpha- scL-rhamnopyranoside, are used to evaluate a protocol for conformational analysis that makes use of molecular dynamics calculations with the CHARMM force field. Dynamics trajectories computed in vacuo and in water are used to calculate time-averaged NMR parameters such as spin-lattice relaxation times (T_1 ), Nuclear Overhauser Enhancements (NOE), and heteronuclear spin-spin coupling constants (^3J _{rm CH}). The calculated NMR parameters are then compared to experimental values and used to evaluate the computational procedure. The energetically accessible conformations are effectively sampled by the simulations. The method has been extended to the conformational analysis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the Streptococcus Group A, and the Shigella flexneri Y O-antigen. The Streptococcus Group A cell-wall polysaccharide is comprised of a backbone of rhamnopyranosyl units connected by alternating alpha- scL-(1 to 3) and alpha- scL -(1 to 2) linkages, to which are attached N-acetyl-beta- scD-glucosamine ( beta- scD-GlcpNAc) residues at the 3 positions of the rhamnose backbone.rm A&rm B^'qquad A^'& rm Bqquad Acr[{-alpha}{-}L{-}Rha {it p}{-}(1to2){-alpha }{-}L{-}Rha{it p} {-}(1to3){-alpha}{ -}L{-}Rha{it p}-(1to2) -alpha-L-Rha{it p}{-}(1 to3){-alpha}{-}L{- }Rha{it p}{-}cr&uparrow(1 to3)&uparrow(1to3)crbeta {-}D{-}&rm Glc{it p }NAcqquadbeta{-}D{-}& rm Glc{it p}NAccr&rm C ^'&rm C] A branched trisaccharide (A^' -(C)B), a tetrasaccharide (A^' -(C)B-A), a pentasaccharide (C^' -B^'-A ^'-(C)B), and two hexasaccharides (C^'-B^ '-A^' -(C)B-A) and (A-(C^')B ^'-A^' -(C)B), have been chosen for study. The Shigella flexneri Y O-antigen is a linear polysaccharide that is composed of rhamnose units linked alpha- scL-(1 to 3) and alpha- scL-(1 to 2), interspersed by N-acetyl-beta - scD-glucosamine (beta- scD -GlcpNAc) to form a periodic repeating unit ABCD. &rm A&rm B&rm C&rm Dcr [{-alpha}{-}L {-}Rha{it p}-(1to2){ -alpha}{-}L{-}Rha{it p}{-}(1to3){-alpha} {-}L{-}Rha{it p}{ -}(1to3){-}beta{-}D {-}Glc{it p}NAc{-}(1 to2){-}]_{it n}A heptasaccharide corresponding to the fragment (ABCDA^'B ^'C^' ) of the Shigella flexneri Y polysaccharide has been investigated. The conformational properties of all of the oligosaccharides have been studied using molecular dynamics simulations. Interproton distances derived from ROESY spectra are used to determine the starting conformations of the oligosaccharides used in the dynamics calculations, and dynamics simulations are computed with proton pairs constrained to the ROESY -derived distances, as well as with the constraints removed. These dynamics trajectories are used to calculate ROESY buildup curves with CROSREL, a program that treats cross relaxation by means of a full matrix relaxation approach. The calculated buildup curves compare favorably with the experimental buildup curves. The study demonstrates that molecular dynamics, in conjunction with NMR spectroscopy, can be a useful tool in the understanding of the conformational behavior of oligosaccharides in solution. The results provide a model for antigen topology that can be used to infer some of the critical features of antibody-antigen interactions.

Reversible Polymer Hydrogels

DTIC Science & Technology

2008-12-01

glucosamine hydrochloride was dissolved in 100 mL of de- ionized water and placed in an ice bath at >5oC and purged with N2 gas for 20 minutes; 3.25...Temperature sensitive hydrogels based on N-isopropyl acrylamide (NIPA) and acryloyl glucosamine (AG) were synthesized using ammonium persulfate (APS) as...hydrogels by copolymerization of poly (N-isopropylacrylamide) (NIPA), and acryloyl glucosamine (AG) a derivative of chi- tosan, a biopolymer from

An ammonium sulfate sensitive chitinase from Streptomyces sp. CS501.

PubMed

Rahman, Md Arifur; Choi, Yun Hee; Pradeep, G C; Yoo, Jin Cheol

2014-12-01

A chitinase from Streptomyces sp. CS501 was isolated from the Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase (Ch501) was purified to 4.60 fold with yield of 28.74 % using Sepharose Cl-6B column. The molecular mass of Ch501 was approximately 43 kDa as estimated by SDS-PAGE and zymography. The enzyme (Ch501) was found to be stable over a broad pH range (5.0-10.0) and temperature (up to 50 °C), and have an optimum temperature of 60 °C. N-terminal sequence of Ch501 was AAYDDAAAAA. Intriguingly, Ch501 was highly sensitive to ammonium sulfate but it's completely suppressed activity was recovered after desalting out. TLC analysis of Ch501 showed the production of N-acetyl D-glucosamine (GlcNAc) and Diacetylchitobiose (GlcNAc)2, as a principal hydrolyzed product. Ch501 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis, which can be used for the biological control of fungus. As has been simple in purification, stable in a broad range of pH, ability to produce oligosaccharides, and antifungal activity showed that Ch501 has potential applications in industries as for chitooligosaccharides production used as prebiotics and/or for the biological control of plant pathogens in agriculture.

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans

PubMed Central

Izano, Era A.; Sadovskaya, Irina; Wang, Hailin; Vinogradov, Evgeny; Ragunath, Chandran; Ramasubbu, Narayanan; Jabbouri, Saïd; Perry, Malcolm B.; Kaplan, Jeffrey B.

2008-01-01

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is PGA, a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. 1H-NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-D-glucosamine residues in β(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix. PMID:17851029

Isolation, purification, and characterization of glucosamine-6-phosphate-N-acetylase from pig liver.

PubMed

Porowski, T S; Porowska, H; Gałasiński, W

1990-08-01

The procedure of isolation, purification, and characterization of glucosamine-6-phosphate acetylase from the pig liver is described. The steps of purification were as follows: adsorption on hydroxylapatite, fractionation with ammonium sulfate, chromatography on cellulose phosphate, electrofocusing, and preparative gel electrophoresis. A highly purified (about 3000-fold) preparation of GlcN-6-P acetylase, with a yield of 23%, was obtained. It was found that GlcN-6-P acetylase from pig liver is heterogeneous and exists in two active forms. The characteristic features of the preparation were established: Mr, about 24 kDa; temperature optimum at 37 degrees; pH optimum at 7.45; and Km (GlcN-6-P) 3.7 x 10(-3) M and Km (AcCoA) 1.4 x 10(-3) M. The ions K+, Na+, NH4+, Mg2+, Mn2+, and CH3COO- do not stimulate the acetylase activity. The product of acetylase reaction (GlcNAc-6-P) inhibits this reaction according to the feedback process. The highly purified preparation of GlcN-6-P acetylase is unstable during storage and it is protected by ampholine or glycine from enzyme inactivation, but it is not protected by 2-mercaptoethanol.

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase.

PubMed

Siala, Wafi; Kucharíková, Soňa; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Dijck, Patrick; Van Bambeke, Françoise

2016-11-03

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

The composition of peptidochitodextrins from sarcophagid puparial cases

PubMed Central

Lipke, H.; Geoghegan, T.

1971-01-01

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal β-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin–protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization. PMID:5145884

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase

PubMed Central

Siala, Wafi; Kucharíková, Soňa; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Dijck, Patrick; Van Bambeke, Françoise

2016-01-01

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections. PMID:27808087

Glucosamine Inhibits Decidualization of Human Endometrial Stromal Cells and Decreases Litter Sizes in Mice1

PubMed Central

Tsai, Jui-He; Schulte, Maureen; O'Neill, Kathleen; Chi, Maggie M.-Y.; Frolova, Antonina I.; Moley, Kelle H.

2013-01-01

ABSTRACT Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 μg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive. PMID:23718985

Influence of glucosamine on glomerular mesangial cell turnover: implications for hyperglycemia and hexosamine pathway flux

PubMed Central

Le, Catherine; Scholey, James W.

2010-01-01

Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) turnover. Compared with physiological glucose (5.6 mM), treatment with high glucose (25 mM) for 24 h stimulated MC proliferation, an effect that was mimicked by exposure to low concentrations of glucosamine (0.05 mM). The percentage of cells in G0/G1 phase of the cell cycle was reduced with a concomitant increase of the number of cells in G2/M phase. Proliferating cell nuclear antigen, phosphorylated mammalian target of rapamycin [phospho-mTOR (Ser2448)], and total regulatory-associated protein of mTOR were increased by high glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (10 μM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher glucosamine concentrations (0.25–10 mM) caused MC apoptosis after 48 h, and, in addition, GFAT overexpression also increased MC apoptosis (TdT-dUTP nick end-labeling-positive cells: 3.8 ± 0.3 vs. 1.1 ± 0.2% for empty vector; P < 0.05). Hence, hexosamine flux is an important determinant of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin-sensitive mTOR complex 1 (mTORC1). PMID:19903862

Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogs for the glmS Ribozyme

PubMed Central

Posakony, Jeffrey J.; Ferré-D'Amaré, Adrian R.

2013-01-01

Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactor of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogs, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz) and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogs. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. PMID:23578404

Green synthesis approach: extraction of chitosan from fungus mycelia.

PubMed

Dhillon, Gurpreet Singh; Kaur, Surinder; Brar, Satinder Kaur; Verma, Mausam

2013-12-01

Chitosan, copolymer of glucosamine and N-acetyl glucosamine is mainly derived from chitin, which is present in cell walls of crustaceans and some other microorganisms, such as fungi. Chitosan is emerging as an important biopolymer having a broad range of applications in different fields. On a commercial scale, chitosan is mainly obtained from crustacean shells rather than from the fungal sources. The methods used for extraction of chitosan are laden with many disadvantages. Alternative options of producing chitosan from fungal biomass exist, in fact with superior physico-chemical properties. Researchers around the globe are attempting to commercialize chitosan production and extraction from fungal sources. Chitosan extracted from fungal sources has the potential to completely replace crustacean-derived chitosan. In this context, the present review discusses the potential of fungal biomass resulting from various biotechnological industries or grown on negative/low cost agricultural and industrial wastes and their by-products as an inexpensive source of chitosan. Biologically derived fungal chitosan offers promising advantages over the chitosan obtained from crustacean shells with respect to different physico-chemical attributes. The different aspects of fungal chitosan extraction methods and various parameters having an effect on the yield of chitosan are discussed in detail. This review also deals with essential attributes of chitosan for high value-added applications in different fields.

Catalytic properties and heat stabilities of novel recombinant human N-acetyltransferase 2 allozymes support existence of genetic heterogeneity within the slow acetylator phenotype.

PubMed

Hein, David W; Doll, Mark A

2017-08-01

Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p < 0.0001) lower when catalyzed by the novel recombinant human NAT2 allozymes compared to NAT2 4. SMZ and INH N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.001) than catalyzed by NAT2 6C and NAT2 14E. N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2'-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p < 0.001) lower while their apparent K m values did not differ significantly (p > 0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant human NAT2 14C, 14D, 14E, and 18 were significantly (p < 0.05) higher than NAT2 4. These results provide further evidence of genetic heterogeneity within the NAT2 slow acetylator phenotype.

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

PubMed Central

Cywes-Bentley, Colette; Skurnik, David; Zaidi, Tanweer; Roux, Damien; DeOliveira, Rosane B.; Garrett, Wendy S.; Lu, Xi; O’Malley, Jennifer; Kinzel, Kathryn; Zaidi, Tauqeer; Rey, Astrid; Perrin, Christophe; Fichorova, Raina N.; Kayatani, Alexander K. K.; Maira-Litràn, Tomas; Gening, Marina L.; Tsvetkov, Yury E.; Nifantiev, Nikolay E.; Bakaletz, Lauren O.; Pelton, Stephen I.; Golenbock, Douglas T.; Pier, Gerald B.

2013-01-01

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology. PMID:23716675

Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

PubMed Central

Tsujimura, Mitsushi; Ishida, Chuzo; Sagara, Yasuko; Miyazaki, Takashi; Murakami, Koichi; Shiraki, Hiroshi; Okochi, Kazuo; Maeda, Yoshiaki

2001-01-01

Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose, N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG. PMID:11238239

Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

PubMed

Upton, Rosie; Bell, Leonard; Guy, Colin; Caldwell, Paul; Estdale, Sian; Barran, Perdita E; Firth, David

2016-10-18

In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

Evaluation of thermophilic fungal consortium for paddy straw composting.

PubMed

Kumar, Adesh; Gaind, Sunita; Nain, Lata

2008-06-01

Out of 10 thermophilic fungi isolated from wheat straw, farm yard manure, and soil, only three showed highest cellobiase, carboxymethyl cellulase, xylanase, and FPase activities. They were identified as Aspergillus nidulans (Th(4)), Scytalidium thermophilum (Th(5)), and Humicola sp. (Th(10)). A fungal consortium of these three fungi was used to compost a mixture (1:1) of silica rich paddy straw and lignin rich soybean trash. The composting of paddy straw for 3 months, during summer period in North India, resulted in a product with C:N ratio 9.5:1, available phosphorus 0.042% and fungal biomass 6.512 mg of N-acetyl glucosamine/100 mg of compost. However, a C:N ratio of 10.2:1 and highest humus content of 3.3% was achieved with 1:1 mixture of paddy straw and soybean trash. The fungal consortium was effective in converting high silica paddy straw into nutritionally rich compost thereby leading to economical and environment friendly disposal of this crop residue.

Improved fluorescent labeling of chitin oligomers: Chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions.

PubMed

Wakita, Satoshi; Kimura, Masahiro; Kato, Naoki; Kashimura, Akinori; Kobayashi, Shunsuke; Kanayama, Naoto; Ohno, Misa; Honda, Shotaro; Sakaguchi, Masayoshi; Sugahara, Yasusato; Bauer, Peter O; Oyama, Fumitaka

2017-05-15

Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing. AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH 8. Here we analyzed chitin degradation by AMCase in weak acidic to neutral conditions by fluorophore-assisted carbohydrate electrophoresis established originally for oligosaccharides analysis. We found that specific fragments with slower-than-expected mobility as defined by chitin oligosaccharide markers were generated at pH 5.0∼8.0 as by-products of the reaction. We established an improved method for chitin oligosaccharides suppressing this side reaction by pre-acidification of the fluorophore-labeling reaction mixture. Our improved method specifically detects chitin oligosaccharides and warrants quantification of up to 50nmol of the material. Using this strategy, we found that AMCase produced dimer of N-acetyl-d-glucosamine (GlcNAc) at strong acidic to neutral condition. Moreover, we found that AMCase generates (GlcNAc) 2 as well as (GlcNAc) 3 under physiological conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins*

PubMed Central

Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan; Yang, Yi-Hu; Shao, Qiu-Yan; Yang, Hong-Bo; Ren, Yan-Min; Wu, Hui; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides. PMID:24936067

Complex Mixture Analysis of Organic Compounds in Yogurt by NMR Spectroscopy

PubMed Central

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

2016-01-01

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

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells.

PubMed

Fan, Xian; Roy, Eileen M; Murphy, Tamara C; Nanes, Mark S; Kim, Sungtae; Pike, J Wesley; Rubin, Janet

2004-11-01

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.

ANALYSIS OF RICIN TOXIN PREPARATIONS FOR CARBOHYDRATE AND FATTY ACID ABUNDANCE AND ISOTOPE RATIO INFORMATION

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

Wunschel, David S.; Kreuzer-Martin, Helen W.; Antolick, Kathryn C.

2009-12-01

This report describes method development and preliminary evaluation for analyzing castor samples for signatures of purifying ricin. Ricin purification from the source castor seeds is essentially a problem of protein purification using common biochemical methods. Indications of protein purification will likely manifest themselves as removal of the non-protein fractions of the seed. Two major, non-protein, types of biochemical constituents in the seed are the castor oil and various carbohydrates. The oil comprises roughly half the seed weight while the carbohydrate component comprises roughly half of the remaining “mash” left after oil and hull removal. Different castor oil and carbohydrate componentsmore » can serve as indicators of specific toxin processing steps. Ricinoleic acid is a relatively unique fatty acid in nature and is the most abundant component of castor oil. The loss of ricinoleic acid indicates a step to remove oil from the seeds. The relative amounts of carbohydrates and carbohydrate-like compounds, including arabinose, xylose, myo-inositol fucose, rhamnose, glucosamine and mannose detected in the sample can also indicate specific processing steps. For instance, the differential loss of arabinose relative to mannose and N-acetyl glucosamine indicates enrichment for the protein fraction of the seed using protein precipitation. The methods developed in this project center on fatty acid and carbohydrate extraction from castor samples followed by derivatization to permit analysis by gas chromatography-mass spectrometry (GC-MS). Method descriptions herein include: the source and preparation of castor materials used for method evaluation, the equipment and description of procedure required for chemical derivatization, and the instrument parameters used in the analysis. Two types of derivatization methods describe analysis of carbohydrates and one procedure for analysis of fatty acids. Two types of GC-MS analysis is included in the method development, one employing a quadrupole MS system for compound identification and an isotope ratio MS for measuring the stable isotope ratios of deuterium and hydrogen (D/H) in fatty acids. Finally, the method for analyzing the compound abundance data is included. This study indicates that removal of ricinoleic acid is a conserved consequence of each processing step we tested. Furthermore, the stable isotope D/H ratio of ricinoleic acid distinguished between two of the three castor seed sources. Concentrations of arabinose, xylose, mannose, glucosamine and myo-inositol differentiated between crude or acetone extracted samples and samples produced by protein precipitation. Taken together these data illustrate the ability to distinguish between processes used to purify a ricin sample as well as potentially the source seeds.« less

Evaluation of the anti-inflammatory actions of various functional food materials including glucosamine on synovial cells.

PubMed

Yamagishi, Yoshie; Someya, Akimasa; Imai, Kensuke; Nagao, Junji; Nagaoka, Isao

2017-08-01

The anti-inflammatory actions of glucosamine (GlcN) on arthritic disorders involve the suppression of inflammatory mediator production from synovial cells. GlcN has also been reported to inhibit the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The present study aimed to determine the cooperative and anti‑inflammatory actions of functional food materials and evaluated the production of interleukin (IL)‑8 and phosphorylation of p38 MAPK in IL-1β-activated synovial cells, incubated with the combination of GlcN and various functional food materials containing L‑methionine (Met), undenatured type II collagen (UC‑II), chondroitin sulfate (CS), methylsulfonylmethane (MSM) and agaro-oligosaccharide (AO). The results indicated that Met, UC‑II, CS, MSM and AO slightly or moderately suppressed the IL-1β-stimulated IL‑8 production by human synovial MH7A cells. The same compounds further decreased the IL‑8 level lowered by GlcN. Similarly, they slightly suppressed the phosphorylation level of p38 MAPK and further reduced the phosphorylation level lowered by GlcN. These observations suggest a possibility that these functional food materials exert an anti‑inflammatory action (inhibition of IL‑8 production) in combination with GlcN by cooperatively suppressing the p38 MAPK signaling (phosphorylation).

N-acetyl-L-tryptophan, a substance-P receptor antagonist attenuates aluminum-induced spatial memory deficit in rats.

PubMed

Fernandes, Joylee; Mudgal, Jayesh; Rao, Chamallamudi Mallikarjuna; Arora, Devinder; Basu Mallik, Sanchari; Pai, K S R; Nampoothiri, Madhavan

2018-06-01

Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease. Neurokinin substance P is a key mediator which modulates neuroinflammation through neurokinin receptor. Involvement of substance P in Alzheimer's disease is still plausible and various controversies exist in this hypothesis. Preventing the deleterious effects of substance P using N-acetyl-L-tryptophan, a substance P antagonist could be a promising therapeutic strategy. This study was aimed to evaluate the effect of N-acetyl-L-tryptophan on aluminum induced spatial memory alterations in rats. Memory impairment was induced using aluminum chloride (AlCl 3 ) at a dose of 10 mg/kg for 42 d. After induction of dementia, rats were exposed to 30 and 50 mg/kg of N-acetyl-L-tryptophan for 28 d. Spatial memory alterations were measured using Morris water maze. Acetylcholinesterase activity and antioxidant enzyme glutathione level were assessed in hippocampus, frontal cortex and striatum. The higher dose of N-acetyl-L-tryptophan (50 mg/kg) significantly improved the aluminum induced memory alterations. N-acetyl-L-tryptophan exposure resulted in significant increase in acetylcholinesterase activity and glutathione level in hippocampus. The neuroprotective effect of N-acetyl-L-tryptophan could be due to its ability to block substance P mediated neuroinflammation, reduction in oxidative stress and anti-apoptotic properties. To conclude, N-acetyl-L-tryptophan may be considered as a novel neuroprotective therapy in Alzheimer's disease.

15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

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

Engdahl, Ryan; Monroy, M. Alexandra; Temple University School of Medicine, Department of Anatomy and Cell Biology, 3400 North Broad Street, Philadelphia, PA 19140

2007-07-20

Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1more » cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.« less

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

PubMed

Toegel, S; Wu, S Q; Piana, C; Unger, F M; Wirth, M; Goldring, M B; Gabor, F; Viernstein, H

2008-10-01

To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Detection of occupational lead nephropathy using early renal markers.

PubMed

Kumar, B D; Krishnaswamy, K

1995-01-01

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

The effect of nonenzymatic glycation on the stability and conformation of two deoxyoligonucleotide duplexes: a spectroscopic analysis by circular dichroism.

PubMed

Dutta, Udayan; Cohenford, Menashi A; Dain, Joel A

2007-01-15

Advanced glycation end products (AGEs) play a significant role in the pathophysiology of diabetes leading to such conditions as atherosclerosis, cataract formation, and renal dysfunction. While the formation of nucleoside AGEs was previously demonstrated, no extensive studies have been performed to assess the effect of AGEs on DNA structure and folding. The objective of this study was to investigate the nonenzymatic glycation of two DNA oligonucleotide duplexes with one duplex consisting of deoxy-poly(A)15 and deoxy-poly(T)15 and the other consisting of deoxy-poly(GA)15 and deoxy-poly(CT)15. With D-glucose, D-galactose, D/L-glyceraldehyde, and D-glucosamine serving as the model glycating carbohydrates, D-glucosamine was found to exhibit the greatest effect on the stability and structure of the oligonucleotide duplexes, a finding that was confirmed by circular dichroism. The nonenzymatic glycation of deoxy-poly(AT) by D-glucosamine destabilized the deoxy-poly(AT) structure and changed its conformation from A form to X form. D-glucosamine also altered the conformation of deoxy-poly(GA)15 and deoxy-poly(CT)15 from A form to B form. Capillary electrophoresis and ultraviolet and fluorescence spectroscopy revealed that, of the various purines and pyrimidines, 2'-deoxyguanosine and guanine were most reactive with D-glucosamine. The nonenzymatic modification of nucleic acids warrants further investigation because this phenomenon may occur in vivo, altering DNA structure and/or function.

A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase

PubMed Central

Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R.; Yang, Shaoqing; Jiang, Zhengqiang

2015-01-01

Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins. PMID:26669854

A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase.

PubMed

Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R; Yang, Shaoqing; Jiang, Zhengqiang

2015-12-16

Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions--a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins.

[Safety Assessment regarding use of glucosamine sulfate by patients whose dietary potassium intake is restricted].

PubMed

Asahina, Yasuko; Hori, Satoko; Sawada, Yasufumi

2010-02-01

Hyperkalemia is common in patients with renal disease, and is sometimes caused by dietary potassium intake. We aimed to determine and compare the content of potassium in nine brands of glucosamine supplements sold in the Japanese market and via the Internet. The potassium content was 0.165-3 mg per daily dose in Japanese products, which contained glucosamine hydrochloride or N-acetylglucosamine, while the content in foreign products, in which glucosamine was sulfated, was 197-280 mg. Our results show that the potassium content in glucosamine sulfate supplements can correspond to 20% of the maximum daily intake of potassium by patients on hemodialysis, because the products sometimes contain glucosamine as glucosamine sulfate potassium chloride for stabilization. Although it is not permitted to sell glucosamine sulfate as food in Japan, consumers can easily buy foreign products that contain glucosamine sulfate via the Internet, and those products rarely indicate the potassium content. Health professionals should pay attention to patients' use of glucosamine supplements, especially when patients' dietary potassium intake needs to be restricted.

Anti-inflammatory and anti-angiogenic activities in vitro of eight diterpenes from Daphne genkwa based on hierarchical cluster and principal component analysis.

PubMed

Wang, Ling; Lan, Xin-Yi; Ji, Jun; Zhang, Chun-Feng; Li, Fei; Wang, Chong-Zhi; Yuan, Chun-Su

2018-06-01

Rheumatoid arthritis (RA) is one of the most prevalent chronic inflammatory and angiogenic diseases. The aim of this study was to evaluate the anti-inflammatory and anti-angiogenic activities in vitro of eight diterpenoids isolated from Daphne genkwa. LC-MS was used to identify diterpenes isolated from D. genkwa. The anti-inflammatory and anti-angiogenic activities of eight diterpenoids were evaluated on LPS-induced macrophage RAW264.7 cells and TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) using hierarchical cluster analysis (HCA) and principal component analysis (PCA). The eight diterpenes isolated from D. genkwa were identified as yuanhuaphnin, isoyuanhuacine, 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, yuanhuagine, isoyuanhuadine, yuanhuadine, yuanhuaoate C and yuanhuacine. All the eight diterpenes significantly down-regulated the excessive secretion of TNF-α, IL-6, IL-1β and NO in LPS-induced RAW264.7 macrophages. However, only 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl markedly reduced production of VEGF, MMP-3, ICAM and VCAM in TNF-α-stimulated HUVECs. HCA obtained 4 clusters, containing 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, isoyuanhuacine, isoyuanhuadine and five other compounds. PCA showed that the ranking of diterpenes sorted by efficacy from highest to lowest was 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl, yuanhuaphnin, isoyuanhuacine, yuanhuacine, yuanhuaoate C, yuanhuagine, isoyuanhuadine, yuanhuadine. In conclusion, eight diterpenes isolated from D. genkwa showed different levels of activity in LPS-induced RAW264.7 cells and TNF-α-stimulated HUVECs. The comprehensive evaluation of activity by HCA and PCA indicated that of the eight diterpenes, 12-O-(2'E,4'E-decadienoyl)-4-hydroxyphorbol-13-acetyl was the best, and can be developed as a new drug for RA therapy.

A little sugar goes a long way: The cell biology of O-GlcNAc

PubMed Central

2015-01-01

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

Enrichment of gilthead seabream (Sparus aurata L.) diet with palm fruit extracts and probiotics: Effects on skin mucosal immunity.

PubMed

Cerezuela, Rebeca; Guardiola, Francisco A; Cuesta, Alberto; Esteban, M Ángeles

2016-02-01

Fish skin mucus contains numerous immune substances still poorly studied. To date, there are no studies regarding the possible influence of dietary supplements on such important substances. In the present work, a commercial diet used as control diet was enriched with: 1) probiotic Shewanella putrefaciens (Pdp11 diet, 10(9) cfu g(-1)); 2) probiotic Bacillus sp. (Bacillus diet, 10(9) ufc g(-1)); 3) aqueous date palm fruits extracts (DPE diet, 4%), and 4) a combination of Pdp11 + Bacillus sp + aqueous DPE (Mix diet). After 2 and 4 weeks of the feeding trial, enzymatic activities (proteases, antiproteases and peroxidases), IgM levels and terminal carbohydrates abundance were determined in skin mucus. In addition, the expression of certain immune related genes was evaluated in the skin. Our results demonstrated the significant alteration of the terminal carbohydrate abundance in skin mucus. Carbohydrates more affected by experimental diets were N-acetyl-galactosamine, N-acetyl-glucosamine, galactose, mannose, glucose and fucose. IgM, peroxidase activity and protease were also significantly higher in fish fed enriched diets. For last, an important up-regulation on the immune related gene studied on the skin was also detected. Present findings provide robust evidence that fish skin mucosal immunity can be improved by the diet. Copyright © 2015 Elsevier Ltd. All rights reserved.

Photoaffinity labeling of opiate receptors with /sup 3/H-etorphine: possible species differences in glycosylation

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

Bowen, W.D.; Kooper, G.

1986-01-01

Opiate receptors from whole rat brain (minus cerebellum) and cow striatum were labeled irreversibly using the intrinsic photolability of /sup 3/H-etorphine. After incubation with 2 nM /sup 3/H-etorphine and centrifugal washing, membranes were irradiated with light of 254 nm. Non-specific binding was determined by carrying out incubations in presence and absence of 10 microM levallorphan. Specific binding in photolabeled membranes was 75-80%, with a photo-incorporation yield of approximately 50%. Photolabeled membranes were extracted with CHAPS/Lubrol and unbound /sup 3/H-etorphine was removed by dialysis and passage over Sephadex G-25. Solubilized proteins were then subjected to chromatography on wheat germ agglutinin, andmore » retained proteins were eluted with N-acetyl D-glucosamine (NAG). Protein profiles from rat brain and cow striatum were identical, with 89% of the total protein flowing through unretained and 11% eluted by NAG. However, the profile of radioactivity was markedly different in the two species. With rat, the specific activity (cpm/A280) was the same for flow-through and NAG-eluate. With cow, the specific activity of the NAG-eluate was 17 times greater than the flow-through. These results indicate that cow striatum and rat whole brain contain populations of opiate receptors which are glycosylated differently.« less

Poly-N-acetylglucosamine matrix polysaccharide impedes fluid convection and transport of the cationic surfactant cetylpyridinium chloride through bacterial biofilms.

PubMed

Ganeshnarayan, Krishnaraj; Shah, Suhagi M; Libera, Matthew R; Santostefano, Anthony; Kaplan, Jeffrey B

2009-03-01

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-beta(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.

Subgroup analyses of the effectiveness of oral glucosamine for knee and hip osteoarthritis: a systematic review and individual patient data meta-analysis from the OA trial bank.

PubMed

Runhaar, Jos; Rozendaal, Rianne M; van Middelkoop, Marienke; Bijlsma, Hans J W; Doherty, Michael; Dziedzic, Krysia S; Lohmander, L Stefan; McAlindon, Timothy; Zhang, Weiya; Bierma Zeinstra, Sita

2017-11-01

To evaluate the effectiveness of oral glucosamine in subgroups of people with hip or knee osteoarthritis (OA) based on baseline pain severity, body mass index (BMI), sex, structural abnormalities and presence of inflammation using individual patient data. After a systematic search of the literature and clinical trial registries, all randomised controlled trials (RCTs) evaluating the effect of any oral glucosamine substance in patients with clinically or radiographically defined hip or knee OA were contacted. As a minimum, pain, age, sex and BMI at baseline and pain as an outcome measure needed to be assessed. Of 21 eligible studies, six (n=1663) shared their trial data with the OA Trial Bank. Five trials (all independent of industry, n=1625) compared glucosamine with placebo, representing 55% of the total number of participants in all published placebo-controlled RCTs. Glucosamine was no better than placebo for pain or function at short (3 months) and long-term (24 months) follow-up. Glucosamine was also no better than placebo among the predefined subgroups. Stratification for knee OA and type of glucosamine did not alter these results. Although proposed and debated for several years, open trial data are not widely made available for studies of glucosamine for OA, especially those sponsored by industry. Currently, there is no good evidence to support the use of glucosamine for hip or knee OA and an absence of evidence to support specific consideration of glucosamine for any clinically relevant OA subgroup according to baseline pain severity, BMI, sex, structural abnormalities or presence of inflammation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine

PubMed Central

Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

2013-01-01

B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source. PMID:23667565

Phytosterols from Dunaliella tertiolecta Reduce Cell Proliferation in Sheep Fed Flaxseed during Post Partum

PubMed Central

Ciliberti, Maria Giovanna; Francavilla, Matteo; Intini, Simona; Albenzio, Marzia; Marino, Rosaria; Santillo, Antonella; Caroprese, Mariangela

2017-01-01

The post partum period is characterized by immunosuppression and increased disease susceptibility. Both phytosterols from microalga Dunaniella tertiolecta and dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) influence cell proliferation and cytokine release during inflammation. The objective of this paper was the evaluation of the effects of physterols, extracted and purified from D. tertiolecta, on the in vitro immune responses of ewes supplemented with flaxseed during post partum. Twenty Comisana parturient ewes were divided in two balanced groups, and supplemented with flaxseed (FS, 250 g/day) or fed with a conventional diet (CON). Blood samples (15 mL) were collected for five weeks, starting from lambing, in order to isolate peripheral blood mononuclear cells (PBMC). Stimulated PBMC were treated with a total sterols fraction from D. tertiolecta (TS), a mix of ergosterol and 7-dehydroporiferasterol (purified extract, PE), and a mix of acetylated ergosterol and 7-dehydroporiferasterol (acetylated purified extract, AcPE), extracted and purified from D. tertiolecta at two concentrations (0.4 and 0.8 mg/mL). Results of the experiment demonstrated that n-3 PUFA from flaxseed induced an anti-inflammatory cytokine profile, with an increase of both IL-10, IL-6 and a decrease of IL-1β. TS, PE, and AcPE purified from D. tertiolecta showed an anti-proliferative effect on sheep PBMC regardless their chemical composition and concentration. PMID:28684702

Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae

PubMed Central

Izano, Era A.; Sadovskaya, Irina; Vinogradov, Evgeny; Mulks, Martha H.; Velliyagounder, Kabilan; Ragunath, Chandran; Kher, William B.; Ramasubbu, Narayanan; Jabbouri, Saïd; Perry, Malcolm B.; Kaplan, Jeffrey B.

2007-01-01

Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl-D-glucosamine (GlcNAc) residues in β(1,6) linkage (poly-β-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and S. epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1-12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs. PMID:17412552

Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (Dionaea muscipula Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

PubMed Central

Libiaková, Michaela; Floková, Kristýna; Novák, Ondřej; Slováková, L'udmila; Pavlovič, Andrej

2014-01-01

The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH4Cl, KH2PO4, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed. PMID:25153528

Randomized trial of glucosamine and chondroitin supplementation on inflammation and oxidative stress biomarkers and plasma proteomics profiles in healthy humans.

PubMed

Navarro, Sandi L; White, Emily; Kantor, Elizabeth D; Zhang, Yuzheng; Rho, Junghyun; Song, Xiaoling; Milne, Ginger L; Lampe, Paul D; Lampe, Johanna W

2015-01-01

Glucosamine and chondroitin are popular non-vitamin dietary supplements used for osteoarthritis. Long-term use is associated with lower incidence of colorectal and lung cancers and with lower mortality; however, the mechanism underlying these observations is unknown. In vitro and animal studies show that glucosamine and chondroitin inhibit NF-kB, a central mediator of inflammation, but no definitive trials have been done in healthy humans. We conducted a randomized, double-blind, placebo-controlled, cross-over study to assess the effects of glucosamine hydrochloride (1500 mg/d) plus chondroitin sulfate (1200 mg/d) for 28 days compared to placebo in 18 (9 men, 9 women) healthy, overweight (body mass index 25.0-32.5 kg/m2) adults, aged 20-55 y. We examined 4 serum inflammatory biomarkers: C-reactive protein (CRP), interleukin 6, and soluble tumor necrosis factor receptors I and II; a urinary inflammation biomarker: prostaglandin E2-metabolite; and a urinary oxidative stress biomarker: F2-isoprostane. Plasma proteomics on an antibody array was performed to explore other pathways modulated by glucosamine and chondroitin. Serum CRP concentrations were 23% lower after glucosamine and chondroitin compared to placebo (P = 0.048). There were no significant differences in other biomarkers. In the proteomics analyses, several pathways were significantly different between the interventions after Bonferroni correction, the most significant being a reduction in the "cytokine activity" pathway (P = 2.6 x 10-16), after glucosamine and chondroitin compared to placebo. Glucosamine and chondroitin supplementation may lower systemic inflammation and alter other pathways in healthy, overweight individuals. This study adds evidence for potential mechanisms supporting epidemiologic findings that glucosamine and chondroitin are associated with reduced risk of lung and colorectal cancer. ClinicalTrials.gov NCT01682694.

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes - Implications for osteoarthritis

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

Imagawa, Kei, E-mail: k.Imagawa@soton.ac.uk; Tohoku University School of Medicine, Sendai; Andres, MC de

Research highlights: {yields} Glucosamine and a NF-kB inhibitor reduce inflammation in OA. {yields} Cytokine induced demethylation of CpG site in IL1{beta} promoter prevented by glucosamine. {yields} Glucosamine and NF-kB inhibitor have epigenetic effects on human chondrocytes. -- Abstract: Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OAmore » is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1{beta}, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1{beta} and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma-Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1{beta}, 2.5 ng/ml OSM and 2 mM GlcN, (v) cultured with 1.0 {mu}M BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1{beta}, 2.5 ng/ml OSM and 1.0 {mu}M BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1{beta} and MMP13 proximal promoter were quantified by pyrosequencing. Result:IL1{beta} expression was enhanced over 580-fold in articular chondrocytes treated with IL-1{beta} and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1{beta} expression by 3-fold. In the presence of BAY, IL-1{beta} induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1{beta} promoter was 65% in control cultures and decreased to 36% in the presence of IL-1{beta}/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1{beta}/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. Conclusion: We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1{beta} promoter and this was associated with decreased expression of IL1{beta}. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.« less

The role of diet and exercise and of glucosamine sulfate in the prevention of knee osteoarthritis: Further results from the PRevention of knee Osteoarthritis in Overweight Females (PROOF) study.

PubMed

Runhaar, Jos; Deroisy, Rita; van Middelkoop, Marienke; Barretta, Francesco; Barbetta, Beatrice; Oei, Edwin H; Vroegindeweij, Dammis; Giacovelli, Giampaolo; Bruyère, Olivier; Rovati, Lucio C; Reginster, Jean-Yves; Bierma-Zeinstra, Sita M A

2016-02-01

The PRevention of knee Osteoarthritis in Overweight Females (PROOF) study (ISRCTN 42823086) described a trend for a decrease in the incidence of knee osteoarthritis (OA) by a tailored diet and exercise program (DEP) or by oral glucosamine sulfate in women at risk for the disease, using a composite clinical and/or radiological outcome. The aim of this updated post-hoc analysis was to re-assess the results according to more precise techniques and take advantage of the 2×2 factorial design. A total of 407 overweight (BMI ≥ 27kg/m(2)) women of 50-60 years of age with no diagnosis of knee OA were randomized to: (1) no DEP + placebo (Control, N = 102), (2) DEP + placebo (DEP, N = 101), (3) glucosamine sulfate + no DEP (GS, N = 102), and (4) DEP + glucosamine sulfate (DEP + GS, N =102) and followed for 2.5 years, with standardized postero-anterior, semiflexed (MTP) view knee radiographs at baseline and end of the study. DEP consisted of a tailored low fat and/or low caloric diet and easy to implement physical activities. Glucosamine was given as oral crystalline glucosamine sulfate 1500mg once daily, double-blinded vs. placebo. Incident knee OA was defined as radiographic progression of ≥1mm minimum joint space narrowing (mJSN) in the medial tibiofemoral compartment, as previously assessed by the visual (manual) technique and by a new semi-automated method. Logistic regression analysis was used to calculate the odds ratio for the effect of the interventions. After 2.5 years, 11.8% of control subjects developed knee OA. This incidence was decreased with glucosamine sulfate, either alone or in combination with the DEP, but not by the DEP alone. Since there was no statistical interaction between treatments, the 2×2 factorial design allowed analysis of patients receiving glucosamine sulfate (N = 204) vs. those not receiving it (N = 203), similarly for those on the DEP (N = 203) or not (N = 204). Glucosamine sulfate significantly decreased the risk of developing knee OA: odds ratio (OR) = 0.41 (95% CI: 0.20-0.85, P = 0.02) by the manual JSN assessment method and OR = 0.42 (95% CI: 0.20-0.92, P = 0.03) by the semi-automated technique. Conversely, there was no decrease in risk with the DEP. Glucosamine sulfate decreased the risk of developing radiographic knee OA over 2.5 years in overweight, middle-aged women at risk, as determined by medial mJSN progression. Conversely a tailored diet and exercise program exerted no preventive effect, possibly because of the lower than expected effect on weight loss. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

PubMed

Esposito, Diego; Günster, Regina A; Martino, Luigi; El Omari, Kamel; Wagner, Armin; Thurston, Teresa L M; Rittinger, Katrin

2018-04-06

The Salmonella -secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N -acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating D X D motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N -acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N -glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N -glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors. © 2018 Esposito et al.

Ultraviolet light and laser irradiation enhances the antibacterial activity of glucosamine-functionalized gold nanoparticles

PubMed Central

Govindaraju, Saravanan; Ramasamy, Mohankandhasamy; Baskaran, Rengarajan; Ahn, Sang Jung; Yun, Kyusik

2015-01-01

Here we report a novel method for the synthesis of glucosamine-functionalized gold nanoparticles (GlcN-AuNPs) using biocompatible and biodegradable glucosamine for antibacterial activity. GlcN-AuNPs were prepared using different concentrations of glucosamine. The synthesized AuNPs were characterized for surface plasmon resonance, surface morphology, fluorescence spectroscopy, and antibacterial activity. The minimum inhibitory concentrations (MICs) of the AuNPs, GlcN-AuNPs, and GlcN-AuNPs when irradiated by ultraviolet light and laser were investigated and compared with the MIC of standard kanamycin using Escherichia coli by the microdilution method. Laser-irradiated GlcN-AuNPs exhibited significant bactericidal activity against E. coli. Flow cytometry and fluorescence microscopic analysis supported the cell death mechanism in the presence of GlcN-AuNP-treated bacteria. Further, morphological changes in E. coli after laser treatment were investigated using atomic force microscopy and transmission electron microscopy. The overall results of this study suggest that the prepared nanoparticles have potential as a potent antibacterial agent for the treatment of a wide range of disease-causing bacteria. PMID:26345521

Acetylation of NDPK-D Regulates Its Subcellular Localization and Cell Survival

PubMed Central

Fujita, Yuki; Fujiwara, Kei; Zenitani, Shigetake; Yamashita, Toshihide

2015-01-01

Nucleoside diphosphate kinases (NDPK) are ubiquitous enzymes that catalyze the reversible phosphotransfer of γ-phosphates between di- and triphosphonucleosides. NDPK-D (Nm23-H4) is the only member of the NDPK family with a mitochondrial targeting sequence. Despite the high expression of NDPK-D in the developing central nervous system, its function remains to be determined. In this study, we show that NDPK-D knockdown induces apoptosis in neuroblastoma cells as well as in mouse cortex, suggesting that NDPK-D is required for neuronal survival. We identified NDPK-D as a binding partner of NAD+-dependent histone deacetylase, SIRT1, by yeast two-hybrid screening. NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation. Inhibition of SIRT1 increases the acetylation of NDPK-D. Overexpression of NDPK-D along with SIRT1, or mutation in the acetylated lysine residues in NDPK-D, increases its nuclear accumulation. Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells. Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis. PMID:26426123

Single Laboratory Validation of a Method for Determination of Glucosamine in Raw Materials and Dietary Supplements Containing Glucosamine Sulfate and/or Glucosamine Hydrochloride by High-Performance Liquid Chromatography with FMOC-Su Derivatization

PubMed Central

Zhou, Joseph ZiQi; Waszkuc, Ted; Mohammed, Felicia

2008-01-01

Single laboratory validation of a method for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by with high-performance liquid Chromatography FMOC-Su derivatization. Tests with 2 blank matrixes containing SAMe, vitamin C, citric acid, chondroitin sulfates, methylsulfonylmethane, lemon juice concentrate, and other potential interferents showed the method to be selective and specific. Eight calibration curves prepared over 7 working days indicated excellent reproducibility with the linear range at least over 2.0–150 μg/mL, and determination coefficients >0.9999. Average spike recovery from the blank matrix (n = 8 over 2 days) was 93.5, 99.4, and 100.4% at respective spike levels of 15,100, and 150%, and from the sample matrix containing glucosamine (n = 3) was 99.9 and 102.8% at respective levels of 10 and 40%, with relative standard deviations <0.9%. The method was also applied to 12 various glucosamine finished products and raw materials. The stability tests confirmed that glucosamine–FMOC-Su derivative once formed is stable at room temperature for at least 5 days. Limit of quantitation was 1 μg/mL and limit of detection was 0.3 μg/mL. The method is ready to proceed for the collaborative study. PMID:15493664

Novel Inhibitors of Protein-Protein Interaction for Prostate Cancer Therapy

DTIC Science & Technology

2011-04-01

medical need. 7 REFERENCES 1. Babbar N, Hacker A, Huang Y, Casero RA Jr. Tumor necrosis factor alpha induces spermidine /spermine N-acetyl...PCa development and progression. We have published that activated androgen receptor (AR)-JunD complex induces spermidine /spermine N1-acetyl transferase

A modeling study for structure features of β-N-acetyl-D-hexosaminidase from Ostrinia furnacalis and its novel inhibitor allosamidin: species selectivity and multi-target characteristics.

PubMed

Wang, Yanli; Liu, Tian; Yang, Qing; Li, Zhong; Qian, Xuhong

2012-04-01

Insect β-N-acetyl-D-hexosaminidase, a chitin degrading enzyme, is physiologically important during the unique life cycle of the insect. OfHex1, a β-N-acetyl-D-hexosaminidase from the insect, Ostrinia furna, which was obtained by our laboratory (Gen Bank No.: ABI81756.1), was studied by molecular modeling as well as by molecular docking with its inhibitor, allosamidin. 3D model of OfHex1 was built through the ligand-supported homology modeling approach. The binding modes of its substrate and inhibitor were proposed through docking and cluster analysis. The pocket's size and shape of OfHex1 differ from that of human β-N-acetyl-D-hexosaminidase, which determined that allosamidin can selectively inhibit OfHex1 instead of human β-N-acetyl-D-hexosaminidase. Moreover, the multi-target characteristics of allosamidin that inhibit enzymes from different families, OfHex1 (EC 3.2.1.52; GH20) and chitinase (EC 3.2.1.14; GH18), were compared. The common -1/+1 sugar-binding site of chitinase and OfHex1, and the -2/-3 sugar-binding site in chitinase contribute to the binding of allosamidin. This work, at molecular level, proved that OfHex1 could be a potential species-specific target for novel green pesticide design and also provide the possibility to develop allosamidin or its derivatives as a new type of insecticide to 'hit two birds with one stone', which maybe become a novel strategy in pest control. © 2011 John Wiley & Sons A/S.

Glucosamine for the Treatment of Osteoarthritis: The Time Has Come for Higher-Dose Trials.

PubMed

McCarty, Mark F; O'Keefe, James H; DiNicolantonio, James J

2018-04-18

Although clinical trials with glucosamine in osteoarthritis have yielded mixed results, leading to doubts about its efficacy, the utility of glucosamine for preventing joint destruction and inflammation is well documented in rodent models of arthritis, including models of spontaneous osteoarthritis. The benefit of oral glucosamine in adjuvant arthritis is markedly dose dependent, likely reflecting a modulation of tissue levels of UDP-N-acetylglucosamine that in turn influences mucopolysaccharide synthesis and the extent of protein O-GlcNAcylation. Importantly, the minimal oral dose of glucosamine that exerts a detectible benefit in adjuvant arthritis achieves plasma glucosamine levels similar to those achieved when the standard clinical dose of glucosamine, 1.5 g daily, is administered as a bolus. The response of plasma glucosamine levels to an increase in glucosamine intake is nearly linear. Remarkably, every published clinical trial with glucosamine has employed the same 1.5 g dose that Rottapharm recommended for its proprietary glucosamine sulfate product decades ago, yet there has never been any published evidence that this dose is optimal with respect to efficacy and side effects. If this dose is on the edge of demonstrable clinical efficacy when experimental design is ideal, then variations in the patient populations targeted, the assessment vehicles employed, and the potency of glucosamine preparations tested could be expected to yield some null results. Failure to employ bolus dosing may also be a factor in the null results observed in the GAIT study and other trials. Clinical studies evaluating the dose dependency of glucosamine's influence on osteoarthritis are long overdue.

Reaction mechanism of chitosanase from Streptomyces sp. N174.

PubMed Central

Fukamizo, T; Honda, Y; Goto, S; Boucher, I; Brzezinski, R

1995-01-01

Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft. PMID:7487871

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy.

PubMed

Peláez, Jesús; Long, Julie A

2007-01-01

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

Perspective: Dendrimer drugs for infection and inflammation.

PubMed

Shaunak, Sunil

2015-12-18

Biologists are dissecting complex biological pathways at breath taking speed. It is opening up new opportunities for the therapeutic evaluation of novel dendrimer drugs. This review focuses on studies of small dendrimers decorated with sulfate, phosphonate, N-acetyl-cysteine, glucosamine and mannose in animal model studies of infection and inflammation. It highlights those animal model studies which have demonstrated the most promising dendrimer drug constructs as potential new medicines. The issues relating to their analytical chemistry that are slowing the progress of dendrimer drugs into the clinic are highlighted. It should be possible to solve these with additional analytical expertise because it is small dendrimers with only 16-32 peripheral groups that make for the best infection and inflammation related medicines. Public-private partnerships are now needed to progress these dendrimer drugs into proof-of-concept clinical trials. Copyright © 2015 Elsevier Inc. All rights reserved.

Dried Fruit of the Luffa Sponge as a Source of Chitin for Applications as Skin Substitutes

PubMed Central

Chien, Mei-Yin; Sheu, Ming-Thau; Huang, Yi-You; Chen, Meng-Hsun; Su, Ching-Hua; Liu, Der-Zen

2014-01-01

LUFFACHITIN obtained from the residue of the sponge-like dried fruit of Luffa aegyptiaca was developed as a weavable skin substitute in this study. A chemical analysis revealed that LUFFACHITIN was composed of a copolymer containing N-acetyl-glucosamine (~40%) as a major monomer with a filamentary structure as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue of the sponge-like dried fruit of Luffa aegyptiaca after treatment was then woven into a thin, porous membrane by filtration and lyophilization as a skin substitute for conducting wound-healing study on rats. The results indicated that the LUFFACHITIN membrane showed significant wound-healing enhancement (25 days to complete healing) compared to cotton gauze (>30 days), but not inferior to that of SACCHACHITIN. Furthermore, the LUFFACHITIN membrane had advantages of having a high yield, better physical properties for fabrication, and a more attractive appearance. PMID:24812618

Dried fruit of the Luffa sponge as a source of chitin for applications as skin substitutes.

PubMed

Jiang, Ping-Lun; Chien, Mei-Yin; Sheu, Ming-Thau; Huang, Yi-You; Chen, Meng-Hsun; Su, Ching-Hua; Liu, Der-Zen

2014-01-01

LUFFACHITIN obtained from the residue of the sponge-like dried fruit of Luffa aegyptiaca was developed as a weavable skin substitute in this study. A chemical analysis revealed that LUFFACHITIN was composed of a copolymer containing N-acetyl-glucosamine (~40%) as a major monomer with a filamentary structure as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue of the sponge-like dried fruit of Luffa aegyptiaca after treatment was then woven into a thin, porous membrane by filtration and lyophilization as a skin substitute for conducting wound-healing study on rats. The results indicated that the LUFFACHITIN membrane showed significant wound-healing enhancement (25 days to complete healing) compared to cotton gauze (>30 days), but not inferior to that of SACCHACHITIN. Furthermore, the LUFFACHITIN membrane had advantages of having a high yield, better physical properties for fabrication, and a more attractive appearance.

Skin lightening preparations and the hydroquinone controversy.

PubMed

Draelos, Zoe Diana

2007-01-01

Skin lightening preparations are widely used in dermatology by persons of all Fitzpatrick skin types. Fitzpatrick skin types I-III require local pigment lightening for the treatment of hormonally induced melasma and postinflammatory hyperpigmentation caused by acne and trauma. Fitzpatrick skin types IV and darker have an even greater need for skin lightening for social reasons, as well as pigmentary changes that occur around the eyes, in the intertriginous areas, following dermatitis, or with acne and trauma. The gold standard dermatologic agent for skin lightening was hydroquinone, until regulatory agencies in Japan, Europe, and most recently in the United States questioned the safety of this substance. This has encouraged research into alternative agents to inhibit skin pigmentation such as retinoids, mequinol, azelaic acid, arbutin, kojic acid, aleosin, licorice extract, ascorbic acid, soy proteins, and N-acetyl glucosamine. The efficacy and safety of each of these ingredients is examined as possible topical alternatives to hydroquinone.

Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system.

PubMed

Ohno, Misa; Kimura, Masahiro; Miyazaki, Haruko; Okawa, Kazuaki; Onuki, Riho; Nemoto, Chiyuki; Tabata, Eri; Wakita, Satoshi; Kashimura, Akinori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Nukina, Nobuyuki; Bauer, Peter O; Oyama, Fumitaka

2016-11-24

Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc) 2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc) 2 , a source of carbon, nitrogen and energy.

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

PubMed Central

Shimizu, S; Sabsay, B; Veis, A; Ostrow, J D; Rege, R V; Dawes, L G

1989-01-01

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones. Images PMID:2592569

The pgaABCD Locus of Escherichia coli Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation

PubMed Central

Wang, Xin; Preston, James F.; Romeo, Tony

2004-01-01

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched β-1,6-N-acetyl-d-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-β-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria. PMID:15090514

A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis.

PubMed

Wan, Jinrong; Zhang, Xue-Cheng; Neece, David; Ramonell, Katrina M; Clough, Steve; Kim, Sung-Yong; Stacey, Minviluz G; Stacey, Gary

2008-02-01

Chitin, a polymer of N-acetyl-d-glucosamine, is found in fungal cell walls but not in plants. Plant cells can perceive chitin fragments (chitooligosaccharides) leading to gene induction and defense responses. We identified a LysM receptor-like protein (LysM RLK1) required for chitin signaling in Arabidopsis thaliana. The mutation in this gene blocked the induction of almost all chitooligosaccharide-responsive genes and led to more susceptibility to fungal pathogens but had no effect on infection by a bacterial pathogen. Additionally, exogenously applied chitooligosaccharides enhanced resistance against both fungal and bacterial pathogens in the wild-type plants but not in the mutant. Together, our data indicate that LysM RLK1 is essential for chitin signaling in plants (likely as part of the receptor complex) and is involved in chitin-mediated plant innate immunity. The LysM RLK1-mediated chitin signaling pathway is unique, but it may share a conserved downstream pathway with the FLS2/flagellin- and EFR/EF-Tu-mediated signaling pathways. Additionally, our work suggests a possible evolutionary relationship between the chitin and Nod factor perception mechanisms due to the similarities between their potential receptors and between the signal molecules perceived by them.

Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

PubMed Central

Ohno, Misa; Kimura, Masahiro; Miyazaki, Haruko; Okawa, Kazuaki; Onuki, Riho; Nemoto, Chiyuki; Tabata, Eri; Wakita, Satoshi; Kashimura, Akinori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Nukina, Nobuyuki; Bauer, Peter O.; Oyama, Fumitaka

2016-01-01

Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy. PMID:27883045

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

PubMed Central

Erickson, D. L.

2013-01-01

Transmission by fleabite is a relatively recent evolutionary adaptation of Yersinia pestis, the bacterial agent of bubonic plague. To produce a transmissible infection, Y. pestis grows as an attached biofilm in the foregut of the flea vector. Biofilm formation both in the flea foregut and in vitro is dependent on an extracellular matrix (ECM) synthesized by the Yersinia hms gene products. The hms genes are similar to the pga and ica genes of Escherichia coli and Staphylococcus epidermidis, respectively, that act to synthesize a poly-β-1,6-N-acetyl-d-glucosamine ECM required for biofilm formation. As with extracellular polysaccharide production in many other bacteria, synthesis of the Hms-dependent ECM is controlled by intracellular levels of cyclic-di-GMP. Yersinia pseudotuberculosis, the food- and water-borne enteric pathogen from which Y. pestis evolved recently, possesses identical hms genes and can form biofilm in vitro but not in the flea. The genetic changes in Y. pestis that resulted in adapting biofilm-forming capability to the flea gut environment, a critical step in the evolution of vector-borne transmission, have yet to be identified. During a flea bite, Y. pestis is regurgitated into the dermis in a unique biofilm phenotype, and this has implications for the initial interaction with the mammalian innate immune response. PMID:18453279

Complete Genome Sequence of the Cystic Fibrosis Pathogen Achromobacter xylosoxidans NH44784-1996 Complies with Important Pathogenic Phenotypes

PubMed Central

Jakobsen, Tim Holm; Hansen, Martin Asser; Jensen, Peter Østrup; Hansen, Lars; Riber, Leise; Cockburn, April; Kolpen, Mette; Rønne Hansen, Christine; Ridderberg, Winnie; Eickhardt, Steffen; Hansen, Marlene; Kerpedjiev, Peter; Alhede, Morten; Qvortrup, Klaus; Burmølle, Mette; Moser, Claus; Kühl, Michael; Ciofu, Oana; Givskov, Michael; Sørensen, Søren J.; Høiby, Niels; Bjarnsholt, Thomas

2013-01-01

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen. PMID:23894309

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-β-D-glucosamine-Based Artificial Ion-Transport Systems.

PubMed

Roy, Arundhati; Saha, Tanmoy; Gening, Marina L; Titov, Denis V; Gerbst, Alexey G; Tsvetkov, Yury E; Nifantiev, Nikolay E; Talukdar, Pinaki

2015-11-23

Cyclo-oligo-(1→6)-β-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

1975-09-22

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

Glycosidases in Brachionus plicatilis (Rotifera).

PubMed

Kühle, K; Kleinow, W

1990-01-01

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

Electronic structure calculations toward new potentially AChE inhibitors

NASA Astrophysics Data System (ADS)

de Paula, A. A. N.; Martins, J. B. L.; Gargano, R.; dos Santos, M. L.; Romeiro, L. A. S.

2007-10-01

The main purpose of this study was the use of natural non-isoprenoid phenolic lipid of cashew nut shell liquid from Anacardium occidentale as lead material for generating new potentially candidates of acetylcholinesterase inhibitors. Therefore, we studied the electronic structure of 15 molecules derivatives from the cardanol using the following groups: methyl, acetyl, N, N-dimethylcarbamoyl, N, N-dimethylamine, N, N-diethylamine, piperidine, pyrrolidine, and N-benzylamine. The calculations were performed at RHF level using 6-31G, 6-31G(d), 6-31+G(d) and 6-311G(d,p) basis functions. Among the proposed compounds we found that the structures with substitution by acetyl, N, N-dimethylcarbamoyl, N, N-dimethylamine, and pyrrolidine groups were better correlated to rivastigmine indicating possible activity.

Stimulation of aortic smooth muscle cell mitogenesis by serotonin

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

Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.

1986-02-01

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ..mu..M serotonin with increased incorporation of (/sup 3/H)thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ..mu..M. At a concentration of 1 ..mu..M, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approx. = 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine weremore » inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.« less

Randomized Trial of Glucosamine and Chondroitin Supplementation on Inflammation and Oxidative Stress Biomarkers and Plasma Proteomics Profiles in Healthy Humans

PubMed Central

Navarro, Sandi L.; White, Emily; Kantor, Elizabeth D.; Zhang, Yuzheng; Rho, Junghyun; Song, Xiaoling; Milne, Ginger L.; Lampe, Paul D.; Lampe, Johanna W.

2015-01-01

Background Glucosamine and chondroitin are popular non-vitamin dietary supplements used for osteoarthritis. Long-term use is associated with lower incidence of colorectal and lung cancers and with lower mortality; however, the mechanism underlying these observations is unknown. In vitro and animal studies show that glucosamine and chondroitin inhibit NF-kB, a central mediator of inflammation, but no definitive trials have been done in healthy humans. Methods We conducted a randomized, double-blind, placebo-controlled, cross-over study to assess the effects of glucosamine hydrochloride (1500 mg/d) plus chondroitin sulfate (1200 mg/d) for 28 days compared to placebo in 18 (9 men, 9 women) healthy, overweight (body mass index 25.0–32.5 kg/m2) adults, aged 20–55 y. We examined 4 serum inflammatory biomarkers: C-reactive protein (CRP), interleukin 6, and soluble tumor necrosis factor receptors I and II; a urinary inflammation biomarker: prostaglandin E2-metabolite; and a urinary oxidative stress biomarker: F2-isoprostane. Plasma proteomics on an antibody array was performed to explore other pathways modulated by glucosamine and chondroitin. Results Serum CRP concentrations were 23% lower after glucosamine and chondroitin compared to placebo (P = 0.048). There were no significant differences in other biomarkers. In the proteomics analyses, several pathways were significantly different between the interventions after Bonferroni correction, the most significant being a reduction in the “cytokine activity” pathway (P = 2.6 x 10-16), after glucosamine and chondroitin compared to placebo. Conclusion Glucosamine and chondroitin supplementation may lower systemic inflammation and alter other pathways in healthy, overweight individuals. This study adds evidence for potential mechanisms supporting epidemiologic findings that glucosamine and chondroitin are associated with reduced risk of lung and colorectal cancer. Trial Registration ClinicalTrials.gov NCT01682694 PMID:25719429

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

PubMed

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

2001-12-01

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

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

PubMed

Veronesi, F; Giavaresi, G; Maglio, M; Scotto d'Abusco, A; Politi, L; Scandurra, R; Olivotto, E; Grigolo, B; Borzì, R M; Fini, M

2017-04-01

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Mucin glycan foraging in the human gut microbiome

PubMed Central

Tailford, Louise E.; Crost, Emmanuelle H.; Kavanaugh, Devon; Juge, Nathalie

2015-01-01

The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and β- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonize the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unraveling the molecular strategies used by mucin-degrading bacteria to utilize host glycans, adapt to the mucosal environment, and influence human health. PMID:25852737

Antioxidant activity in vitro and in vivo of the polysaccharides from different varieties of Auricularia auricula.

PubMed

Xu, Siqi; Zhang, Yongjun; Jiang, Kan

2016-09-14

In this study, five different kinds of polysaccharides (AAP1, AAP2, AAP3, AAP4, and AAP5) were extracted from different varieties of Auricularia auricula through an alkali extraction process. Furthermore, the crude polysaccharides were deproteinized by the Sevag method. Auricularia auricula produced in the Shanxi province had the highest content of polysaccharide, 53.02%. The monosaccharide composition was determined by the GC method. Their antioxidant capacities in vitro were assessed by radical-scavenging capacity (DPPH, superoxide, and hydroxyl radicals), metal chelating ability and reducing-power methods. In addition, the evaluation of their antioxidant effects in vivo was performed using the C. elegans model. The yield of crude polysaccharides, monosaccharide composition and antioxidant activity of Auricularia auricula polysaccharides (AAPs) were different among samples from various sources. Among them, the strongest antioxidant activity was shown for AAP1, consisting of arabinose, xylose, 2-deoxy-d-glucose, mannose, glucose, and N-acetyl-d-glucosamine with the molar ratio of 1 : 0.44 : 0.33 : 1.67 : 1 : 0.17. It could scavenge free radicals, up-regulate stress-resistance-related enzymes including superoxide dismutase (SOD) by 70.04 ± 8.75% and CAT by 117.32 ± 8.06% and reduce the level of reactive oxygen species (ROS) in C. elegans under oxidative stress. The present results suggested that variety was an important factor that affects the antioxidant activity of A. auricula polysaccharides.

Nonsteroidal anti-inflammatory drugs (NSAID) sparing effects of glucosamine hydrochloride through N-glycosylation inhibition; strategy to rescue stomach from NSAID damage.

PubMed

Park, S H; Hong, H; Han, Y M; Kangwan, N; Kim, S J; Kim, E H; Hahm, K B

2013-04-01

Gastrointestinal or cardiovascular complications limit nonsteroidal anti-inflammatory drugs (NSAID) prescription. Glucosamine hydrochloride (GS-HCl) alternatively chosen, but debates still exist in its clinical efficiency. COX-2 instability through inhibiting COX-2 N-glycosylation of GS-HCl raised the possibility of NSAID sparing effect. Study was done to determine whether combination treatment of glucosamine and NSAID contributes to gastric safety through NSAID sparing effect. IEC-6 cells were stimulated with TNF-α and compared the expressions of inflammatory mediators after indomethacin alone or combination of indomethacin and GS-HCl by Western blotting and RT-PCR. C57BL/6 mice injected with type II collagen to induce arthritis were treated with indomethacin alone or combination of reduced dose of indomethacin and GS-HCl after 3 weeks. TNF-α increased the expression of COX-2, iNOS and inflammatory cytokines, but GS-HCl significantly attenuated TNF-α-induced COX-2 expression. Decreased COX-2 after GS-HCl was caused by N-glycosylation inhibition as much as tunicamycin. Combination of reduced dose of indomethacin and GS-HCl significantly reduced the expressions of ICAM-1, VCAM-1, IL-8, IL-1β, MMP-2, MMP-7, MMP-9, and MMP-11 mRNA as well as NF-κB activation better than high dose indomethacin alone. These NSAID sparing effect of GS-HCl was further proven in collagen-induced arthritis model. Combination of GS-HCl and 2.5 mg/kg indomethacin showed significant protection from gastric damages as well as efficacious anti-arthritic effect. Taken together, COX-2 N-glycosylation inhibition by GS-HCl led to indomethacin sparing effects, based on which combination of GS-HCl and reduced dose of NSAID can provide the strategy to secure stomach from NSAID-induced gastric damage as well as excellent anti-arthritic effects.

Synthesis of methyl 3-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 3-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

PubMed

Dubey, R; Jain, R K; Abbas, S A; Matta, K L

1987-08-01

Methyl 2-O-benzyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha- D-mannopyranoside (4) and methyl 2-O-benzyl-3-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (6) were prepared from a common intermediate, namely, methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- mannopyranosyl)-alpha-D-mannopyranoside. On treatment with tert-butylchlorodiphenylsilane, in N,N-dimethylformamide in the presence of imidazole, 4 and 6 afforded methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (7), and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert- butyldiphenylsilyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8), respectively. Compound 8 was converted into its 2,3-O-isopropylidene derivative (9), and oxidation of 7 and 9 with pyridinium chlorochromate, and reduction of the resulting carbonyl intermediates gave methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-talopyranoside and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert-butyldiphe nylsilyl- 2,3-O-isopropylidene-alpha-D-talopyranosyl)-alpha-D-talopyranoside , respectively. Removal of the protecting groups furnished the title disaccharides.

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

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

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

Tandem Mass Spectrometry of Heparan Sulfate Negative Ions: Sulfate Loss Patterns and Chemical Modification Methods for Improvement of Product Ion Profiles

NASA Astrophysics Data System (ADS)

Shi, Xiaofeng; Huang, Yu; Mao, Yang; Naimy, Hicham; Zaia, Joseph

2012-09-01

Heparan sulfate (HS) is a polysaccharide modified with sulfation, acetylation, and epimerization that enable its binding with protein ligands and regulation of important biological processes. Tandem mass spectrometry has been employed to sequence linear biomolecules e.g., proteins and peptides. However, its application in structural characterization of HS is limited due to the neutral loss of sulfate (SO3) during collisional induced dissociation (CID). In this report, we studied the dissociation patterns of HS disaccharides and demonstrate that the N-sulfate (N-S) bond is especially facile during CID. We identified factors that influence the propensities of such losses from precursor ions and proposed a Free Proton Index (FPI) to help select ions that are able to produce meaningful backbone dissociations. We then investigated the thermodynamics and kinetics of SO3 loss from sulfates that are protonated, deprotonated, and metal-adducted using density functional theory computations. The calculations showed that sulfate loss from a protonated site was much more facile than that from a deprotonated or metal-adducted site. Further, the loss of SO3 from N-sulfate was energetically favored by 3-8 kcal/mol in transition states relative to O-sulfates, making it more prone to this process by a substantial factor. In order to reduce the FPI, representing the number of labile sulfates in HS native chains and oligosaccharides, we developed a series of chemical modifications to selectively replace the N-sulfates of the glucosamine with deuterated acetyl group. These modifications effectively reduced the sulfate density on the HS oligosaccharides and generated considerably more backbone dissociation using on-line LC/tandem MS.

Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

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

Pampa, K.J., E-mail: sagarikakj@gmail.com; Lokanath, N.K.; Girish, T.U.

Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme bymore » X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.« less

Can Glucosamine Supplements Protect My Knee Cartilage from Osteoarthritis?

MedlinePlus

... Can glucosamine supplements protect my knee cartilage from osteoarthritis? Answers from Brent A. Bauer, M.D. Study results on this question have ... build cartilage. The most common type of arthritis, osteoarthritis wears away the slick cartilage that covers the ...

Use of Glucosamine and Chondroitin and Lung Cancer Risk in the VITamins And Lifestyle (VITAL) Cohort

PubMed Central

Brasky, Theodore M.; Lampe, Johanna W.; Slatore, Christopher G.; White, Emily

2011-01-01

Objective Inflammation plays an important role in lung carcinogenesis. Epidemiologic studies have reported inverse associations of non-steroidal anti-inflammatory drug (NSAID) use and lung cancer risk. Previously, we found that ever use of glucosamine and chondroitin, which have anti-inflammatory properties, were inversely associated with lung cancer risk. After an additional year of follow-up, we further examined the association including frequency/duration of use, interaction with factors associated with inflammation, and lung cancer histology. Methods Participants were members of the VITamins And Lifestyle (VITAL) Cohort. Adults, ages 50–76 years, who were residents of western Washington State, completed a baseline questionnaire in 2000–2002 (n=76,904). Participants were queried on their use of glucosamine and chondroitin, over the 10 years prior to baseline, and categorized as nonuser, low use <4 days/week or <3 years, or high use ≥4 days/week and ≥3 years. Lung cancer cases (n=808) were ascertained through linkage to the Surveillance, Epidemiology, and End Results cancer registry. Results High 10-year use of glucosamine [Hazard Ratio (HR) 0.77, 95% CI: 0.56–1.07; P-trend=0.04] but not chondroitin was associated with a reduction in lung cancer risk. The association with glucosamine was limited to adenocarcinoma (HR 0.49, 95% CI: 0.27–0.90; Ptrend-<0.01), and was not modified by NSAID use or smoking status. Conclusions Our results for glucosamine use are similar to the prior human studies of NSAID use and lung cancer, both in magnitude and the limitation of the association to adenocarcinoma. Unlike NSAIDs, glucosamine has no known adverse-effects. Although confirmatory studies are needed, glucosamine is an attractive candidate for lung cancer chemoprevention. PMID:21706174

Total synthesis and structural revision of TMG-chitotriomycin, a specific inhibitor of insect and fungal beta-N-acetylglucosaminidases.

PubMed

Yang, You; Li, Yao; Yu, Biao

2009-09-02

TMG-chitotriomycin, a potent and selective inhibitor of the beta-N-acetylglucosaminidases that possesses an unique N,N,N-trimethyl-d-glucosamine (TMG) residue, is revised to be the TMG-beta-(1-->4)-chitotriose instead of the originally proposed alpha-anomer via its total synthesis, for which a highly convergent approach was developed in which the sterically demanding (1-->4)-glycosidic linkages are efficiently constructed by the Au(I)-catalyzed glycosylation protocol with glycosyl o-hexynylbenzoates as donors.

Oral acetate supplementation attenuates N-methyl D-aspartate receptor hypofunction-induced behavioral phenotypes accompanied by restoration of acetyl-histone homeostasis.

PubMed

Singh, Seema; Choudhury, Arnab; Gusain, Priya; Parvez, Suhel; Palit, Gautam; Shukla, Shubha; Ganguly, Surajit

2016-04-01

Aberrations in cellular acetate-utilization processes leading to global histone hypoacetylation have been implicated in the etiology of neuropsychiatric disorders like schizophrenia. Here, we investigated the role of acetate supplementation in the form of glyceryl triacetate (GTA) for the ability to restore the N-methyl D-aspartate (NMDA) receptor-induced histone hypoacetylation and to ameliorate associated behavioral phenotypes in mice. Taking cues from the studies in SH-SY5Y cells, we monitored acetylation status of specific lysine residues of histones H3 and H4 (H3K9 and H4K8) to determine the impact of oral GTA supplementation in vivo. Mice treated chronically with MK-801 (10 days; 0.15 mg/kg daily) induced hypoacetylation of H3K9 and H4K8 in the hippocampus. Daily oral supplementation of GTA (2.9 g/kg) was able to prevent this MK801-induced hypoacetylation significantly. Though MK-801-stimulated decreases in acetyl-H3K9 and acetyl-H4K8 were found to be associated with ERK1/2 activation, GTA seemed to act independent of this pathway. Simultaneously, GTA administration was able to attenuate the chronic MK-801-induced cognitive behavior phenotypes in elevated plus maze and novel object recognition tests. Not only MK-801, GTA also demonstrated protective effects against behavioral phenotypes generated by another NMDA receptor antagonist, ketamine. Acute (single injection) ketamine-mediated hyperactivity phenotype and chronic (10 days treatment) ketamine-induced phenotype of exaggerated immobility in forced swim test were ameliorated by GTA. The signature behavioral phenotypes induced by acute and chronic regimen of NMDA receptor antagonists seemed to be attenuated by GTA. This study thus provides a therapeutic paradigm of using dietary acetate supplement in psychiatric disorders.

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells.

PubMed

Klimaszewska-Łata, Joanna; Gul-Hinc, Sylwia; Bielarczyk, Hanna; Ronowska, Anna; Zyśk, Marlena; Grużewska, Katarzyna; Pawełczyk, Tadeusz; Szutowicz, Andrzej

2015-04-01

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways. © 2014 International Society for Neurochemistry.

Comparison of Glucosamine-Chondroitin Sulfate with and without Methylsulfonylmethane in Grade I-II Knee Osteoarthritis: A Double Blind Randomized Controlled Trial.

PubMed

Lubis, Andri M T; Siagian, Carles; Wonggokusuma, Erick; Marsetyo, Aldo F; Setyohadi, Bambang

2017-04-01

Glucosamine, chondroitinsulfate are frequently used to prevent further joint degeneration in osteoarthritis (OA). Methylsulfonylmethane (MSM) is a supplement containing organic sulphur and also reported to slow anatomical joint progressivity in the knee OA. The MSM is often combined with glucosamine and chondroitin sulfate. However, there are controversies whether glucosamine-chondroitin sulfate or their combination with methylsulfonylmethane could effectively reduce pain in OA. This study is aimed to compare clinical outcome of glucosamine-chondroitin sulfate (GC), glucosamine-chondroitin sulfate-methylsulfonylmethane (GCM), and placeboin patients with knee osteoarthritis (OA) Kellgren-Lawrence grade I-II. a double blind, randomized controlled clinical trial was conducted on 147 patients with knee OA Kellgren-Lawrence grade I-II. Patients were allocated by permuted block randomization into three groups: GC (n=49), GCM (n=50), or placebo (n=48) groups. GC group received 1500 mg of glucosamine + 1200 mg of chondroitin sulfate + 500 mg of saccharumlactis; GCM group received 1500 mg of glucosamine + 1200 mg of chondroitin sulfate + 500 mg of MSM; while placebo group received three matching capsules of saccharumlactis. The drugs were administered once daily for 3 consecutive months VAS and WOMAC scores were measured before treatment, then at 4th, 8th and 12th week after treatment. on statistical analysis it was found that at the 12th week, there are significant difference between three treatment groups on the WOMAC score (p=0.03) and on the VAS score (p=0.004). When analyzed between weeks, GCM treatment group was found statistically significant on WOMAC score (p=0.01) and VAS score (p<0.001). Comparing the score difference between weeks, WOMAC score analysis showed significant difference between GC, GCM, and placebo in week 4 (p=0.049) and week 12 (p=0.01). In addition, VAS score also showed significant difference between groups in week 8 (p=0.006) and week 12 (p<0.001). combination of glucosamine-chondroitinsulfate-methylsulfonylmethane showed clinical benefit for patients with knee OAK ellgren-Lawrence grade I-II compared with GC and placebo. GC did not make clinical improvement in overall groups of patients with knee OA Kellgren Lawrence grade I-II.

Phloem Transport of d,l-Glufosinate and Acetyl-l-Glufosinate in Glufosinate-Resistant and -Susceptible Brassica napus1

PubMed Central

Beriault, Jennifer N.; Horsman, Geoff P.; Devine, Malcolm D.

1999-01-01

Phloem transport of d,l-[14C]glufosinate, d-[14C]glufosinate, and acetyl-l-[14C]glufosinate was examined in the susceptible Brassica napus cv Excel and a glufosinate-resistant genotype (HCN27) derived by transformation of cv Excel with the phosphinothricin-N-acetyltransferase (pat) gene. Considerably more 14C was exported from an expanded leaf in HCN27 than in cv Excel following application of d,l-[14C]glufosinate (25% versus 6.3% of applied, respectively, 72 h after treatment). The inactive isomer, d-glufosinate, was much more phloem mobile in cv Excel than racemic d,l-glufosinate. Foliar or root supplementation with 1 mm glutamine increased d,l-[14C]glufosinate translocation in cv Excel but only transiently, suggesting that glutamine depletion is not the major cause of the limited phloem transport. Acetyl-l-[14C]glufosinate (applied as such or derived from l-glufosinate in pat transformants) was translocated extensively in the phloem of both genotypes. Acetyl-l-[14C]glufosinate was readily transported into the floral buds and flowers, and accumulated in the anthers in both genotypes. These results suggest that phloem transport of d,l-glufosinate is limited by rapid physiological effects of the l-isomer in source leaf tissue. The accumulation of acetyl-l-glufosinate in the anthers indicates that it is sufficiently phloem mobile to act as a foliar-applied chemical inducer of male sterility in plants expressing a deacetylase gene in the tapetum, generating toxic concentrations of l-glufosinate in pollen-producing tissues. PMID:10517854

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

PubMed Central

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

1983-01-01

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

Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study.

PubMed

Wang, Xianhuo; Chen, Xiang; Chen, Lijuan; Wang, Biqin; Peng, Cheng; He, Chunmei; Tang, Minghai; Zhang, Fan; Hu, Jia; Li, Rui; Zhao, Xia; Wei, Yuquan

2008-11-01

A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.

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

PubMed

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

2007-12-27

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

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Novel Micro/Nano Approaches for Glucose Measurement Using pH-Sensitive

DTIC Science & Technology

2006-06-01

step, the anionic enzyme was assembled in multilayers in alternation with poly(allylamine hydrochloride ) (PAH) (polycation). A total of three...group of D- glucosamine residues in chitosan can be conjugated with amine-reactive dyes, such as succinimidyl esters (Alexa Fluor 647™, CY5...measured values for FITC-chitosan were found to be: A494nm=0.153276 MW=161 [D- glucosamine ]=0.77g/L εdye=68000cm-1M-1

Catabolism and Detoxification of 1-Aminoalkylphosphonic Acids: N-Acetylation by the phnO Gene Product

PubMed Central

Hove-Jensen, Bjarne; McSorley, Fern R.; Zechel, David L.

2012-01-01

In Escherichia coli uptake and catabolism of organophosphonates are governed by the phnCDEFGHIJKLMNOP operon. The phnO cistron is shown to encode aminoalkylphosphonate N-acetyltransferase, which utilizes acetylcoenzyme A as acetyl donor and aminomethylphosphonate, (S)- and (R)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate as acetyl acceptors. Aminomethylphosphonate, (S)-1-aminoethylphosphonate, 2-aminoethyl- and 3-aminopropylphosphonate are used as phosphate source by E. coli phn+ strains. 2-Aminoethyl- or 3-aminopropylphosphonate but not aminomethylphosphonate or (S)-1-aminoethylphosphonate is used as phosphate source by phnO strains. Neither phn+ nor phnO strains can use (R)-1-aminoethylphosphonate as phosphate source. Utilization of aminomethylphosphonate or (S)-1-aminoethylphosphonate requires the expression of phnO. In the absence of phnO-expression (S)-1-aminoethylphosphonate is bacteriocidal and rescue of phnO strains requires the simultaneous addition of d-alanine and phosphate. An intermediate of the carbon-phosphorus lyase pathway, 5′-phospho-α-d-ribosyl 1′-(2-N-acetamidoethylphosphonate), a substrate for carbon-phosphorus lyase, was found to accumulate in cultures of a phnP mutant strain. The data show that the physiological role of N-acetylation by phnO-specified aminoalkylphosphonate N-acetyltransferase is to detoxify (S)-1-aminoethylphosphonate, an analog of d-alanine, and to prepare (S)-1-aminoethylphosphonate and aminomethylphosphonate for utilization of the phosphorus-containing moiety. PMID:23056305

Observational study: daily treatment with a new compound “tradamixina” plus serenoa repens for two months improved the lower urinary tract symptoms

PubMed Central

2012-01-01

Background Lower urinary tract symptoms (LUTS) are associated with great emotional costs to individuals and substantial economic costs to society. This study seeks to evaluate the effect of a new natural compound “Tradamixina plus Serenoa Repens” in order to improve lower urinary tract symptoms. Methods 100 patients (≥45years) who had had LUTS/BPH for >6 mo at screening and with IPSS -The international Prostate symptom scores- ≥13 and maximum urinary flow rate (Qmax) ≥4 to ≤15 ml/s. were recruited. The compound “Tradamixina plus Serenoa Repens” (80 mg of Alga Ecklonia Bicyclis, 100 mg of Tribulus Terrestris and 100 mg of D-Glucosamine and N-Acetyl-D-Glucosamine plus 320 mg of Serenoa Repens) was administered daily for 2 months. At visit and after 60 days of treatment patients were evaluated by means of detailed medical urological history, clinical examination, laboratory investigations (total PSA), and instrumental examination like urolfowmetry. Efficacy measures included IPSS-International Prostate Sympto, BPH Impact Index (BII), Quality-of-Life (QoL) Index. Measures were assessed at baseline and end point (12 wk or end of therapy) and also at screening, 1 and 4 wk for IPSS, and 4 wk for BII. Statistical significance was interpreted only if the results of the preceding analysis were significant at the 0.05 level. Results After 2 months of treatment the change from baseline to week 12 relative to “Tradamixina plus Seronea Repens” in total IPSS and Qol was statistically significant. Differences from baseline in BII were statistically significant for “Tradamixina plus Seronea Repens” above all differences in BII were also significant at 4 wk (LSmean ± SE: -0.8 ± 0.2). In the distribution of subjects over the PGI-I and CGI-I response categories were significant for”Tradamixina plus Seronea Repens” (PGI-I: p = 0.001; CGI-I). We also observed a decrease of total PSA. Conclusion The daily treatment with a new compound “Tradamixina plus Serenoa Repens” for 2 months improved the male sexual function , it improved the bother symptoms which affect the patient’s quality of life , improved uroflowmetric parameters, and we also observed a decrease of serum PSA level. PMID:23173650

Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

PubMed Central

Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

2000-01-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640

Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones.

PubMed

Galasinski, S K; Lively, T N; Grebe De Barron, A; Goodrich, J A

2000-03-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression.

N-Acetyl Cysteine and Vitamin D Supplementation in Treatment Resistant Obsessive- compulsive Disorder Patients: a General Review.

PubMed

di Michele, Flavia; Siracusano, Alberto; Talamo, Alessandra; Niolu, Cinzia

2018-04-17

Obsessive-compulsive disorder (OCD) is a disabling mental illness for which pharmacological and psychosocial interventions are all too often inadequate. This demonstrates the need for more targeted therapeutics. Recent preclinical and clinical studies have implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of OCD. Moreover there are studies suggesting that neuroimmune abnormalities may play an important role in the pathogenesis of OCD. N-acetyl cysteine (NAC) is a safe and readily available agent that would modify the synaptic release of glutamate in subcortical brain regions via modulation of the cysteine-glutamate antiporter. The modulation of inflammatory pathways may also play a role in the benefits seen following NAC treatment. Therefore NAC can be considered a neuroprotective agent. This paper explores the role of NAC in the treatment of OCD conditions refractory to first-line pharmacological interventions, reviewing the clinical studies published in the last decade. The possible benefit mechanisms of NAC for this disorder will be discussed, as well as the role of vitamin D supplementation, given its specific property of stimulating the formation of glutathione in the brain. Nutraceutical supplementation in treatment resistance OCD may be important not only for improving obsessive-compulsive symptomatology, but also from a psychological perspective, given its better acceptance by the patients compared to pharmacological treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

PubMed

Borodkin, Vladimir S; Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-18

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host

PubMed Central

Echeverz, Maite; García, Begoña; Sabalza, Amaia; Valle, Jaione; Lasa, Iñigo

2017-01-01

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host. PMID:28542593

Functional Characterization of Corynebacterium glutamicum Mycothiol S-Conjugate Amidase

PubMed Central

Si, Meiru; Long, Mingxiu; Chaudhry, Muhammad Tausif; Xu, Yixiang; Zhang, Pan; Zhang, Lei; Shen, Xihui

2014-01-01

The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. PMID:25514023

Synthesis and evaluation of 68Ga-labeled DOTA-2-deoxy-D-glucosamine as a potential radiotracer in μPET imaging

PubMed Central

Yang, Zhi; Xiong, Chiyi; Zhang, Rui; Zhu, Hua; Li, Chun

2012-01-01

The purposes of this study were to develop an efficient method of labeling D-glucosamine hydrochloride with gallium 68 (68Ga) and investigate the imaging properties of the resulting radiotracer in a human tumor xenograft model using micro-positron emission tomography (μPET). The precursor compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-2-deoxy-D-glucosamine (DOTA-DG) was synthesized from D-glucosamine hydrochloride and 2-(4-isothiocyanatobenzyl)-DOTA. Radiolabeling of DOTA-DG with 68Ga was achieved in 10 minutes using microwave heating. The labeling efficiency a nd radiochemical purity after purification of 68Ga-DOTA-DG were ~85% and greater than 98%, respectively. In A431 cells, the percentages of 68Ga-DOTA-DG and 18F-FDG uptakes after 60 min incubation were 15.7% and 16.2%, respectively. In vivo, the mean ± standard deviation of 68Ga-DOTADG uptake values in A431 tumors were 2.38±0.30, 0.75±0.13, and 0.39±0.04 percent of the injected dose per gram of tissue at 10, 30, and 60 minutes after intravenous injection, respectively. μPET imaging of A431-bearing mice clearly delineated tumors at 60 minutes after injection of 68Ga-DOTA-DG at a dose of 3.7 MBq. 68Ga-DOTA-DG displayed significantly higher tumor-to-heart, tumor-to-brain, and tumor-to-muscle ratios than 18F-FDG did. Further studies are needed to identify the mechanism of tumor uptake of this new glucosamine-based PET imaging tracer. PMID:23145365

Synthesis and evaluation of (68)Ga-labeled DOTA-2-deoxy-D-glucosamine as a potential radiotracer in μPET imaging.

PubMed

Yang, Zhi; Xiong, Chiyi; Zhang, Rui; Zhu, Hua; Li, Chun

2012-01-01

The purposes of this study were to develop an efficient method of labeling D-glucosamine hydrochloride with gallium 68 ((68)Ga) and investigate the imaging properties of the resulting radiotracer in a human tumor xenograft model using micro-positron emission tomography (μPET). The precursor compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-2-deoxy-D-glucosamine (DOTA-DG) was synthesized from D-glucosamine hydrochloride and 2-(4-isothiocyanatobenzyl)-DOTA. Radiolabeling of DOTA-DG with (68)Ga was achieved in 10 minutes using microwave heating. The labeling efficiency a nd radiochemical purity after purification of (68)Ga-DOTA-DG were ~85% and greater than 98%, respectively. In A431 cells, the percentages of (68)Ga-DOTA-DG and (18)F-FDG uptakes after 60 min incubation were 15.7% and 16.2%, respectively. In vivo, the mean ± standard deviation of (68)Ga-DOTADG uptake values in A431 tumors were 2.38±0.30, 0.75±0.13, and 0.39±0.04 percent of the injected dose per gram of tissue at 10, 30, and 60 minutes after intravenous injection, respectively. μPET imaging of A431-bearing mice clearly delineated tumors at 60 minutes after injection of (68)Ga-DOTA-DG at a dose of 3.7 MBq. (68)Ga-DOTA-DG displayed significantly higher tumor-to-heart, tumor-to-brain, and tumor-to-muscle ratios than (18)F-FDG did. Further studies are needed to identify the mechanism of tumor uptake of this new glucosamine-based PET imaging tracer.

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

NASA Astrophysics Data System (ADS)

Elywa, M.

2015-07-01

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

PREVALENCE, BIOCHEMICAL CHARACTERISTICS, AND ANTIBIOTIC SUSCEPTIBILITY OF AEROMONADS, VIBRIOS, AND PLESIOMONADS ISOLATED FROM DIFFERENT SOURCES AT A ZOO.

PubMed

Kim, Kyoo-Tae; Lee, Seung-Hun; Kwak, Dongmi

2015-06-01

Aeromonas spp., Vibrio parahaemolyticus , and Plesiomonas shigelloides are commonly implicated in foodborne and waterborne diarrheal illnesses of humans and other animals. The present study assessed the prevalence, biochemical characteristics, and antibiotic susceptibility of Aeromonas spp., V. parahaemolyticus , and P. shigelloides by analyzing samples from 729 sources at a zoo, including animal feces (n=607), watering facilities (n=104), and pond water samples (n=18). Of the 729 samples collected, 40 (5.5%) contained one of these four species of bacteria: A. hydrophila (n=16; 2.2%), A. sobria (n=12; 1.6%), V. parahaemolyticus (n=10; 1.4%), and P. shigelloides (n=2; 0.3%). The 16 isolates of A. hydrophila came from three fecal samples, eight watering facilities, and five pond water samples. The 12 isolates of A. sobria came from four fecal samples, three watering facilities, and five pond water samples. The 10 isolates of V. parahaemolyticus came from one fecal sample and nine watering facilities. The two isolates of P. shigelloides came from one watering facility and one pond water sample. Of the 40 isolates, 16 (40.0%), 21 (52.5%), and three (7.5%) originated from mammals, birds, and reptiles, respectively. All isolates tested positive for NO3, tryptophan, p-nitrophenyl-β-D-galactopyranoside, glucose assimilation, N-acetyl-glucosamine, maltose, gluconate, malate, and oxidase. Aeromonas spp. and V. parahaemolyticus exhibited similar biochemical characteristics, whereas P. shigelloides exhibited distinct fermentation characteristics. All the isolated strains exhibited hemolytic activity; variable results of DNase, protease, and Congo red uptake tests; and resistance to ampicillin, bacitracin, novobiocin, penicillin, and vancomycin. All the strains were sensitive to amikacin, chloramphenicol, colistin, gentamicin, kanamycin, norfloxacin, and trimethoprim-sulfadimethoxazole. Because of the high proportion of asymptomatic carriers of these potentially pathogenic bacteria and their wide distribution, consistent monitoring of food and water sources is necessary to prevent disease outbreaks.

Crowning: a novel Escherichia coli colonizing behaviour generating a self-organized corona

PubMed Central

2014-01-01

Background Encased in a matrix of extracellular polymeric substances (EPS) composed of flagella, adhesins, amyloid fibers (curli), and exopolysaccharides (cellulose, β-1,6-N-acetyl-D-glucosamine polymer-PGA-, colanic acid), the bacteria Escherichia coli is able to attach to and colonize different types of biotic and abiotic surfaces forming biofilms and colonies of intricate morphological architectures. Many of the biological aspects that underlie the generation and development of these E. coli’s formations are largely poorly understood. Results Here, we report the characterization of a novel E. coli sessile behaviour termed "crowning" due to the bacterial generation of a new 3-D architectural pattern: a corona. This bacterial pattern is formed by joining bush-like multilayered "coronal flares or spikes" arranged in a ring, which self-organize through the growth, self-clumping and massive self-aggregation of cells tightly interacting inside semisolid agar on plastic surfaces. Remarkably, the corona’s formation is developed independently of the adhesiveness of the major components of E. coli’s EPS matrix, the function of chemotaxis sensory system, type 1 pili and the biofilm master regulator CsgD, but its formation is suppressed by flagella-driven motility and glucose. Intriguingly, this glucose effect on the corona development is not mediated by the classical catabolic repression system, the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Thus, corona formation departs from the canonical regulatory transcriptional core that controls biofilm formation in E. coli. Conclusions With this novel "crowning" activity, E. coli expands its repertoire of colonizing collective behaviours to explore, invade and exploit environments whose critical viscosities impede flagella driven-motility. PMID:24568619

A metabolomic investigation into the effects of temperature on Streptococcus agalactiae from Nile tilapia (Oreochromis niloticus) based on UPLC-MS/MS.

PubMed

Hu, Wen-Ting; Guo, Wei-Liang; Meng, Ai-Yun; Sun, Yun; Wang, Shi-Feng; Xie, Zhen-Yu; Zhou, Yong-Can; He, Chaozu

2017-10-01

Streptococcosis caused by Streptococcus agalactiae is one of the most serious diseases in farmed tilapia, and temperature is one of the most important environmental factors related to its outbreak. To elucidate the influence of temperature variation on the pathogen from a metabolic perspective, the global metabolomics of 2 pathogenic strains of S. agalactiae from sick tilapia were analyzed at 35°C and 25°C using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with pattern recognition approaches and pathway analysis. The result showed that the metabolic status of S. agalactiae was extensively affected by its culture temperature. Based on the results of metabolites contributing to these differences, a large number of nucleotides and their ramifications were markedly elevated at 35°C. Various energy substances, components of the cell wall and substances associated with stress regulation such as glyceraldehyde 3-phosphate, pyroglutamic acid, glutamate, d-Alanyl-d-alanine, glycerophosphocholine, dephospho-CoA, and oxidized glutathione increased when the strains were cultured at 35°C. Additionally, a general decrease in various precursors of capsule, antigen, and virulence protein formation were detected including mannose, maltotriose, N-acetyl-d-glucosamine 6-phosphate, uracil, proline, and citrulline. These metabolic changes indicated that metabolic activity decreased, while adaptive ability to environment and pathogenicity to host increased at high temperature. This study is the first to determine the metabolomic responses of S. agalactiae to temperature, and the results are useful to reveal its pathogenic mechanism and find effective disease control. Copyright © 2017 Elsevier B.V. All rights reserved.

[Carbon Source Utilization Characteristics of Soil Microbial Community for Apple Orchard with Interplanting Herbage].

PubMed

Du, Yi-fei; Fang, Kai-kai; Wang, Zhi-kang; Li, Hui-ke; Mao, Peng-juan; Zhang, Xiang-xu; Wang, Jing

2015-11-01

As soil fertility in apple orchard with clean tillage is declined continuously, interplanting herbage in orchard, which is a new orchard management model, plays an important role in improving orchard soil conditions. By using biolog micro-plate technique, this paper studied the functional diversity of soil microbial community under four species of management model in apple orchards, including clear tillage model, interplanting white clover model, interplanting small crown flower model and interplanting cocksfoot model, and the carbon source utilization characteristics of microbial community were explored, which could provide a reference for revealing driving mechanism of ecological process of orchard soil. The results showed that the functional diversity of microbial community had a significant difference among different treatments and in the order of white clover > small crown flower > cocksfoot > clear tillage. The correlation analysis showed that the average well color development (AWCD), Shannon index, Richness index and McIntosh index were all highly significantly positively correlated with soil organic carbon, total nitrogen, microbial biomass carbon, and Shannon index was significantly positively correlated with soil pH. The principal component analysis and the fingerprints of the physiological carbon metabolism of the microbial community demonstrated that grass treatments improved carbon source metabolic ability of soil microbial community, and the soil microbes with perennial legumes (White Clover and small crown flower) had a significantly higher utilization rate in carbohydrates (N-Acetyl-D-Glucosamine, D-Mannitol, β-Methyl-D-Glucoside), amino acids (Glycyl-L-Glutamic acid, L-Serine, L-Threonine) and polymers (Tween 40, Glycogen) than the soil microbes with clear tillage. It was considered that different treatments had the unique microbial community structure and peculiar carbon source utilization characteristics.

Some further studies on the synthesis of glycopeptide derivatives: 2-acetamido-2-deoxy-β-d-glucopyranosylamine derivatives

PubMed Central

Bolton, C. H.; Hough, L.; Khan, M. Y.

1966-01-01

1. The isolation, characterization and properties of two by-products in the preparation of 2-acetamido-3,4,6-tri-O- acetyl-2-deoxy-β-d-glucopyranosylamine are described. They are bis(2-acetamido-2-deoxy-d-glucopyranosyl)amines. 2. An independent synthesis of the bis-glycopyranosylamines is reported and conditions are given for their preparation in high yield. 3. Further improvements are given for the synthesis of 2-acetamido-1-N-(β-l- aspartyl)-2-deoxy-β-d-glucopyranosylamine and the α-l-aspartyl isomer. 4. The synthesis of 2-acetamido-1-N-acetyl-2-deoxy-β-d-glucopyranosylamine is described. PMID:5971780

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

PubMed

Otsubo, N; Ishida, H; Kiso, M

2001-01-15

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

Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Mercer, Natalia; Ramakrishnan, Boopathy; Boeggeman, Elizabeth; Qasba, Pradman K

2011-01-01

Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17-amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.

Too sweet to resist: Control of immune cell function by O-GlcNAcylation.

PubMed

de Jesus, Tristan; Shukla, Sudhanshu; Ramakrishnan, Parameswaran

2018-06-02

O-linked β-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer's disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Novel mutations of the carbohydrate sulfotransferase-6 (CHST6) gene causing macular corneal dystrophy in India.

PubMed

Sultana, Afia; Sridhar, Mittanamalli S; Jagannathan, Aparna; Balasubramanian, Dorairajan; Kannabiran, Chitra; Klintworth, Gordon K

2003-12-22

Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive central haze, confluent punctate opacities and abnormal deposits in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, encoding corneal N-acetyl glucosamine-6-O-sulfotransferase (C-GlcNAc-6-ST). We screened the CHST6 gene for mutations in Indian families with MCD, in order to determine the range of pathogenic mutations. Genomic DNA was isolated from peripheral blood leukocytes of patients with MCD and normal controls. The coding regions of the CHST6 gene were amplified using three pairs of primers and amplified products were directly sequenced. We identified 22 (5 nonsense, 5 frameshift, 2 insertion, and 10 missense) mutations in 36 patients from 31 families with MCD, supporting the conclusion that loss of function of this gene is responsible for this corneal disease. Seventeen of these mutations are novel. These data highlight the allelic heterogeneity of macular corneal dystrophy in Indian patients.

Epitope mapping of histo blood group antigens bound to norovirus VLPs using STD NMR experiments reveals fine details of molecular recognition.

PubMed

Fiege, Brigitte; Leuthold, Mila; Parra, Francisco; Dalton, Kevin P; Meloncelli, Peter J; Lowary, Todd L; Peters, Thomas

2017-10-01

Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be critical for the infection process. Therefore, we have determined binding epitopes of synthetic type 1 to 6 blood group A- and B-tetrasaccharides binding to GII.4 human Norovirus virus like particles (VLPs) using STD NMR experiments. So far, little information is available from crystal structure analysis studies on the interactions of the reducing-end sugars with the protruding domain (P-domain) of the viral coat protein VP1. Here, we show that the reducing-end sugars make notable contacts with the protein surface. The type of glycosidic linkage, and the identity of the sugar at the reducing end modulate HBGA recognition. Most strikingly, type 2 structures yield only very poor saturation transfer indicating impeded binding. This observation is in accordance with previous mass spectrometry based affinity measurements, and can be understood based on recent crystal structure data of a complex of highly homologous GII.4 P-dimers with H-type 2 trisaccharide where the N-acetyl group of the reducing N-acetyl glucosamine residue points towards a loop comprising amino acids Q390 to H395. We suggest that in our case, binding of type 2 A- and B-tetrasaccharides leads to steric conflicts with this loop. In order to identify factors determining L-Fuc recognition, we also synthesized GII.4 VLPs with point mutations D391A and H395A. Prior studies had suggested that these residues, located in a second shell around the L-Fuc binding site, assist L-Fuc binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes suggesting that these two mutations do not significantly alter HBGA recognition. Our study emphasizes that recognition of α-(1→2)-linked L-Fuc residues is a conserved feature of GII.4 noroviruses. However, structural variation of the HBGA core structures clearly modulates molecular recognition depending on the genotype.

Toxicokinetics of novel psychoactive substances: characterization of N-acetyltransferase (NAT) isoenzymes involved in the phase II metabolism of 2C designer drugs.

PubMed

Meyer, Markus R; Robert, Anja; Maurer, Hans H

2014-06-05

The 2,5-dimethoxyphenethylamine-derived designer drugs (so-called "2Cs") recently became of great importance on the illicit drug market as stimulating hallucinogens. They are distributed and consumed as "novel psychoactive substances" (NPS) without any safety testing at the forefront. As previous studies have shown, the 2Cs are mainly metabolized by O-demethylation, N-acetylation, or deamination. Therefore, the aim of this study was to elucidate the role of the recombinant human N-acetyltransferase (NAT) isoforms 1 and 2 in the phase II metabolism of 2Cs. For these studies, cDNA-expressed recombinant human NATs were used and formation of metabolites after incubation was measured using GC-MS. NAT2 could be shown to be the only isoform catalyzing the reaction in vitro, hence it should be the only relevant enzyme for in vivo acetylation. In general, all metabolite formation reactions followed classic Michaelis-Menten kinetics and the affinity to human NAT2 was increasing with the volume of the 4-substituent. In consequence, a slow acetylator phenotype or inhibition of NAT2 could lead to decreased N-acetylation and might lead to an increased risk of side effects caused by these novel psychoactive substances. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

1H-Magnetic resonance spectroscopy study of stimulant medication effect on brain metabolites in French Canadian children with attention deficit hyperactivity disorder

PubMed Central

Benamor, Leila

2014-01-01

Background Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder in school aged children. Functional abnormalities have been reported in brain imaging studies in ADHD populations. Psychostimulants are considered as the first line treatment for ADHD. However, little is known of the effect of stimulants on brain metabolites in ADHD patients. Objectives To compare the brain metabolite concentrations in children with ADHD and on stimulants with those of drug naïve children with ADHD, versus typically developed children, in a homogenous genetic sample of French Canadians. Methods Children with ADHD on stimulants (n=57) and drug naïve children with ADHD (n=45) were recruited, as well as typically developed children (n=38). The presence or absence of ADHD diagnosis (Diagnostic and Statistical Manual of Mental Disorders IV criteria) was based on clinical evaluation and The Diagnostic Interview Schedule for Children IV. All children (n=140) underwent a proton magnetic resonance spectroscopy session to measure the ratio of N-acetyl-aspartate, choline, glutamate, and glutamate–glutamine to creatine, respectively, in the left and right prefrontal and striatal regions of the brain, as well as in the left cerebellum. Results When compared with drug naïve children with ADHD, children with ADHD on stimulants and children typically developed were found to have higher choline ratios in the left prefrontal region (P=0.04) and lower N-acetyl-aspartate ratios in the left striatum region (P=0.01), as well as lower glutamate–glutamine ratios in the left cerebellum (P=0.05). In these three regions, there was no difference between children with ADHD on stimulants and typically developed children. Conclusion Therapeutic psychostimulant effects in children with ADHD may be mediated by normalization of brain metabolite levels, particularly in the left fronto-striato-cerebellar regions. PMID:24476627

Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins.

PubMed

Villers, C; Cacan, R; Mir, A M; Labiau, O; Verbert, A

1994-02-15

The N-glycosylation of proteins is accompanied by the release of soluble oligosaccharide material. Besides oligosaccharide phosphates originating from the cleavage of lipid intermediates, neutral free oligosaccharides represent the major part of this material and are heterogeneous depending on whether the reducing end has one or two N-acetylglucosamine residues. The present study focuses on the intracellular origin of neutral free oligosaccharides in a CHO cell line. Kinetic and pulse-chase experiments clearly indicate that oligosaccharides possessing a chitobiosyl unit are derived from oligosaccharide pyrophosphodolichol, whereas oligosaccharides possessing one N-acetyl-glucosamine residue are derived from newly synthesized glycoprotein. This relationship is confirmed by comparing the glycosylation pattern of lipid donors and glycoproteins with those of neutral free oligosaccharides under various incubation conditions (inhibition of protein synthesis, presence of processing inhibitors, presence or absence of glucose). Degradation of newly synthesized glycoprotein and formation of neutral oligosaccharides with one N-acetylglucosamine residue are inhibited at 16 degrees C but not affected by lysosomotropic agents such as leupeptin or NH4Cl. Together with the fact that the degradation of newly synthesized glycoproteins and the subsequent release of the glycan are recovered in permeabilized cells, these results suggest that this phenomenon occurs in the rough endoplasmic reticulum or in a closely related compartment.

A novel member of the GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans preferentially catalyses the N-acetylation of thialysine [S-(2-aminoethyl)-L-cysteine

PubMed Central

2004-01-01

The putative diamine N-acetyltransferase D2023.4 has been cloned from the model nematode Caenorhabditis elegans. The 483 bp open reading frame of the cDNA encodes a deduced polypeptide of 18.6 kDa. Accordingly, the recombinantly expressed His6-tagged protein forms an enzymically active homodimer with a molecular mass of approx. 44000 Da. The protein belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily, and its amino acid sequence exhibits considerable similarity to mammalian spermidine/spermine-N1-acetyltransferases. However, neither the polyamines spermidine and spermine nor the diamines putrescine and cadaverine were efficiently acetylated by the protein. The smaller diamines diaminopropane and ethylenediamine, as well as L-lysine, represent better substrates, but, surprisingly, the enzyme most efficiently catalyses the N-acetylation of amino acids analogous with L-lysine. As determined by the kcat/Km values, the C. elegans N-acetyltransferase prefers thialysine [S-(2-aminoethyl)-L-cysteine], followed by O-(2-aminoethyl)-L-serine and S-(2-aminoethyl)-D,L-homocysteine. Reversed-phase HPLC and mass spectrometric analyses revealed that N-acetylation of L-lysine and L-thialysine occurs exclusively at the amino moiety of the side chain. Remarkably, heterologous expression of C. elegans N-acetyltransferase D2023.4 in Escherichia coli, which does not possess a homologous gene, results in a pronounced resistance against the anti-metabolite thialysine. Furthermore, C. elegans N-acetyltransferase D2023.4 exhibits the highest homology with a number of GNATs found in numerous genomes from bacteria to mammals that have not been biochemically characterized so far, suggesting a novel group of GNAT enzymes closely related to spermidine/spermine-N1-acetyltransferase, but with a distinct substrate specificity. Taken together, we propose to name the enzyme ‘thialysine Nε-acetyltransferase’. PMID:15283700

Structure and Active Stie Residues of Pg1D, an N-Acetyltransferase from the Bacillosamine Synthetic Pathway Required for N-Glycan Synthesis in Campylobacter jejuni

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

Rangarajan,E.; Ruane, K.; Sulea, T.

2008-01-01

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2, 4-diacetamido-2, 4,6-trideoxy-d-Glc (QuiNAc4NAc or N, N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2, 4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Angstroms resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal {alpha}/{beta}-domain and a C-terminal left-handed {beta}-helix. Few structural differencesmore » accompany CoA binding, except in the C-terminal region following the {beta}-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the {beta}-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an 'L-shape', compared with the 'in-line' orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.« less

Acetylation mediates Cx43 reduction caused by electrical stimulation

PubMed Central

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

2015-01-01

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

Novel Micro/Nano Approaches for Glucose Measurement Using pH-Sensitive Hydrogels

DTIC Science & Technology

2005-06-01

hydrochloride ) (PAH) (polycation). A total of three bilayers of GOx/PAH were assembled, and enzyme activity was confirmed by colorimetric assay...D- glucosamine residues in chitosan can be conjugated with amine-reactive dyes, such as succinimidyl esters (Alexa Fluor 647TM, CY5®), isothiocyanates...were found to be: A 4 94 nm=0.153276 MW=161 [D- glucosamine ]=0.77g/L Sdye=68000cm-M-1 DOL=4.71 x 10-’ Therefore, the labeling ratio of FITC:chitosan

Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

PubMed

Kanabar, V; Page, C P; Simcock, D E; Karner, C; Mahn, K; O'Connor, B J; Hirst, S J

2008-06-01

The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

The folding of acetyl(Ala)28NH2 and acetyl(Ala)40NH2 extended strand peptides into antiparallel β-sheets. A density functional theory study of β-sheets with β-turns.

PubMed

Ali-Torres, Jorge; Dannenberg, J J

2012-12-06

We report ONIOM calculations using B3LYP/D95** and AM1 on β-sheet formation from acetyl(Ala)(N)NH(2) (N = 28 or 40). The sheets contain from one to four β-turns for N = 28 and up to six for N = 40. We have obtained four types of geometrically optimized structures. All contain only β-turns. They differ from each other in the types of β-turns formed. The unsolvated sheets containing two turns are most stable. Aqueous solvation (using the SM5.2 and CPCM methods) reduces the stabilities of the folded structures compared to the extended strands.

Total mortality risk in relation to use of less-common dietary supplements123

PubMed Central

Pocobelli, Gaia; Kristal, Alan R; Patterson, Ruth E; Potter, John D; Lampe, Johanna W; Kolar, Ann; Evans, Ilonka; White, Emily

2010-01-01

Background: Dietary supplement use is common in older US adults; however, data on health risks and benefits are lacking for a number of supplements. Objective: We evaluated whether 10-y average intakes of 13 vitamin and mineral supplements and glucosamine, chondroitin, saw palmetto, Ginko biloba, garlic, fish-oil, and fiber supplements were associated with total mortality. Design: We conducted a prospective cohort study of Washington State residents aged 50–76 y during 2000–2002. Participants (n = 77,719) were followed for mortality for an average of 5 y. Results: A total of 3577 deaths occurred during 387,801 person-years of follow-up. None of the vitamin or mineral 10-y average intakes were associated with total mortality. Among the nonvitamin-nonmineral supplements, only glucosamine and chondroitin were associated with total mortality. The hazard ratio (HR) when persons with a high intake of supplements (≥4 d/wk for ≥3 y) were compared with nonusers was 0.83 (95% CI: 0.72, 0.97; P for trend = 0.009) for glucosamine and 0.83 (95% CI: 0.69, 1.00; P for trend = 0.011) for chondroitin. There was also a suggestion of a decreased risk of total mortality associated with a high intake of fish-oil supplements (HR: 0.83; 95% CI: 0.70, 1.00), but the test for trend was not statistically significant. Conclusions: For most of the supplements we examined, there was no association with total mortality. Use of glucosamine and use of chondroitin were each associated with decreased total mortality. PMID:20410091

Functional modification of chitosan for biomedical application

NASA Astrophysics Data System (ADS)

Tang, Ruogu

Chitosan is a linear polysaccharide. Normally commercial chitosan consists of randomly distributed beta-(1-4)-linked D-glucosamine (deacetylated proportion) and N-acetyl-D-glucosamine (acetylated proportion) together. Chitosan has been proved to be a multifunctional biopolymer that presents several unique properties due to free amino groups in the repeating unit therefore chitosan has been widely applied in various areas. To be specific, provided by the excellent biocompatibility, chitosan is expected to be used in biological and medical applications including wound dressing, implants, drug carrier/delivery, etc. In this thesis, we worked on chitosan functionalization for biomedical application. The thesis are composed of three parts: In the first part, we focused on modifying the chitosan thin film, chemically introducing the nitric oxide functional groups on chitosan film. We covalently bonded small molecule diazeniumdiolates onto the chitosan films and examined the antimicrobial function and biocompatibility. Commercial chitosan was cast into films from acidic aqueous solutions. Glutaraldehyde reacted with the chitosan film to introduce aldehyde groups onto the chitosan film (GA-CS film). GA-CS reacted with a small molecule NO donor, NOC-18, to covalently immobilize NONO groups onto the polymer (NO-CS film). The-CHO and [NONO] group were verified by FT IR, UV and Griess reagent. The NO releasing rate in aqueous solution and and thermal stability were studied quantitatively to prove its effectiveness. A series of antimicrobial tests indicated that NO-CS films have multiple functions: 1. It could inhibit the bacteria growth in nutrient rich environment; 2. It could directly inactivate bacteria and biofilm; 3. It could reduce the bacteria adherence on the film surface as well as inhibit biofilm formation. In addition, the NO-CS film was proved to be biocompatible with cell and it was also compatible with other antibiotics like Amoxicillin. In the second part, we focused on chitosan treatment on titanium surface. We have covalently immobilized chitosan onto titanium (Ti), a widely used implant material, to manage implant-related infection and poor osseointegration that are two of most serious orthopedic implants. The Ti surface was first treated with sulfuric acid and then covalently reacted with chitosan. Surface properties including roughness, contact angle and zeta potential of the samples were markedly increased by the sulfuric acid treatment and the subsequent chitosan immobilization. We have cooperated with the Dr. Ying Deng group's and demonstrated that the chitosan-immobilized Ti showed two novel antimicrobial roles: It prevented the invasion and internalization of bacteria into the osteoblast-like cells; on the other hand, it significantly increased the susceptibility of adherent bacteria to antibiotics. In addition, the SA-Ti and CS-Ti led to a significantly increased osteoblast-likecell attachment, enhanced cell proliferation, and better osteogenic differentiation and mineralization of cells. Chitosan based nanoparticle for drug loading and delivery is also reported in this thesis. By adopting the self-assembly approach, we have prepared alginate/chitosan nanoparticles where the chlorhexidine/cyclodextrin complex is loaded on. The nanoparticles have been proved to be antimicrobial effective and it can bind on cells.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C

PubMed Central

2013-01-01

Background The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. Results Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. Conclusions Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway. PMID:23634833

Activating and Attenuating the Amicoumacin Antibiotics.

PubMed

Park, Hyun Bong; Perez, Corey E; Perry, Elena Kim; Crawford, Jason M

2016-06-24

The amicoumacins belong to a class of dihydroisocoumarin natural products and display antibacterial, antifungal, anticancer, and anti-inflammatory activities. Amicoumacins are the pro-drug activation products of a bacterial nonribosomal peptide-polyketide hybrid biosynthetic pathway and have been isolated from Gram-positive Bacillus and Nocardia species. Here, we report the stimulation of a "cryptic" amicoumacin pathway in the entomopathogenic Gram-negative bacterium Xenorhabdus bovienii, a strain not previously known to produce amicoumacins. X. bovienii participates in a multi-lateral symbiosis where it is pathogenic to insects and mutualistic to its Steinernema nematode host. Waxmoth larvae are common prey of the X. bovienii-Steinernema pair. Employing a medium designed to mimic the amino acid content of the waxmoth circulatory fluid led to the detection and characterization of amicoumacins in X. bovienii. The chemical structures of the amicoumacins were supported by 2D-NMR, HR-ESI-QTOF-MS, tandem MS, and polarimeter spectral data. A comparative gene cluster analysis of the identified X. bovienii amicoumacin pathway to that of the Bacillus subtilis amicoumacin pathway and the structurally-related Xenorhabdus nematophila xenocoumacin pathway is presented. The X. bovienii pathway encodes an acetyltransferase not found in the other reported pathways, which leads to a series of N-acetyl-amicoumacins that lack antibacterial activity. N-acetylation of amicoumacin was validated through in vitro protein biochemical studies, and the impact of N-acylation on amicoumacin's mode of action was examined through ribosomal structural analyses.

Absence of preserved glucosamine and amino acids in fossil crustacean exoskeletons

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

Schimmelmann, A.; Krause, R.G.F.; DeNiro, M.J.

1988-01-01

No glucosamine and only traces of amino acids were detected in kerogen prepared from fossil crustacean exoskeletons. The elemental C/N ratios of the kerogen samples were above 20, indicating that most of the organic nitrogen was eliminated from the chitin biopolymer during diagenesis. The results contradict earlier reports of the stability of chitin during fossilization.

Validated high-performance anion-exchange chromatography with pulsed amperometric detection method for the determination of residual keratan sulfate and other glucosamine impurities in sodium chondroitin sulfate.

PubMed

Bottelli, Susanna; Grillo, Gianluca; Barindelli, Edoardo; Nencioni, Alessandro; Di Maria, Alessandro; Fossati, Tiziano

2017-07-07

An efficient and sensitive analytical method based on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was devised for the determination of glucosamine (GlcN) in sodium chondroitin sulfate (CS). Glucosamine (GlcN) is intended as marker of residual keratan sulfate (KS) and other impurities generating glucosamine by acidic hydrolyzation. The latter brings CS and KS to their respective monomers. Since GlcN is present only in KS we developed a method that separates GlcN from GalN, the principal hydrolytic product of CS, and then we validated it in order to quantify GlcN. Method validation was performed by spiking CS raw material with known amounts of KS. Detection limit was 0.5% of KS in CS (corresponding to 0.1μg/ml), and the linear range was 0.5-5% of KS in CS (corresponding to 0.1-1μg/ml). The optimized analysis was carried out on an ICS-5000 system (Dionex, Sunnyvale, CA, USA) equipped with a Dionex Amino Trap guard column (3mm×30mm), Dionex CarboPac-PA20 (3mm×30mm) and a Dionex CarboPac-PA20 analytical column (3mm×150mm) using gradient elution at a 0.5ml/min flow rate. Regression equations revealed good linear relationship (R 2 =0.99, n=5) within the test ranges. Quality parameters, including precision and accuracy, were fully validated and found to be satisfactory. The fully validated HPAEC-PAD method was readily applied for the quantification of residual KS in CS in several raw materials and USP/EP reference substance. Results confirmed that the HPAEC-PAD method is more specific than the electrophoretic method for related substance reported in EP and provides sensitive determination of KS in acid-hydrolyzed CS samples, enabling the quantitation of KS and other impurities (generating glucosamine) in CS. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a simple analytical methodology for determination of glucosamine release from modified release matrix tablets.

PubMed

Wu, Yunqi; Hussain, Munir; Fassihi, Reza

2005-06-15

A simple spectrophotometric method for determination of glucosamine release from sustained release (SR) hydrophilic matrix tablet based on reaction with ninhydrin is developed, optimized and validated. The purple color (Ruhemann purple) resulted from the reaction was stabilized and measured at 570 nm. The method optimization was essential as many procedural parameters influenced the accuracy of determination including the ninhydrin concentration, reaction time, pH, reaction temperature, purple color stability period, and glucosamine/ninhydrin ratio. Glucosamine tablets (600 mg) with different hydrophilic polymers were formulated and manufactured on a rotary press. Dissolution studies were conducted (USP 26) using deionized water at 37+/-0.2 degrees C with paddle rotation of 50 rpm, and samples were removed manually at appropriate time intervals. Under given optimized reaction conditions that appeared to be critical, glucosamine was quantitatively analyzed and the calibration curve in the range of 0.202-2.020 mg (r=0.9999) was constructed. The recovery rate of the developed method was 97.8-101.7% (n=6). Reproducible dissolution profiles were achieved from the dissolution studies performed on different glucosamine tablets. The developed method is easy to use, accurate and highly cost-effective for routine studies relative to HPLC and other techniques.

The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

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

Van Bueren, A.; Ghinet, M; Gregg, K

2009-01-01

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

The Structural Basis of Substrate Recognition in an exo-beta-d-Glucosaminidase Involved in Chitosan Hydrolysis

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

Lammerts van Bueren, A.; Ghinet, M; Gregg, K

2009-01-01

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

Protein-Ligand Interaction Detection with a Novel Method of Transient Induced Molecular Electronic Spectroscopy (TIMES): Experimental and Theoretical Studies.

PubMed

Zhang, Tiantian; Wei, Tao; Han, Yuanyuan; Ma, Heng; Samieegohar, Mohammadreza; Chen, Ping-Wei; Lian, Ian; Lo, Yu-Hwa

2016-11-23

Protein-ligand interaction detection without disturbances (e.g., surface immobilization, fluorescent labeling, and crystallization) presents a key question in protein chemistry and drug discovery. The emergent technology of transient induced molecular electronic spectroscopy (TIMES), which incorporates a unique design of microfluidic platform and integrated sensing electrodes, is designed to operate in a label-free and immobilization-free manner to provide crucial information for protein-ligand interactions in relevant physiological conditions. Through experiments and theoretical simulations, we demonstrate that the TIMES technique actually detects protein-ligand binding through signals generated by surface electric polarization. The accuracy and sensitivity of experiments were demonstrated by precise measurements of dissociation constant of lysozyme and N -acetyl-d-glucosamine (NAG) ligand and its trimer, NAG 3 . Computational fluid dynamics (CFD) computation is performed to demonstrate that the surface's electric polarization signal originates from the induced image charges during the transition state of surface mass transport, which is governed by the overall effects of protein concentration, hydraulic forces, and surface fouling due to protein adsorption. Hybrid atomistic molecular dynamics (MD) simulations and free energy computation show that ligand binding affects lysozyme structure and stability, producing different adsorption orientation and surface polarization to give the characteristic TIMES signals. Although the current work is focused on protein-ligand interactions, the TIMES method is a general technique that can be applied to study signals from reactions between many kinds of molecules.

Effects of glucosamine and chondroitin on treating knee osteoarthritis: an analysis with marginal structural models

PubMed Central

Yang, Shibing; Eaton, Charles B.; McAlindon, Timothy E.; Lapane, Kate L.

2014-01-01

Objective The purpose of this study was to estimate the effectiveness of glucosamine and chondroitin in relieving knee symptoms and slowing disease progression among patients with knee osteoarthritis (OA). Methods The 4-year follow-up data from Osteoarthritis Initiative were analyzed. We used a “new-user” design, for which only participants who were not using glucosamine/chondroitin at baseline were included in analyses (n=1,625). Cumulative exposure was calculated as the number of visits when participants reported use of glucosamine/chondroitin. Knee symptoms were measured with WOMAC scale and structural progression was measured with joint space width (JSW). To control for the time-varying confounders that might be influenced by prior treatments, we used marginal structural models to estimate the effects of using glucosamine/chondroitin for three years, two years and one year on treating OA. Results During the study period, 18% of the participants initiated treatment with glucosamine/chondroitin. After adjustment for potential confounders with marginal structural models, we found no clinically significant differences between users at all assessments and never-users of glucosamine/chondroitin in WOMAC Pain: 0.68 (95% CI: -0.16 to 1.53); WOMAC Stiffness: 0.41 (95% CI: 0 to 0.82); WOMAC Function: 1.28 (95% CI: -1.23 to 3.79); or JSW: 0.11 (95% CI: -0.21 to 0.44). Conclusions Use of glucosamine/chondroitin did not appear to relieve symptoms or modify disease progression among patients with radiographically confirmed OA. Our findings, which are consistent with meta-analyses of clinical trials, extend the results to a more general population with knee OA. PMID:25369761

Glucosamine enhances body weight gain and reduces insulin response in mice fed chow diet but mitigates obesity, insulin resistance and impaired glucose tolerance in mice high-fat diet.

PubMed

Hwang, Ji-Sun; Park, Ji-Won; Nam, Moon-Suk; Cho, Hyeongjin; Han, Inn-Oc

2015-03-01

This study investigated the potential of glucosamine (GlcN) to affect body weight gain and insulin sensitivity in mice normal and at risk for developing diabetes. Male C57BL/6J mice were fed either chow diet (CD) or a high fat diet (HFD) and the half of mice from CD and HFD provided with a solution of 10% (w/v) GlcN. Total cholesterol and nonesterified free fatty acid levels were determined. Glucose tolerance test and insulin tolerance test were performed. HepG2 human hepatoma cells or differentiated 3T3-L1 adipocytes were stimulated with insulin under normal (5 mM) or high glucose (25 mM) conditions. Effect of GlcN on 2-deoxyglucose (2-DG) uptake was determined. JNK and Akt phosphorylation and nucleocytoplasmic protein O-GlcNAcylation were assayed by Western blotting. GlcN administration stimulated body weight gain (6.58±0.82 g vs. 11.1±0.42 g), increased white adipose tissue fat mass (percentage of bodyweight, 3.7±0.32 g vs. 5.61±0.34 g), and impaired the insulin response in livers of mice fed CD. However, GlcN treatment in mice fed HFD led to reduction of body weight gain (18.02±0.66 g vs. 16.22±0.96 g) and liver weight (2.27±0.1 vs. 1.85±0.12 g). Furthermore, obesity-induced insulin resistance and impaired Akt insulin signaling in the liver were alleviated by GlcN administration. GlcN inhibited the insulin response under low (5 mM) glucose conditions, whereas it restored the insulin response for Akt phosphorylation under high (25 mM) glucose conditions in HepG2 and 3T3-L1 cells. Uptake of 2-DG increased upon GlcN treatment under 5 mM glucose compared to control, whereas insulin-stimulated 2-DG uptake decreased under 5 mM and increased under 25 mM glucose in differentiated 3T3-L1 cells. Our results show that GlcN increased body weight gain and reduced the insulin response for glucose maintenance when fed to normal CD mice, whereas it alleviated body weight gain and insulin resistance in HFD mice. Therefore, the current data support the integrative function of the HBP reflecting the nutrient status of lipids or glucose and further implicate the importance of the pathway in insulin signaling for the regulation of metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

Low-dose D-methionine and N-acetyl-L-cysteine for protection from permanent noise-induced hearing loss in chinchillas.

PubMed

Clifford, Royce E; Coleman, John K M; Balough, Ben J; Liu, Jianzhong; Kopke, Richard D; Jackson, Ronald L

2011-12-01

Despite efforts at public health awareness and stringent industrial standards for hearing protection, noise-induced hearing loss (NIHL) remains a formidable public health concern. Although many antioxidants have proven to be beneficial in the laboratory for prevention of permanent NIHL, low-dose combinations of compounds with different biochemical mechanisms of action may allow long-term administration with fewer side effects and equal efficacy. The mixture of D-methionine and N-acetyl-L-cysteine administered at levels less than 10% of standard dosing has not been previously reported. Twenty-six female adult Chinchilla laniger were placed in 4 study groups, consisting of (1) a group receiving combination 12.5 mg/kg each D-methionine and N-acetyl-L-cysteine (DMET/NAC group), (2) a group receiving 12.5 mg/kg D-methionine (DMET-only group), (3) a group receiving 12.5 mg/kg N-acetyl-L-cysteine (NAC-only group), and (4) saline controls. Laboratory. All groups received twice-daily intraperitoneal injections 2 days prior to noise exposure, 1 hour before and after exposure on day 3, and for 2 days subsequently, totaling 10 doses of 125 mg/kg for each antioxidant over 5 days. Although NAC-only animals paralleled saline control recovery during 3 weeks, the DMET-only group revealed gradual improvement with statistically significant recovery in the middle frequencies. The DMET/NAC group showed significant improvement at most frequencies compared with controls (P < .001 and P < .05). Significant recovery of hearing was observed following continuous noise exposure with either DMET only or a combination of low-dose DMET/NAC, demonstrating a considerably lower dose of antioxidants required than previously reported for hearing recovery following acoustic trauma.

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

PubMed

Jean, M; Smaoui, F; Lavertu, M; Méthot, S; Bouhdoud, L; Buschmann, M D; Merzouki, A

2009-09-01

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Tumor-targeted polymeric nanostructured lipid carriers with precise ratiometric control over dual-drug loading for combination therapy in non-small-cell lung cancer.

PubMed

Liang, Yan; Tian, Baocheng; Zhang, Jing; Li, Keke; Wang, Lele; Han, Jingtian; Wu, Zimei

2017-01-01

Gemcitabine (GEM) and paclitaxel (PTX) are effective combination anticancer agents against non-small-cell lung cancer (NSCLC). At the present time, a main challenge of combination treatment is the precision of control that will maximize the combined effects. Here, we report a novel method to load GEM (hydrophilic) and PTX (hydrophobic) into simplex tumor-targeted nanostructured lipid carriers (NLCs) for accurate control of the ratio of the two drugs. We covalently preconjugated the dual drugs through a hydrolyzable ester linker to form drug conjugates. N -acetyl-d-glucosamine (NAG) is a glucose receptor-targeting ligand. We added NAG to the formation of NAG-NLCs. In general, synthesis of poly(6- O -methacryloyl-d-galactopyranose)-GEM/PTX (PMAGP-GEM/PTX) conjugates was demonstrated, and NAG-NLCs were prepared using emulsification and solvent evaporation. NAG-NLCs displayed sphericity with an average diameter of 120.3±1.3 nm, a low polydispersity index of 0.233±0.04, and accurate ratiometric control over the two drugs. A cytotoxicity assay showed that the NAG-NLCs had better antitumor activity on NSCLC cells than normal cells. There was an optimal ratio of the two drugs, exhibiting the best cytotoxicity and combinatorial effects among all the formulations we tested. In comparison with both the free-drug combinations and separately nanopackaged drug conjugates, PMAGP-GEM/PTX NAG-NLCs (3:1) exhibited superior synergism. Flow cytometry and confocal laser scanning microscopy showed that NAG-NLCs exhibited higher uptake efficiency in A549 cells via glucose receptor-mediated endocytosis. This combinatorial delivery system settles problems with ratiometric coloading of hydrophilic and hydrophobic drugs for tumor-targeted combination therapy to achieve maximal anticancer efficacy in NSCLC.

D-Glucosamine Conjugation Accelerates the Labeling Efficiency of Quantum Dots in Osteoblastic Cells

PubMed Central

Xie, Ming-Fang

2014-01-01

Quantum dots (QDs) are useful imaging tools in the medical and biological fields due to their optical properties, such as a high fluorescence intensity, remarkable resistance to photobleaching, broad absorption spectra, and narrow emission spectra. This is the first study to investigate the uptake of carboxylated QDs conjugated with D-glucosamine (core size: approximately 3 nm, final modified size: 20–30 nm) into cultured osteoblastic cells. The QDs attached to the cell surface and were transported into the cytoplasm within approximately three hours of culture, whose process was clearly demonstrated using specific fluorescent staining of the cell membrane. Although the intranuclear distribution was not observed, a dramatic decrease in the transfer of quantum dots into the cytoplasm was recognized after approximately seven days of culture. Other interesting phenomena include the escape of the quantum dots from lysosomes in the cytoplasm, as confirmed by the merging of both QD fluorescence and specific fluorescent staining of lysosomes in the cytoplasm. These findings suggest that D-glucosamine conjugation enhances proton absorption in acid organelles and promotes the lysosomal escape of QDs. PMID:24818156

Physiological role of D-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of D-amino acids.

PubMed

Yow, Geok-Yong; Uo, Takuma; Yoshimura, Tohru; Esaki, Nobuyoshi

2006-03-01

Saccharomyces cerevisiae is sensitive to D-amino acids: those corresponding to almost all proteinous L-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that D-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of D-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to D-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to D-amino acids than the wild type. We further confirmed that, upon cultivation with D-phenylalanine, N-acetyl-D-phenylalanine was accumulated in the culture but not in the wild type and hpa3Delta cells overproducing DNT cells. Thus, D-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.

Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications.

PubMed

Wang, Limin; Detamore, Michael S

2009-01-01

Temporomandibular joint (TMJ) condylar cartilage is a distinct cartilage that has both fibrocartilaginous and hyaline-like character, with a thin proliferative zone that separates the fibrocartilaginous fibrous zone at the surface from the hyaline-like mature and hypertrophic zones below. In this study, we compared the effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), and glucosamine sulphate on porcine TMJ condylar cartilage and ankle cartilage cells in monolayer culture. In general, TMJ condylar cartilage cells proliferated faster than ankle cartilage cells, while ankle cells produced significantly greater amounts of glycosaminoglycans (GAGs) and collagen than TMJ condylar cartilage cells. IGF-I and bFGF were potent stimulators of TMJ cell proliferation, while no signals statistically outperformed controls for ankle cell proliferation. IGF-I was the most effective signal for GAG production with ankle cells, and the most potent upregulator of collagen synthesis for both cell types. Glucosamine sulphate promoted cell proliferation and biosynthesis at specific concentrations and outperformed growth factors in certain instances. In conclusion, hyaline cartilage cells had lower cell numbers and superior biosynthesis compared to TMJ condylar cartilage cells, and we have found IGF-I at 100 ng/mL and glucosamine sulphate at 100 microg/mL to be the most effective signals for these cells under the prescribed conditions.

Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

PubMed

Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

2016-01-01

Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC 50 >1 mM) on the activities of five major isoforms of human CYP in vitro.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

PubMed

Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

2018-05-05

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases.

PubMed

Decker, Daniel; Kleczkowski, Leszek A

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP- N -acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P ( K m values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P ( K m of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P ( K m of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P ( K m of 1 mM) and, to some extent, D-Glc-1-P ( K m of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

PubMed Central

Decker, Daniel; Kleczkowski, Leszek A.

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products. PMID:28970843

Separation and purification of four flavonol diglucosides from the flower of Meconopsis integrifolia by high-speed counter-current chromatography.

PubMed

Huang, Yanfei; Han, Yatao; Chen, Keli; Huang, Bisheng; Liu, Yuan

2015-12-01

Flavonoids are the main components of Meconopsis integrifolia (Maxim.) Franch, which is a traditional Tibetan medicine. However, traditional chromatography separation requires a large quantity of raw M. integrifolia and is very time consuming. Herein, we applied high-speed counter-current chromatography in the separation and purification of flavonoids from the ethanol extracts of M. integrifolia flower. Ethyl acetate/n-butanol/water (2:3:5, v/v/v) was selected as the optimum solvent system to purify the four components, namely quercetin-3-O-β-d-glucopyrannosy-(1→6)-β-d-glucopyranoside (compound 1, 60 mg), quercetin 3-O-[2'''-O-acetyl-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (compound 2, 40 mg), quercetin 3-O-[3'''-O-acetyl-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (compound 3, 11 mg), and quercetin 3-O-[6'''-O-acetyl-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (compound 4, 16 mg). Among the four compounds, 3 and 4 were new acetylated flavonol diglucosides. After the high-speed counter-current chromatography separation, the purities of the four flavonol diglucosides were 98, 95, 90, and 92%, respectively. The structures of these compounds were identified by mass spectrometry and NMR spectroscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sexual asthenia: Tradamixina versus Tadalafil 5 mg daily

PubMed Central

2012-01-01

Background Reduced libido is widely considered the most prominent symptomatic reflection of low testosterone (T) levels in men. Testosterone deficiency (TD) afflicts approximately 30% of men aged 40-79 years. This study seeks to evaluate the effect of a new natural compound “tradamixina “in order to improve male sexual function in elderly men, particularly libido and possible erectile dysfunction, versus administration of tadalafil 5 mg daily. Methods Seventy patients (67.3± 3.7 years) with stable marital relations and affected by reduced libido, with or without erectile dysfunction were recruited. They were randomly separated in 2 groups A-B of 35. Group A was administered twice a day a new compound “Tradamixina” (150 mg of Alga Ecklonia Bicyclis, 396 mg of Tribulus Terrestris and 144 mg of D-Glucosamine and N-Acetyl-D-Glucosamine) for two months, while Group B was administered tadalafil 5 mg daily, for two months. At visit and after 60 days of treatment patients were evaluated by means of detailed medical and sexual history, clinical examination, laboratory investigations (Total and Free T), instrumental examination (NPTR- nocturnal penile tumescence and rigidity test- with Rigiscan). Patients completed a self-administered IIEF questionnaire (The international index of erectile function) and SQoLM questionnaire (Sexual quality of life Questionnarie-Male). The results pre and post treatment were compared by Student t test (p<0.005). Results After 2 months of treatment in group A serum TT levels (230±18 ng/dl vs 671±14 ng/dl ) and FT levels(56± 2.4 pg/ml vs 120± 3.9pg/ml) increased, while in group B serum TT levels (245±12 ng/dl vs 247±15 ng/dl ) and FT levels(53± 0.3 pg/ml vs 55± 0.5pg/ml) increased not statistically significant. The patient’s numbers with negative NPTR improved after treatment in group A and B (15 vs 18 and 13 vs 25 respectively). The IIEF total score in group A increased after treatment with tradamixina (15±1.5 vs 29.77±1.2); the IIEF total score in group B increased slightly (12±1.3 vs 23.40±1.2). The SQoLM total score improved in both groups (A:16±2,3 vs 33±4,1 and B: 16±3,4 vs 31±2,1). Conclusion The treatment twice a day with “Tradamixina” for 2 months improved libido in elderly men without side effects of Tadalafil. PMID:23173697

A myosin II ATPase inhibitor reduces force production, glucose transport, and phosphorylation of AMPK and TBC1D1 in electrically stimulated rat skeletal muscle.

PubMed

Blair, David R; Funai, Katsuhiko; Schweitzer, George G; Cartee, Gregory D

2009-05-01

Contraction-stimulated glucose transport by skeletal muscle appears to be caused by the cumulative effects of multiple inputs [potentially including AMP-activated protein kinase (AMPK), Ca(2+) flux, and force production], making it challenging to isolate the roles of these putative regulatory factors. To distinguish the effects of force production from the direct consequences of Ca(2+) flux, the predominantly type II rat epitrochlearis muscle was incubated without (vehicle) or with N-benzyl-p-toluenesulfonamide (BTS), a highly specific myosin II ATPase inhibitor that prevents force production by electrically stimulated (ES) type II fibers without altering cytosolic Ca(2+). In ES muscles, BTS vs. vehicle had an 84% reduction in force production and a 57% decrement in contraction-stimulated 3-O-methylglucose transport (3MGT). BTS did not alter the ES increase in phosphorylation of CaMKII (indicative of cytosolic Ca(2+)) or the amount of glycogen depletion. ES caused significant reductions in ATP (48%) and phosphocreatine (67%) concentrations for vehicle-treated muscles. For BTS-treated muscles, ES did not reduce ATP and caused only a 42% decrease in phosphocreatine. There was an ES increase in phosphorylation of AMPK, acetyl-CoA carboxylase (an AMPK substrate), and TBC1D1 for vehicle-treated muscles but not for BTS-treated muscles. These results point toward an essential role for tension-related events, including AMPK activation, in the 57% contraction-stimulated increase in 3MGT that was inhibited by BTS and further suggest a possible role for TBC1D1 phosphorylation. Non-tension-related events (e.g., increased cytosolic Ca(2+) rather than increased AMPK and TBC1D1 phosphorylation) are implicated in the contraction-stimulated increase in 3MGT that persisted in the presence of BTS.

Influence of Biochemical Features of Burkholderia pseudomallei Strains on Identification Reliability by Vitek 2 System.

PubMed

Zakharova, Irina B; Lopasteyskaya, Yana A; Toporkov, Andrey V; Viktorov, Dmitry V

2018-01-01

Burkholderia pseudomallei is a Gram-negative saprophytic soil bacterium that causes melioidosis, a potentially fatal disease endemic in wet tropical areas. The currently available biochemical identification systems can misidentify some strains of B. pseudomallei . The aim of the present study was to identify the biochemical features of B. pseudomallei , which can affect its correct identification by Vitek 2 system. The biochemical patterns of 40 B. pseudomallei strains were obtained using Vitek 2 GN cards. The average contribution of biochemical tests in overall dissimilarities between correctly and incorrectly identified strains was assessed using nonmetric multidimensional scaling. It was found ( R statistic of 0.836, P = 0.001) that a combination of negative N-acetyl galactosaminidase, β-N-acetyl glucosaminidase, phosphatase, and positive D-cellobiase (dCEL), tyrosine arylamidase (TyrA), and L-proline arylamidase (ProA) tests leads to low discrimination of B. pseudomallei , whereas a set of positive dCEL and negative N-acetyl galactosaminidase, TyrA, and ProA determines the wrong identification of B. pseudomallei as Burkholderia cepacia complex. The further expansion of the Vitek 2 identification keys is needed for correct identification of atypical or regionally distributed biochemical profiles of B. pseudomallei .

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

NASA Astrophysics Data System (ADS)

Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

2015-11-01

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Management of hyperpigmentation in darker racial ethnic groups.

PubMed

Grimes, Pearl E

2009-06-01

Dyschromias, in particular hyperpigmentation, are major issues of concern for people of color. Pigmentary disorders such as melasma and postinflammatory hyperpigmentation (PIH) can cause psychological and emotional distress and can pose a negative impact on a person's health-related quality of life. The precise etiology of these conditions is unknown. Therapies for melasma and PIH target various points during the cycle of melanin production and degradation. Therapies for these conditions include topical agents and resurfacing procedures. Hydroquinone remains the gold standard of topical agents. Other efficacious agents include kojic acid, azelaic acid, mequinol, and retinoids. Cosmeceutical agents include licorice, arbutin, soy, N-acetyl glucosamine, and niacinamide. Resurfacing procedures that have been used to treat melasma and PIH include chemical peels, microdermabrasion, lasers, and intense pulsed light. These procedures are best used in combination with topical bleaching agents. Given the propensity of darker skin to hyperpigment, resurfacing procedures should be used with care and caution. Maximal results are best achieved with repetitive, superficial, resurfacing modalities. In addition, ultraviolet protective measures such as broad-spectrum sunscreens are fundamental to the successful management of these conditions.

Postinflammatory hyperpigmentation: a review of the epidemiology, clinical features, and treatment options in skin of color.

PubMed

Davis, Erica C; Callender, Valerie D

2010-07-01

Postinflammatory hyperpigmentation is a common sequelae of inflammatory dermatoses that tends to affect darker skinned patients with greater frequency and severity. Epidemiological studies show that dyschromias, including postinflammatory hyperpigmentation, are among the most common reasons darker racial/ethnic groups seek the care of a dermatologist. The treatment of postinflammatory hyperpigmentation should be started early to help hasten its resolution and begins with management of the initial inflammatory condition. First-line therapy typically consists of topical depigmenting agents in addition to photoprotection including a sunscreen. Topical tyrosinase inhibitors, such as hydroquinone, azelaic acid, kojic acid, arbutin, and certain licorice extracts, can effectively lighten areas of hypermelanosis. Other depigmenting agents include retinoids, mequinol, ascorbic acid, niacinamide, N-acetyl glucosamine, and soy with a number of emerging therapies on the horizon. Topical therapy is typically effective for epidermal postinflammatory hyperpigmentation; however, certain procedures, such as chemical peeling and laser therapy, may help treat recalcitrant hyperpigmentation. It is also important to use caution with all of the above treatments to prevent irritation and worsening of postinflammatory hyperpigmentation.

Determination of Glucosamine in Raw Materials and Dietary Supplements Containing Glucosamine Sulfate and/or Glucosamine Hydrochloride by High-Performance Liquid Chromatography with FMOC-Su Derivatization: Collaborative Study

PubMed Central

Zhou, Joseph Ziqi; Waszkuc, Ted; Mohammed, Felicia

2008-01-01

A collaborative study was conducted for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by high-performance liquid Chromatography (HPLC) with N-(9-fluorenyl-methoxycarbonyloxy) succinimide (FMOC-Su) derivatization. Thirteen blind materials, one pair of which were duplicates, were tested by 12 collaborating laboratories. The test samples consisted of various commercial products, including tablets, capsules, drink mix, and liquids as well as raw materials, blanks, and those for spike recovery analyses. The tests with blank products and products spiked with glucosamine showed good specificity of the method. The average recoveries at spike levels of 100 and 150% of the declared amount were 99.0% with a relative standard deviation (RSD) of 2.1%, and 101% with an RSD of 2.3%, respectively. The test results between laboratories on each commercial product were reproducible with RSD values of no more than 4.0%, and the results were repeatable in the same laboratory with an average RSD of 0.7%. HorRat values ranged from 0.5 to 1.7 on both tests of spike recovery and reproducibility between laboratories on commercial products. The average determination coefficient of the calibration curves from the laboratories was 0.9995 with an RSD of 0.03%. All of the 12 collaborating laboratories succeeded in the study and none of their reported test results were outliers, partly indicating the robustness of the method. It is recommended that the method be accepted by AOAC INTERNATIONAL as Official First Action. PMID:16152919

Comparison of glucosamine sulfate and a polyherbal supplement for the relief of osteoarthritis of the knee: a randomized controlled trial [ISRCTN25438351

PubMed Central

Mehta, Komal; Gala, Jayesh; Bhasale, Surendra; Naik, Sattayasheel; Modak, Millind; Thakur, Harshad; Deo, Nivedita; Miller, Mark JS

2007-01-01

Background The efficacy and safety of a dietary supplement derived from South American botanicals was compared to glucosamine sulfate in osteoarthritis subjects in a Mumbai-based multi-center, randomized, double-blind study. Methods Subjects (n = 95) were screened and randomized to receive glucosamine sulfate (n = 47, 1500 mg/day) or reparagen (n = 48, 1800 mg/day), a polyherbal consisting of 300 mg of vincaria (Uncaria guianensis) and 1500 mg of RNI 249 (Lepidium meyenii) administered orally, twice daily. Primary efficacy variable was response rate based on a 20% improvement in WOMAC pain scores. Additional outcomes were WOMAC scores for pain, stiffness and function, visual analog score (VAS) for pain, with assessments at 1, 2, 4, 6 and 8 weeks. Tolerability, investigator and subject global assessments and rescue medication consumption (paracetamol) were measured together with safety assessments including vital signs and laboratory based assays. Results Subject randomization was effective: age, gender and disease status distribution was similar in both groups. The response rates (20% reduction in WOMAC pain) were substantial for both glucosamine (89%) and reparagen (94%) and supported by investigator and subject assessments. Using related criteria response rates to reparagen were favorable when compared to glucosamine. Compared to baseline both treatments showed significant benefits in WOMAC and VAS outcomes within one week (P < 0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (45–62% reduction in WOMAC or VAS scores). Tolerability was excellent, no serious adverse events were noted and safety parameters were unchanged. Rescue medication use was significantly lower in the reparagen group (p < 0.01) at each assessment period. Serum IGF-1 levels were unaltered by treatments. Conclusion Both reparagen and glucosamine sulfate produced substantial improvements in pain, stiffness and function in subjects with osteoarthritis. Response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. Reparagen represents a new natural productive alternative in the management of joint health. Trial registration Current Controlled Trials ISRCTN25438351. PMID:17974032

The Folding of Acetyl(Ala)28NH2 and Acetyl(Ala)40NH2 Extended Strand Peptides into Antiparallel β-Sheets. A Density Functional Theory Study of β-Sheets with β-Turns

PubMed Central

Ali-Torres, Jorge

2012-01-01

We report ONIOM calculations using B3LYP/D95** and AM1 on β-sheet formation from acetyl(Ala)NNH2 (N=28 or 40). The sheets contain from one to four β-turns for N=28 and up to six for N=40. We have obtained four types of geometrically optimized structures. All contain only β-turns. They differ from each other in the types of β-turns formed. The unsolvated sheets containing two turns are most stable. Aqueous solvation (using the SM5.2 and CPCM methods) reduces the stabilities of the folded structures compared to the extended strands. PMID:23157432

Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth.

PubMed

Carabetta, Valerie J; Greco, Todd M; Tanner, Andrew W; Cristea, Ileana M; Dubnau, David

2016-05-01

N ε -Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. The past decade highlighted N ε -lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis . To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.

Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery.

PubMed

Woodman, Jessica L; Suh, Min Sung; Zhang, Jianxing; Kondaveeti, Yuvabharath; Burgess, Diane J; White, Bruce A; Prestwich, Glenn D; Kuhn, Liisa T

2015-01-01

Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44(+)/CD24(-/low) human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

Esterification of all four monoribonucleotides with acetyl-D-L-valine proceeds with a preference for the D-isomer but the D/L ratio in the products declines as a function of the hydrophobicity of the nucleotide

NASA Technical Reports Server (NTRS)

Wickramasinghe, N. S.; Lacey, J. C. Jr; Lacey JC, J. r. (Principal Investigator)

1992-01-01

We recently reported that esterification of 5'-AMP with N-acetyl amino acids proceeds with a preference for D-amino acids, and the D/L ratio in products declines as the hydrophobicity of the amino acid declines. Using one amino acid, Ac-Val, we now show that esterification of all four nucleotides proceeds with a preference for the D-isomer and the preference declines as the hydrophobicity of the nucleotide declines. So, in both types of experiments, the preferences seem determined by hydrophobic interactions.

Enzymatic synthesis of novel quercetin sialyllactoside derivatives.

PubMed

Darsandhari, Sumangala; Bae, Jae Yoon; Shrestha, Biplav; Yamaguchi, Tokutaro; Jung, Hye Jin; Han, Jang Mi; Rha, Chan-Su; Pandey, Ramesh Prasad; Sohng, Jae Kyung

2018-06-06

Quercetin and its derivatives are important flavonols that show diverse biological activity, such as antioxidant, anticarcinogenic, anti-inflammatory, and antiviral activities. Adding different substituents to quercetin may change the biochemical activity and bioavailability of molecules, when compared to the aglycone. Here, we have synthesised two novel derivatives of quercetin, quercetin-3-O-β-d-glucopyranosyl, 4''-O-d-galactopyranosyl 3'''-O-α-N-acetyl neuraminic acid i.e. 3'-sialyllactosyl quercetin (3'SL-Q) and quercetin-3-O-β-d-glucopyranosyl, 4''-O-β-d-galactopyranosyl 6'''-O-α-N-acetyl neuraminic acid i.e. 6'-sialyllactosyl quercetin (6'SL-Q) with the use of glycosyltransferases and sialyltransferases enzymes. These derivatives of quercetin were characterised by high-resolution quadrupole-time-of-flight electrospray ionisation mass spectrometry (HR-QTOF-ESI/MS) and 1 H and 13 C nuclear magnetic resonance (NMR) analyses.

Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

PubMed

Dempsey, Daniel R; Jeffries, Kristen A; Handa, Sumit; Carpenter, Anne-Marie; Rodriguez-Ospina, Santiago; Breydo, Leonid; Merkler, David J

2015-04-28

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

PubMed

Hadfield, A T; Harvey, D J; Archer, D B; MacKenzie, D A; Jeenes, D J; Radford, S E; Lowe, G; Dobson, C M; Johnson, L N

1994-11-11

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

PubMed Central

2015-01-01

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

[Distribution of acetylator phenotypes in the normal Moscow city population and in chronic alcoholism].

PubMed

Lil'in, E T; Korsunskaia, M P; Meksin, V A; Drozdov, E S; Nazarov, V V

1984-09-01

The distribution of acetylator phenotypes was studied in 169 normal individuals of Moscow Russian population and 75 inhabitants of Moscow suffering from chronic alcoholism. Polymorphism was found by means of acetylation in both groups studied. The proportion of repeatability of rapid and slow acetylators amounts to 48 and 52% among normal individuals, 44 and 56% among those who suffer from chronic alcoholism. The comparative analyses of such repeatability within the classes resulted in authentic increase of the rate of rapid acetylators among the chronic alcoholics (chi 2 = 18.32; p less than 0.01); in comparison with normal individual groups, (the modes being in classes 50-60% and 80-90%, with the antimode 70-80%), a shift of one of the modes from the 50-60% class into the 60-70% class was traced among diseased individuals. It is supposed that chronic alcohol consumption stimulates the process of acetylation; possible reasons for this stimulation are discussed.

Immunomodulatory effects of an acetylated Cyclocarya paliurus polysaccharide on murine macrophages RAW264.7.

PubMed

Liu, Xin; Xie, Jianhua; Jia, Shuo; Huang, Lixin; Wang, Zhijun; Li, Chang; Xie, Mingyong

2017-05-01

Polysaccharides (CP) extracted from the leaves of Cyclocarya paliurus (C. paliurus) have been shown to possess a variety of biological activities. In present study, CP was successfully modified to obtain its acetylated derivative Ac-CP. Its potential immunomodulatory activities on RAW264.7 macrophages were investigated. Results showed that the acetylated polysaccharide Ac-CP could significantly stimulate macrophage proliferation, its actions were significantly stronger than that of the corresponding unmodified polysaccharide, CP. Meanwhile, the NO production activities of macrophages were not significantly enhanced by Ac-CP compared to CP group. In addition, both the phagocytic activity and levels of cytokines TNF-a, IL-1β and IL-6 were enhanced in the RAW264.7 macrophages by stimulation of Ac-CP. These results indicated that the acetylated derivative Ac-CP could enhance the activation of peritoneal macrophages, and acetylation modification can enhance the immunomodulation function of CP, indicating the potential application of acetylated polysaccharide as an immunotherapeutic adjuvant. Copyright © 2017 Elsevier B.V. All rights reserved.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection*

PubMed Central

Melo-Braga, Marcella N.; Verano-Braga, Thiago; León, Ileana R.; Antonacci, Donato; Nogueira, Fábio C. S.; Thelen, Jay J.; Larsen, Martin R.; Palmisano, Giuseppe

2012-01-01

Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection. PMID:22778145

Comparative evaluation of cytotoxicity of a glucosamine-TBA conjugate and a chitosan-TBA conjugate.

PubMed

Guggi, Davide; Langoth, Nina; Hoffer, Martin H; Wirth, Michael; Bernkop-Schnürch, Andreas

2004-07-08

D-glucosamine and chitosan were modified by the immobilization of thiol groups utilizing 2-iminothiolane. The toxicity profile of the resulting D-glucosamine-TBA (4-thiobutylamidine) conjugate, of chitosan-TBA conjugate and of the corresponding unmodified controls was evaluated in vitro. On the one hand, the cell membrane damaging effect of 0.025% solutions of the test compounds was investigated via red blood cell lysis test. On the other hand, the cytotoxity of 0.025, 0.25 and 0.5% solutions of the test compounds was evaluated on L-929 mouse fibroblast cells utilizing two different bioassays: the MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), which assess the mitochondrial metabolic activity of the cells, and the BrdU-based enzyme-linked immunosorbent assay, which measures the incorporation in the DNA of 5-bromo-2'-deoxyuridine and consequently the cell proliferation. Results of the red blood cell lysis test showed that both thiolated compounds displayed a lower membrane damaging effect causing a significantly lower haemoglobine release than the unmodified compounds. Data obtained by the MTT assay and the BrdU assay revealed a concentration dependent relative cytotoxicity for all tested compounds. The covalent linkage of the TBA-substructure to D-glucosamine did not cause a significant increase in cytotoxicity, whereas at higher concentrations a slightly enhanced cytotoxic effect was caused by the derivatisation of chitosan. In conclusion, the -TBA derivatives show a comparable toxicity profile to the corresponding unmodified compounds, which should not compromise their future use as save pharmaceutical excipients.

Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes

PubMed Central

Vignali, Marissa; Steger, David J.; Neely, Kristen E.; Workman, Jerry L.

2000-01-01

We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-specific chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates. PMID:10835360

Role of N-acetyltransferase 2 acetylation polymorphism in 4, 4'-methylene bis (2-chloroaniline) biotransformation.

PubMed

Hein, David W; Zhang, Xiaoyan; Doll, Mark A

2018-02-01

Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the acetylation of arylamine carcinogens. Single nucleotide polymorphisms in the NAT2 coding exon present in NAT2 haplotypes encode allozymes with reduced N-acetyltransferase activity towards the N-acetylation of arylamine carcinogens and the O-acetylation of their N-hydroxylated metabolites. NAT2 acetylator phenotype modifies urinary bladder cancer risk following exposures to arylamine carcinogens such as 4-aminobiphenyl. 4, 4'-methylene bis (2-chloroaniline) (MOCA) is a Group 1 carcinogen for which a role of the NAT2 acetylation polymorphism on cancer risk is unknown. We investigated the role of NAT2 and the genetic acetylation polymorphism on both MOCA N-acetylation and N-hydroxy-MOCA O-acetylation. MOCA N-acetylation exhibited a robust gene dose response in rabbit liver cytosol and in cryopreserved human hepatocytes derived from individuals of rapid, intermediate and slow acetylator NAT2 genotype. MOCA exhibited about 4-fold higher affinity for recombinant human NAT2 than NAT1. Recombinant human NAT2*4 (reference) and 15 variant recombinant human NAT2 allozymes catalyzed both the N-acetylation of MOCA and the O-acetylation of N-hydroxy-MOCA. Human NAT2 5, NAT2 6, NAT2 7 and NAT2 14 allozymes catalyzed MOCA N-acetylation and N-hydroxy-O-acetylation at rates much lower than the reference NAT2 4 allozyme. In conclusion, our results show that NAT2 acetylator genotype has an important role in MOCA metabolism and suggest that risk assessments related to MOCA exposures consider accounting for NAT2 acetylator phenotype in the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Progressive neurologic and somatic disease in a novel mouse model of human mucopolysaccharidosis type IIIC

PubMed Central

Marcó, Sara; Pujol, Anna; Roca, Carles; Motas, Sandra; Ribera, Albert; Garcia, Miguel; Molas, Maria; Villacampa, Pilar; Melia, Cristian S.; Sánchez, Víctor; Sánchez, Xavier; Bertolin, Joan; Ruberte, Jesús; Haurigot, Virginia

2016-01-01

ABSTRACT Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging because of the unfeasibility of cross-correction. In this study, we generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing investigation into sites of endogenous expression, which was particularly prominent in the CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches. PMID:27491071

Heparins from porcine and bovine intestinal mucosa: Are they similar drugs?

PubMed

Aquino, Rafael S; Pereira, Mariana S; Vairo, Bruno C; Cinelli, Leonardo P; Santos, Gustavo R C; Fonseca, Roberto J C; Mourão, Paulo A S

2010-05-01

Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides -->4-alpha-IdoA2S-1-->4-alpha-GlcNS6S-1-->. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.

[Rhizosphere microbial impacts of alleviating faba bean Fusarium wilt with inoculating AM fungi].

PubMed

Dong, Yan; Dong, Kun; Yang, Zhi Xian; Tang, Li; Zheng, Yi

2016-12-01

Greenhouse pot trials were conducted to investigate the effects of arbuscular mycorrhizal fungus (Glomus mosseae, Glomus tortuosum, Glomus intraradices and Glomus etunicatum) inoculation on the seedling growth, occurance of Fusarium wilt, population of Fusarium oxysporum and rhizosphere microbial community functional diversity in faba bean rhizosphere soil. Results showed that after inoculation of G. mosseae, G. tortuosum, G. intraradices and G. etunicatum, the shoot and root fresh mass of faba bean seedlings increased significantly, the disease index of faba bean fusarium wilt decreased significantly by 94.0%, 60.0%, 64.0% and 94.0%, respectively, the amount of F. oxysporum of faba bean rhizosphere decreased significantly by 98.6%, 74.3%, 77.8% and 90.4%, respectively. The best inhibitory effects to Fusarium wilt were with G. mosseae and G. etunicatum treatments. Inoculation of G. mosseae, G. tortuosum and G. etunicatum significantly increased carbon sources utilization ability of carbohydrates, amino acids, carboxylic acids and phenolic acids, with the average well color development (AWCD) value being increased by 34.4%, 31.5% and 50.8% respectively, but such significant differences were not observed with inoculation of G. intraradice. Principal component analyses showed that inoculation of G. mosseae, G. tortuosum and G. etunicatum fungi changed the rhizospheric microbial community composition. Correlation analyses showed that the utilization of carbohydrates carbon sources (β-Methyl-D-glucoside, D-Galacturonic acid, D-Mannitol, N-Acetyl-D-Glucosamine, D-Cellobiose,) and carboxylic acids carbon sources (D-Galactonic acid-γ-Lactone) were significantly increased after inoculation of G. tortuosum, and the utilization of L-Arginine and 4-Hydroxy benzoic acid significantly increased after inoculation of G. mosseae and G. etunicatum. Carbohydrates, carboxylic acids were main carbon sources utilized by rhizosphere microbes after G. tortuosum and G. intraradices inoculation, and amino acids and phenolic acids were main carbon sources utilized by rhizosphere microbes after G. mosseae and G. etunicatum inoculation. Inoculation of AM fungi significantly increased the activities of rhizosphere microbes, changed soil microbe community functional diversity, and thus inhibited the growth of F. oxysporum. The inhibitory impacts of AM fungi inoculations depended on the changes of microbes utilizing carbon sources.

Relationship between carbon and nitrogen mineralization in a subtropical soil

NASA Astrophysics Data System (ADS)

Li, Qianru; Sun, Yue; Zhang, Xinyu; Xu, Xingliang; Kuzyakov, Yakov

2014-05-01

In most soils, more than 90% nitrogen is bonded with carbon in organic forms. This indicates that carbon mineralization should be closely coupled with nitrogen mineralization, showing a positive correlation between carbon and nitrogen mineralization. To test this hypothesis above, we conducted an incubation using a subtropical soil for 10 days at 15 °C and 25 °C. 13C-labeled glucose and 15N-labeled ammonium or nitrate was used to separate CO2 and mineral N released from mineralization of soil organic matter and added glucose or inorganic nitrogen. Phospholipid fatty acid (PLFA) and four exoenzymes (i.e. β-1,4- Glucosaminidase, chitinase, acid phosphatase, β-1,4-N- acetyl glucosamine glycosidase) were also analyzed to detect change in microbial activities during the incubation. Our results showed that CO2 release decreased with increasing nitrogen mineralization rates. Temperature did not change this relationship between carbon and nitrogen mineralization. Although some changes in PLFA and the four exoenzymes were observed, these changes did not contribute to changes in carbon and nitrogen mineralization. These findings indicates that carbon and nitrogen mineralization in soil are more complicated than as previously expected. Future investigation should focus on why carbon and nitrogen mineralization are coupled in a negative correlation not in a positive correlation in many soils for a better understanding of carbon and nitrogen transformation during their mineralization.

Lectin activity in mycelial extracts of Fusarium species.

PubMed

Bhari, Ranjeeta; Kaur, Bhawanpreet; Singh, Ram S

2016-01-01

Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d-mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia

PubMed Central

Fan, Yongjun; Wali, Gautam; Sutharsan, Ratneswary; Bellette, Bernadette; Crane, Denis I.; Sue, Carolyn M.; Mackay-Sim, Alan

2014-01-01

ABSTRACT Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials. PMID:24857849

Synthesis of a D-Glucopyranosyl Azide: Spectroscopic Evidence for Stereochemical Inversion in the S[subscript N]2 Reaction

ERIC Educational Resources Information Center

Adesoye, Olumuyiwa G.; Mills, Isaac N.; Temelkoff, David P.; Jackson, John A.; Norris, Peter

2012-01-01

Stereospecific S[subscript N]2 conversion of configurationally pure acetobromoglucose (2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide) to the corresponding beta-D-glucopyranosyl azide is a useful exercise in the advanced organic undergraduate teaching laboratory. The procedure is safe and suitable for small-scale implementation, and firm…

Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

PubMed Central

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

2015-01-01

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

Glucokinase inhibitor glucosamine stimulates feeding and activates hypothalamic neuropeptide Y and orexin neurons.

PubMed

Zhou, Ligang; Yueh, Chen-Yu; Lam, Daniel D; Shaw, Jill; Osundiji, Mayowa; Garfield, Alastair S; Evans, Mark; Heisler, Lora K

2011-09-12

Maintaining glucose levels within the appropriate physiological range is necessary for survival. The identification of specific neuronal populations, within discreet brain regions, sensitive to changes in glucose concentration has led to the hypothesis of a central glucose-sensing system capable of directly modulating feeding behaviour. Glucokinase (GK) has been identified as a glucose-sensor responsible for detecting such changes both within the brain and the periphery. We previously reported that antagonism of centrally expressed GK by administration of glucosamine (GSN) was sufficient to induce protective glucoprivic feeding in rats. Here we examine a neurochemical mechanism underlying this effect and report that GSN stimulated food intake is highly correlated with the induction of the neuronal activation marker cFOS within two nuclei with a demonstrated role in central glucose sensing and appetite, the arcuate nucleus of the hypothalamus (ARC) and lateral hypothalamic area (LHA). Furthermore, GSN stimulated cFOS within the ARC was observed in orexigenic neurons expressing the endogenous melanocortin receptor antagonist agouti-related peptide (AgRP) and neuropeptide Y (NPY), but not those expressing the anorectic endogenous melanocortin receptor agonist alpha-melanocyte stimulating hormone (α-MSH). In the LHA, GSN stimulated cFOS was found within arousal and feeding associated orexin/hypocretin (ORX), but not orexigenic melanin-concentrating hormone (MCH) expressing neurons. Our data suggest that GK within these specific feeding and arousal related populations of AgRP/NPY and ORX neurons may play a modulatory role in the sensing of and appetitive response to hypoglycaemia. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

PubMed

Jain, R K; Dubey, R; Abbas, S A; Matta, K L

1987-03-15

Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

PubMed Central

Romão-Dumaresq, Aline S.; de Araújo, Welington Luiz; Talbot, Nicholas J.; Thornton, Christopher R.

2012-01-01

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte. PMID:23110120

Influence of Biochemical Features of Burkholderia pseudomallei Strains on Identification Reliability by Vitek 2 System

PubMed Central

Zakharova, Irina B; Lopasteyskaya, Yana A; Toporkov, Andrey V; Viktorov, Dmitry V

2018-01-01

Background: Burkholderia pseudomallei is a Gram-negative saprophytic soil bacterium that causes melioidosis, a potentially fatal disease endemic in wet tropical areas. The currently available biochemical identification systems can misidentify some strains of B. pseudomallei. The aim of the present study was to identify the biochemical features of B. pseudomallei, which can affect its correct identification by Vitek 2 system. Materials and Methods: The biochemical patterns of 40 B. pseudomallei strains were obtained using Vitek 2 GN cards. The average contribution of biochemical tests in overall dissimilarities between correctly and incorrectly identified strains was assessed using nonmetric multidimensional scaling. Results: It was found (R statistic of 0.836, P = 0.001) that a combination of negative N-acetyl galactosaminidase, β-N-acetyl glucosaminidase, phosphatase, and positive D-cellobiase (dCEL), tyrosine arylamidase (TyrA), and L-proline arylamidase (ProA) tests leads to low discrimination of B. pseudomallei, whereas a set of positive dCEL and negative N-acetyl galactosaminidase, TyrA, and ProA determines the wrong identification of B. pseudomallei as Burkholderia cepacia complex. Conclusion: The further expansion of the Vitek 2 identification keys is needed for correct identification of atypical or regionally distributed biochemical profiles of B. pseudomallei. PMID:29563716

Study to elucidate formation pathways of selected roast-smelling odorants upon extrusion cooking.

PubMed

Davidek, Tomas; Festring, Daniel; Dufossé, Thierry; Novotny, Ondrej; Blank, Imre

2013-10-30

The formation pathways of the N-containing roast-smelling compounds 2-acetyl-1-pyrroline, 2-acetyl-1(or 3),4,5,6-tetrahydropyridine, and their structural analogues 2-propionyl-1-pyrroline and 2-propionyl-1(or 3),4,5,6-tetrahydropyridine were studied upon extrusion cooking using the CAMOLA approach. The samples were produced under moderate extrusion conditions (135 °C, 20% moisture, 400 rpm) employing a rice-based model recipe enriched with flavor precursors ([U-(13)C6]-D-glucose, D-glucose, glycine, L-proline, and L-ornithine). The obtained data indicate that the formation of these compounds upon extrusion follows pathways similar to those reported for nonsheared model systems containing D-glucose and L-proline. 2-Acetyl-1-pyrroline is formed (i) by acylation of 1-pyrroline via C2 sugar fragments (major pathway) and (ii) via ring-opening of 1-pyrroline incorporating C3 sugar fragments (minor pathway), whereas 2-propionyl-1-pyrroline incorporates exclusively C3 sugar fragments. 2-Acetyl-1(or 3),4,5,6-tetrahydropyridine and the corresponding propionyl analogue incorporate C3 and C4 sugar fragments, respectively. In addition, it has been shown that the formation of 2-acetyl-1-pyrroline in low-moisture systems depends on the pH value of the reaction mixture.

Spatial Periodicity of Escherichia coli K-12 Biofilm Microstructure Initiates during a Reversible, Polar Attachment Phase of Development and Requires the Polysaccharide Adhesin PGA

PubMed Central

Agladze, Konstantin; Wang, Xin; Romeo, Tony

2005-01-01

Using fast Fourier transform (FFT) analysis, we previously observed that cells within Escherichia coli biofilm are organized in nonrandom or periodic spatial patterns (K. Agladze et al., J. Bacteriol. 185:5632-5638, 2003). Here, we developed a gravity displacement assay for examining cell adherence and used it to quantitatively monitor the formation of two distinct forms of cell attachment, temporary and permanent, during early biofilm development. Temporarily attached cells were mainly surface associated by a cell pole; permanent attachments were via the lateral cell surface. While temporary attachment precedes permanent attachment, both forms can coexist in a population. Exposure of attached cells to gravity liberated an unattached population capable of rapidly reassembling a new monolayer, composed of temporarily attached cells, and possessing periodicity. A csrA mutant, which forms biofilm more vigorously than its wild-type parent, exhibited an increased proportion of permanently attached cells and a form of attachment that was not apparent in the parent strain, permanent polar attachment. Nevertheless, it formed periodic attachment patterns. In contrast, biofilm mutants with altered lipopolysaccharide synthesis (waaG) exhibited increased cell-cell interactions, bypassed the polar attachment step, and produced FFT spectra characteristic of aperiodic cell distribution. Mutants lacking the polysaccharide adhesin β-1,6-N-acetyl-d-glucosamine (ΔpgaC) also exhibited aperiodic cell distribution, but without apparent cell-cell interactions, and were defective in forming permanent attachments. Thus, spatial periodicity of biofilm microstructure is genetically determined and evident during the formation of temporary cell surface attachments. PMID:16321928

Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface.

PubMed

Tremblay, Yannick D N; Lévesque, Cynthia; Segers, Ruud P A M; Jacques, Mario

2013-10-20

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae. In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy. This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

Detection of Metabolism Function of Microbial Community of Corpses by Biolog-Eco Method.

PubMed

Jiang, X Y; Wang, J F; Zhu, G H; Ma, M Y; Lai, Y; Zhou, H

2016-06-01

To detect the changes of microbial community functional diversity of corpses with different postmortem interval （PMI） and to evaluate forensic application value for estimating PMI. The cultivation of microbial community from the anal swabs of a Sus scrofa and a human corpse placed in field environment from 0 to 240 h after death was performed using the Biolog-Eco Microplate and the variations of the absorbance values were also monitored. Combined with the technology of forensic pathology and flies succession, the metabolic characteristics and changes of microbial community on the decomposed corpse under natural environment were also observed. The diversity of microbial metabolism function was found to be negatively correlated with the number of maggots in the corpses. The freezing processing had the greatest impact on average well color development value at 0 h and the impact almost disappeared after 48 h. The diversity of microbial metabolism of the samples became relatively unstable after 192 h. The principal component analysis showed that 31 carbon sources could be consolidated for 5 principal components （accumulative contribution ratio >90%）.The carbon source tsquare-analysis showed that N -acetyl- D -glucosamine and L -serine were the dominant carbon sources for estimating the PMI （0=240 h） of the Sus scrofa and human corpse. The Biolog-Eco method can be used to reveal the metabolic differences of the carbon resources utilization of the microbial community on the corpses during 0-240 h after death, which could provide a new basis for estimating the PMI. Copyright© by the Editorial Department of Journal of Forensic Medicine

O-Linked β-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

PubMed

Yao, Dan; Xu, Lijuan; Xu, Oufan; Li, Rujun; Chen, Mingxing; Shen, Hui; Zhu, Huajiang; Zhang, Fengyi; Yao, Deshang; Chen, Yiu-Fai; Oparil, Suzanne; Zhang, Zhengang; Gong, Kaizheng

2018-06-01

Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury. © 2018 American Heart Association, Inc.

ATP-citrate lyase links cellular metabolism to histone acetylation.

PubMed

Wellen, Kathryn E; Hatzivassiliou, Georgia; Sachdeva, Uma M; Bui, Thi V; Cross, Justin R; Thompson, Craig B

2009-05-22

Histone acetylation in single-cell eukaryotes relies on acetyl coenzyme A (acetyl-CoA) synthetase enzymes that use acetate to produce acetyl-CoA. Metazoans, however, use glucose as their main carbon source and have exposure only to low concentrations of extracellular acetate. We have shown that histone acetylation in mammalian cells is dependent on adenosine triphosphate (ATP)-citrate lyase (ACL), the enzyme that converts glucose-derived citrate into acetyl-CoA. We found that ACL is required for increases in histone acetylation in response to growth factor stimulation and during differentiation, and that glucose availability can affect histone acetylation in an ACL-dependent manner. Together, these findings suggest that ACL activity is required to link growth factor-induced increases in nutrient metabolism to the regulation of histone acetylation and gene expression.

Synthesis of D-glucosamine quaternary ammonium derivatives and evaluation of their antifungal activity together with aminodeoxyglucose derivatives against two wood fungi Coriolus versicolor and Poria placenta: structure-activity relationships.

PubMed

Muhizi, Théoneste; Coma, Véronique; Grelier, Stéphane

2011-03-01

Structure-activity relationships are often reported in scientific studies. These may be employed in searching for new acceptable biocides to use against harmful microorganisms, because the biocides used hitherto encounter various problems, including lack of efficiency, high toxicity and persistence. Nowadays, scientists are trying to find new, environmentally acceptable biocides to replace these earlier biocides. Different compounds from renewable materials have been studied and have shown pronounced antifungal activity against wood fungi. These include aminopolysaccharide derivatives and different quaternary ammonium polymers. A biological study carried out with these products indicated a possible relationship between amino groups and differences in biological activity observed. In this study, an amino group was successively fixed to different carbon atoms of glucose, and glucosamine was also modified by both N-alkylation and quaternisation. The impact of the amino group position on antifungal activity against two wood decay fungi was investigated. The amino group at the anomeric position showed the highest antifungal activity against both Coriolus versicolor Quel. and Poria placenta (Fr.) Cooke. Furthermore, the positive impact of both N-alkylation and quaternisation on the growth of both strains was demonstrated. The anomeric position of the amino group and the N-alkylation and quaternisation of amino sugars considerably increase the antifungal activity of these compounds. Copyright © 2010 Society of Chemical Industry.

Metabolomic Analyses of Blood Plasma after Oral Administration of D-Glucosamine Hydrochloride to Dogs

PubMed Central

Osaki, Tomohiro; Azuma, Kazuo; Kurozumi, Seiji; Takamori, Yoshimori; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Minami, Saburo

2012-01-01

D-Glucosamine hydrochloride (GlcN∙HCl) is an endogenous amino monosaccharide synthesized from glucose that is useful in the treatment of joint diseases in both humans and animals. The aim of this study was to examine amino acid metabolism in dogs after oral administration of GlcN∙HCl. Accelerated fumarate respiration and elevated plasma levels of lactic acid and alanine were observed after administration. These results suggest that oral administration of GlcN∙HCl induces anaerobic respiration and starvation in cells, and we hypothesize that these conditions promote cartilage regeneration. Further studies are required to evaluate the expression of transforming growth factor-beta (TGF-β). PMID:23015778

Alga Ecklonia bicyclis, Tribulus terrestris, and Glucosamine Oligosaccharide Improve Erectile Function, Sexual Quality of Life, and Ejaculation Function in Patients with Moderate Mild-Moderate Erectile Dysfunction: A Prospective, Randomized, Placebo-Controlled, Single-Blinded Study

PubMed Central

Sansalone, Salvatore; Leonardi, Rosario; Antonini, Gabriele; Vitarelli, Antonio; Vespasiani, Giuseppe

2014-01-01

We aimed to evaluate the efficacy of oral therapy with alga Ecklonia bicyclis, Tribulus terrestris, and glucosamine oligosaccharide (Tradamix TX1000) in patients with erectile dysfunction (ED) at 3 months of follow-up. From January 2013 to September 2013, 177 patients diagnosed with mild-moderate ED (IIEF-EF < 26) were enrolled in this multicenter, single-blinded, placebo-controlled study and randomized in Group A (Tradamix, n = 87) and Group B (placebo, n = 90). Penile color Doppler ultrasound measures, IIEF-15 questionnaire, male sexual health questionnaire-ejaculation disorder (MSHQ-EjD), and sexual quality of life (SQoL-M) were collected. We observed significant changes of the IIEF-15 in Group A (mean difference: 11.54; P < 0.05) at 3 months versus Group B (P < 0.05). PSV (P < 0.05), IIEF-intercourse satisfaction (P < 0.05), IIEF-orgasmic function (mean P < 0.05), IIEF-sexual desire (P < 0.05), IIEF-overall satisfaction (P < 0.05), MSHQ-EjD (mean difference: 1.21; P < 0.05), and SQoL-M (mean difference: 10.2; P < 0.05) were significantly changed in Group A versus baseline and Group B. Patients with moderate arterial dysfunction showed significant increase of PSV (P < 0.05), IIEF-EF (P < 0.05), MSHQ-EjD (P < 0.05), and SQoL-M (P < 0.05) in Group A. Therapy with Tradamix improves erectile and ejaculation function and sexual quality of life in patients with mild-moderate ED and in particular for those with moderate arterial dysfunction. PMID:25136552

Alga Ecklonia bicyclis, Tribulus terrestris, and glucosamine oligosaccharide improve erectile function, sexual quality of life, and ejaculation function in patients with moderate mild-moderate erectile dysfunction: a prospective, randomized, placebo-controlled, single-blinded study.

PubMed

Sansalone, Salvatore; Leonardi, Rosario; Antonini, Gabriele; Vitarelli, Antonio; Vespasiani, Giuseppe; Basic, Dragoslav; Morgia, Giuseppe; Cimino, Sebastiano; Russo, Giorgio Ivan

2014-01-01

We aimed to evaluate the efficacy of oral therapy with alga Ecklonia bicyclis, Tribulus terrestris, and glucosamine oligosaccharide (Tradamix TX1000) in patients with erectile dysfunction (ED) at 3 months of follow-up. From January 2013 to September 2013, 177 patients diagnosed with mild-moderate ED (IIEF-EF < 26) were enrolled in this multicenter, single-blinded, placebo-controlled study and randomized in Group A (Tradamix, n = 87) and Group B (placebo, n = 90). Penile color Doppler ultrasound measures, IIEF-15 questionnaire, male sexual health questionnaire-ejaculation disorder (MSHQ-EjD), and sexual quality of life (SQoL-M) were collected. We observed significant changes of the IIEF-15 in Group A (mean difference: 11.54; P < 0.05) at 3 months versus Group B (P < 0.05). PSV (P < 0.05), IIEF-intercourse satisfaction (P < 0.05), IIEF-orgasmic function (mean P < 0.05), IIEF-sexual desire (P < 0.05), IIEF-overall satisfaction (P < 0.05), MSHQ-EjD (mean difference: 1.21; P < 0.05), and SQoL-M (mean difference: 10.2; P < 0.05) were significantly changed in Group A versus baseline and Group B. Patients with moderate arterial dysfunction showed significant increase of PSV (P < 0.05), IIEF-EF (P < 0.05), MSHQ-EjD (P < 0.05), and SQoL-M (P < 0.05) in Group A. Therapy with Tradamix improves erectile and ejaculation function and sexual quality of life in patients with mild-moderate ED and in particular for those with moderate arterial dysfunction.

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed Central

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells. PMID:9003425

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.

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

PubMed

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

2015-03-06

Main objective of this study was to investigate the detailed structural information about O-acetylated sugar residues in Dendronan(®). A water solution (2%, w/w) of Dendronan(®) was treated with endo-β-mannanase to produce oligosaccharides rich in O-acetylated sugar residues. The oligosaccharides were partly recovered by ethanol precipitation (70%, w/w). The recovered sample (designated Hydrolyzed Dendrobium officinale Polysaccharide, HDOP) had a yield of 24.7% based on the dry weight of Dendronan(®) and was highly O-acetylated. A D2O solution of HDOP (6%, w/w) generated strong signals in (1)H, (13)C, 2D (1)H-(1)H COSY, 2D (1)H-(1)H TOCSY, 2D (1)H-(1)H NOESY, 2D (1)H-(13)C HMQC, and 2D (1)H-(13)C HMBC NMR spectra. Results of NMR analyses showed that the majority of O-acetylated mannoses were mono-substituted with acetyl groups at O-2 or O-3 position. There were small amounts of mannose residues with di-O-acetyl substitution at both O-2 and O-3 positions. Minor levels of mannoses with 6-O-acetyl, 2,6-di-O-acetyl, and 3,6-di-O-acetyl substitutions were also identified. Much information about sugar residue sequence was extracted from 2D (1)H-(13)C HMBC and 2D (1)H-(1)H NOESY spectra. (1)J(C-H) coupling constants of major sugar residues were obtained. Evidences for the existence of branches or O-acetylated glucoses in HDOP were not found. The major structure of Dendronan(®) is shown as follows: [Formula: see text] M: β-D-mannopyranose; G: β-D-glucopyranose; a: O-acetyl group. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

The carbohydrate deposits detected by histochemical methods in the molecular layer of the dentate gyrus in the hippocampal formation of patients with schizophrenia, Down's syndrome and dementia, and aged person.

PubMed

Nishimura, A; Ikemoto, K; Satoh, K; Yamamoto, Y; Rand, S; Brinkmann, B; Nishi, K

2000-11-01

Post-mortem brain tissue was obtained from 28 patients with brain disorders, of which 15 had clinically diagnosed schizophrenia, 6 Alzheimer type dementia, 5 dementia with tangles and 2 cases of Down's syndrome. The controls were 22 cases from autopsies without brain disorders or with no known episodes of brain disorder. The tissues were stained for the detection of carbohydrate deposits in the hippocampal formation, using lectin, immunohistochemical and conventional staining methods. The staining revealed the existence of spherical deposits in the inner and middle molecular layers of the dentate gyrus in the hippocampal formation which contained fucose, galactose, N-acetyl galactosamine, N-acetyl glucosamine, sialic acid, mannose and chondroitin sulfate. The number of the deposits was higher in patients with brain disorder such as schizophrenia, Alzheimer type dementia, dementia with tangles or Down's syndrome, and in some aged individuals, in comparison to those in younger individuals. No deposits were detected in a few younger or aged individuals. Spherical deposits 3-10 microm in diameter may be an immature form of the corpora amylacea, since they were similar in the histochemical characteristics with lectin, immunohistochemical and conventional staining methods. However, differing staining ability by hematoxylin, periodic acid Schiff's reagent and antibodies against the intracellular degraded proteins such as ubiquitin and tau-protein was observed. The antibodies against ubiquitin and tau-protein showed clear reactivity with the corpora amylacea and no reactivity with spherical deposits, indicating that the corpora amylacea has an intracellular origin and spherical deposits an extracellular matrix origin. The results obtained in this study indicate that not only neuronal degeneration but also unusual glycometabolism in neurons may disturb the neuronal function and cause brain disorders, and that spherical deposits may cause dysfunction of the neuronal network in the dentate gyrus of the hippocampus which is closely linked with recognition and memory functions.

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

PubMed

Alais, J; David, S

1992-06-04

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

Andrographolide: antibacterial activity against common bacteria of human health concern and possible mechanism of action.

PubMed

Banerjee, Malabika; Parai, Debaprasad; Chattopadhyay, Subrata; Mukherjee, Samir Kumar

2017-05-01

Increasing bacterial resistance to common drugs is a major public health concern for the treatment of infectious diseases. Certain naturally occurring compounds of plant sources have long been reported to possess potential antimicrobial activity. This study was aimed to investigate the antibacterial activity and possible mechanism of action of andrographolide (Andro), a diterpenoid lactone from a traditional medicinal herb Andrographis paniculata. Extent of antibacterial action was assessed by minimal bactericidal concentration method. Radiolabeled N-acetyl glucosamine, leucine, thymidine, and uridine were used to determine the effect of Andro on the biosyntheses of cell wall, protein, DNA, and RNA, respectively. In addition, anti-biofilm potential of this compound was also tested. Andro showed potential antibacterial activity against most of the tested Gram-positive bacteria. Among those, Staphylococcus aureus was found to be most sensitive with a minimal inhibitory concentration value of 100 μg/mL. It was found to be bacteriostatic. Specific inhibition of intracellular DNA biosynthesis was observed in a dose-dependent manner in S. aureus. Andro mediated inhibition of biofilm formation by S. aureus was also found. Considering its antimicrobial potency, Andro might be accounted as a promising lead for new antibacterial drug development.

Effective atomic number and electron density of amino acids within the energy range of 0.122-1.330 MeV

NASA Astrophysics Data System (ADS)

More, Chaitali V.; Lokhande, Rajkumar M.; Pawar, Pravina P.

2016-08-01

Photon attenuation coefficient calculation methods have been widely used to accurately study the properties of amino acids such as n-acetyl-L-tryptophan, n-acetyl-L-tyrosine, D-tryptophan, n-acetyl-L-glutamic acid, D-phenylalanine, and D-threonine. In this study, mass attenuation coefficients (μm) of these amino acids for 0.122-, 0.356-, 0.511-, 0.662-, 0.884-, 1.170, 1.275-, 1.330-MeV photons are determined using the radio-nuclides Co57, Ba133, Cs137, Na22, Mn54, and Co60. NaI (Tl) scintillation detection system was used to detect gamma rays with a resolution of 8.2% at 0.662 MeV. The calculated attenuation coefficient values were then used to determine total atomic cross sections (σt), molar extinction coefficients (ε), electronic cross sections (σe), effective atomic numbers (Zeff), and effective electron densities (Neff) of the amino acids. Theoretical values were calculated based on the XCOM data. Theoretical and experimental values are found to be in a good agreement (error<5%). The variations of μm, σt, ε, σe, Zeff, and Neff with energy are shown graphically. The values of μm, σt, ε, σe are higher at lower energies, and they decrease sharply as energy increases; by contrast, Zeff and Neff were found to be almost constant.

Comparative conformational studies of 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxyhex-1-enitols at the DFT level.

PubMed

Nowacki, Andrzej; Liberek, Beata

2018-06-15

B3LYP and M06-2X optimization and MP2 single point calculations are reported for the 4 H 5 and 5 H 4 conformations of 3,4,6-tri-O-acetyl-D-allal, 3,4,6-tri-O-acetyl-D-galactal, 3,4,6-tri-O-acetyl-D-glucal, and 3,4,6-tri-O-acetyl-D-gulal. Significant discrepancies in predictions of relative energies and conformers' population for B3LYP and M06-2X optimized geometries are observed. Generally, B3LYP overestimates the conformers' energies with respect to MP2, whereas M06-2X slightly underestimates the conformers' energies. B3LYP failed to estimate the 4 H 5 ⇄ 5 H 4 conformational equilibrium for 3,4,6-tri-O-acetyl-D-galactal and 3,4,6-tri-O-acetyl-D-glucal. The M06-2X functional showed good agreement with experimental results for all glycals studied. The 4 H 5 ⇄ 5 H 4 conformational equilibrium for 3,4,6-tri-O-acetyl-D-allal and 3,4,6-tri-O-acetyl-D-gulal is governed by the vinylogous anomeric effect (VAE), whereas competition between the VAE and quasi 1,3-diaxial interactions influence this equilibrium for 3,4,6-tri-O-acetyl-D-galactal and 3,4,6-tri-O-acetyl-D-glucal. The orientation of the 4-OAc group influences the strength of the quasi 1,3-diaxial interactions between the 3-OAc and 5-CH 2 OAc groups. AIM analysis shows weak bonding interaction between the 3-OAc and 5-CH 2 OAc groups. Copyright © 2018 Elsevier Ltd. All rights reserved.

Distinct effects of glucose and glucosamine on vascular endothelial and smooth muscle cells: Evidence for a protective role for glucosamine in atherosclerosis

PubMed Central

Duan, Wenlan; Paka, Latha; Pillarisetti, Sivaram

2005-01-01

Accelerated atherosclerosis is one of the major vascular complications of diabetes. Factors including hyperglycemia and hyperinsulinemia may contribute to accelerated vascular disease. Among the several mechanisms proposed to explain the link between hyperglycemia and vascular dysfunction is the hexosamine pathway, where glucose is converted to glucosamine. Although some animal experiments suggest that glucosamine may mediate insulin resistance, it is not clear whether glucosamine is the mediator of vascular complications associated with hyperglycemia. Several processes may contribute to diabetic atherosclerosis including decreased vascular heparin sulfate proteoglycans (HSPG), increased endothelial permeability and increased smooth muscle cell (SMC) proliferation. In this study, we determined the effects of glucose and glucosamine on endothelial cells and SMCs in vitro and on atherosclerosis in apoE null mice. Incubation of endothelial cells with glucosamine, but not glucose, significantly increased matrix HSPG (perlecan) containing heparin-like sequences. Increased HSPG in endothelial cells was associated with decreased protein transport across endothelial cell monolayers and decreased monocyte binding to subendothelial matrix. Glucose increased SMC proliferation, whereas glucosamine significantly inhibited SMC growth. The antiproliferative effect of glucosamine was mediated via induction of perlecan HSPG. We tested if glucosamine affects atherosclerosis development in apoE-null mice. Glucosamine significantly reduced the atherosclerotic lesion in aortic root. (P < 0.05) These data suggest that macrovascular disease associated with hyperglycemia is unlikely due to glucosamine. In fact, glucosamine by increasing HSPG showed atheroprotective effects. PMID:16207378

The synergistic effects of radiofrequency ablation (RFA) with glycated chitosan for inhibiting the metastasis of breast cancer

NASA Astrophysics Data System (ADS)

Chiu, Hsin-Yu; Leu, Jyh-Der; Chen, Wei R.; Lee, Yi-Jang

2016-03-01

Breast cancer is increasing with years in Taiwan because of dietary style, life behavior and several social-physiological factors. According to the record of Bureau of Health Promotion in Taiwan, the incidence of breast cancer is top one, and the mortality of that is top one cancer type in women. Compared with USA, most of breast cancer cases found in Taiwanese women have reached to stage 2 or 3. Current therapeutic strategies for breast cancer include surgery, radiation therapy, chemotherapy, hormone therapy and targeted therapy. However, these methods used for curing the late-stage breast cancer remains rare. Because the metastasis is the major problem of late-stage breast cancer, it is of interest to investigate whether a systemic therapy can reduce the symptoms of cancer. The immunotherapy, particularly an induction of autoimmune system, is probably important for the treatment of late-stage breast cancer. Glycated chitosan (GC) is derived from chitosan, a linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine through β-(1-4) linkage. Several lines of evidence have shown that GC is an immunoadjuvant that can target on primary and metastatic tumors formed in animal and human patients. In our previous data, GC was demonstrated to decrease the motility and invasion of mammalian breast cancer cells in vitro and in vivo. Radiofrequency ablation (RFA) is dependent on a small generator that delivers high frequency alternating electric current directly to burn a tumor lesion. Therefore, the temperature may reach up to above 60 °C. In this study, we used 4T1 mouse breast cancer cell that is the approximately equal to stage 4 of human breast cancer. And triple modality reporter gene (3R) was delivered into the cells using transfected piggyBac, a transposable element for observation of tumor growth and metastasis in vivo. Data showed that growth and metastasis of tumors smaller than 500mm3 were entirely suppressed by RFA-GC combination treatment. Plasma extraction from this group displayed an inhibitory effect on cultured 4T1 cells, suggesting some immunoreactions were triggered and therefore secreted some cytokines. Protein array data indicated that PF-4 may play a key role in GC-RFA caused tumor suppression but the further effects should be investigated. On the other hand, the survival rate of small tumor-bearing mice under RFA-GC treatment was higher than those of GC or RFA treatment only. In conclusion, this study confirmed that radiofrequency ablation combined with GC could trigger an autoimmune response to inhibit tumor metastasis and tumorigenesis. For metastatic cancers, this combination treatment may become the foundation for a feasible cancer treatment modality.

NAA and NAAG variation in neuronal activation during visual stimulation.

PubMed

Castellano, G; Dias, C S B; Foerster, B; Li, L M; Covolan, R J M

2012-11-01

N-acetyl-aspartyl-glutamate (NAAG) and its hydrolysis product N-acetyl-L-aspartate (NAA) are among the most important brain metabolites. NAA is a marker of neuron integrity and viability, while NAAG modulates glutamate release and may have a role in neuroprotection and synaptic plasticity. Investigating on a quantitative basis the role of these metabolites in brain metabolism in vivo by magnetic resonance spectroscopy (MRS) is a major challenge since the main signals of NAA and NAAG largely overlap. This is a preliminary study in which we evaluated NAA and NAAG changes during a visual stimulation experiment using functional MRS. The paradigm used consisted of a rest period (5 min and 20 s), followed by a stimulation period (10 min and 40 s) and another rest period (10 min and 40 s). MRS from 17 healthy subjects were acquired at 3T with TR/TE = 2000/288 ms. Spectra were averaged over subjects and quantified with LCModel. The main outcomes were that NAA concentration decreased by about 20% with the stimulus, while the concentration of NAAG concomitantly increased by about 200%. Such variations fall into models for the energy metabolism underlying neuronal activation that point to NAAG as being responsible for the hyperemic vascular response that causes the BOLD signal. They also agree with the fact that NAAG and NAA are present in the brain at a ratio of about 1:10, and with the fact that the only known metabolic pathway for NAAG synthesis is from NAA and glutamate.

Capillary electrophoresis determination of glucosamine in nutraceutical formulations after labeling with anthranilic acid and UV detection.

PubMed

Volpi, Nicola

2009-04-05

A new robust CE method for the determination of the glucosamine (GlcN) content in nutraceutical formulations is described after its derivatization with anthranilic acid (2-aminobenzoic acid, AA). The CE separation of derivatized GlcN with AA was performed on an uncoated fused-silica capillary tube (50 microm I.D.) using an operating pH 7.0 buffer of 150 mM boric acid/50 mM NaH2PO4 and UV detection at 214 nm. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for GlcN was linear over the selected concentration range from 240 to 2400 pg (40-400 microg/mL) with a correlation coefficient greater than 0.980. The intra- and inter-day variations (CV%) were between 0.5 and 0.9 for migration time, and between 2.8 and 4.3 for peak area, respectively. The LOD and the LOQ of the method were approximately 200 and 500 pg, respectively. The intra- and inter-day accuracy was estimated to range from 2.8% to 5.1%, while the percent recoveries of GlcN in formulations were calculated to be about 100% after simple centrifugation for 10 min, lyophilization and derivatization with AA. The CE method was applied to the determination of GlcN content, in the form of GlcN-hydrochloride or GlcN-sulfate, of several nutraceutical preparations in the presence of other ingredients, i.e. chondroitin sulfate, vitamin C and/or methylsulfonylmethane (MSM) as well as salts and other agents. The quantitative results obtained were in total conformity with the label claims.

Pharmacokinetics and N-acetylation metabolism of S-methyl-l-cysteine and trans-S-1-propenyl-l-cysteine in rats and dogs.

PubMed

Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

2016-11-01

1. Pharmacokinetics and N-acetylation metabolism of S-methyl-L-cysteine (SMC) and trans-S-1-propenyl-L-cysteine (S1PC) were examined in rats and dogs. SMC and S1PC (2-5 mg/kg) were well absorbed in both species with high bioavailability (88-100%). 2. SMC and S1PC were excreted only to a small extent in the urine of rats and dogs. The small renal clearance values (<0.03 l/h/kg) indicated the extensive renal reabsorption of SMC and S1PC, which potentially contributed to their long elimination half-lives (>5 h) in dogs. 3. S1PC, but not SMC, underwent N-acetylation extensively in vivo, which can be explained by the relative activities of N-acetylation of S1PC/SMC and deacetylation of their N-acetylated forms, N-acetyl-S1PC/N-acetyl-SMC, in the liver and kidney in vitro. The activities for S1PC N-acetylation were similar to or higher than those for N-acetyl-S1PC deacetylation in liver S9 fractions of rat and dog, whereas liver and kidney S9 fractions of rat and dog had little activity for SMC N-acetylation or considerably higher activities for N-acetyl-SMC deacetylation. 4. Our study demonstrated that the pharmacokinetics of SMC and S1PC in rats and dogs was characterized by high bioavailability and extensive renal reabsorption; however, the extent of undergoing the N-acetylation metabolism was extremely different between SMC and S1PC.

N-Acetyl and Glutamatergic Neurometabolites in Perisylvian Brain Regions of Methamphetamine Users.

PubMed

Tang, Jinsong; O'Neill, Joseph; Alger, Jeffry R; Shen, Zhiwei; Johnson, Maritza C; London, Edythe D

2018-05-21

Methamphetamine induces neuronal N-acetyl-aspartate synthesis in preclinical studies. In a preliminary human proton magnetic resonance spectroscopic imaging investigation, we also observed that N-acetyl-aspartate+N-acetyl-aspartyl-glutamate in right inferior frontal cortex correlated with years of heavy methamphetamine abuse. In the same brain region, glutamate+glutamine is lower in methamphetamine users than in controls and is negatively correlated with depression. N-acetyl and glutamatergic neurochemistries therefore merit further investigation in methamphetamine abuse and the associated mood symptoms. Magnetic resonance spectroscopic imaging was used to measure N-acetyl-aspartate+N-acetyl-aspartyl-glutamate and glutamate+glutamine in bilateral inferior frontal cortex and insula, a neighboring perisylvian region affected by methamphetamine, of 45 abstinent methamphetamine-dependent and 45 healthy control participants. Regional neurometabolite levels were tested for group differences and associations with duration of heavy methamphetamine use, depressive symptoms, and state anxiety. In right inferior frontal cortex, N-acetyl-aspartate+N-acetyl-aspartyl-glutamate correlated with years of heavy methamphetamine use (r = +0.45); glutamate+glutamine was lower in methamphetamine users than in controls (9.3%) and correlated negatively with depressive symptoms (r = -0.44). In left insula, N-acetyl-aspartate+N-acetyl-aspartyl-glutamate was 9.1% higher in methamphetamine users than controls. In right insula, glutamate+glutamine was 12.3% lower in methamphetamine users than controls and correlated negatively with depressive symptoms (r = -0.51) and state anxiety (r = -0.47). The inferior frontal cortex and insula show methamphetamine-related abnormalities, consistent with prior observations of increased cortical N-acetyl-aspartate in methamphetamine-exposed animal models and associations between cortical glutamate and mood in human methamphetamine users.

The preparation and antioxidant activity of glucosamine sulfate

NASA Astrophysics Data System (ADS)

Xing, Ronge; Liu, Song; Wang, Lin; Cai, Shengbao; Yu, Huahua; Feng, Jinhua; Li, Pengcheng

2009-05-01

Glucosamine sulfate was prepared from glucosamine hydrochloride that was produced by acidic hydrolysis of chitin by ion-exchange method. Optical rotation and elemental analysis characterized the degree of its purity. In addition, the antioxidant potency of chitosan derivative-glucosamine sulfate was investigated in various established in vitro systems, such as superoxide (O{2/-})/hydroxyl (·OH) radicals scavenging, reducing power, iron ion chelating. The following results are obtained: first, glucosamine sulfate had pronounced scavenging effect on superoxide radical. For example the O{2/-} scavenging activity of glucosamine sulfate was 92.11% at 0.8 mg/mL. Second, the ·OH scavenging activity of glucosamine sulfate was also strong, and was about 50% at 3.2 mg/mL. Third, the reducing power of glucosamine sulfate was more pronounced. The reducing power of glucosamine sulfate was 0.643 at 0.75 mg/mL. However, its potency for ferrous ion chelating was weak. Furthermore, except for ferrous ion chelating potency, the scavenging rate of radical and reducing power of glucosamine sulfate were concentration-dependent and increased with their increasing concentrations, but its ferrous ion chelating potency decreased with the increasing concentration. The multiple antioxidant activities of glucosamine sulfate were evidents of reducing power and superoxide/hydroxyl radicals scavenging ability. These in vitro results suggest the possibility that glucosamine sulfate could be used effectively as an ingredient in health or functional food, to alleviate oxidative stress.

Glucosamine-anchored doxorubicin-loaded targeted nano-niosomes: pharmacokinetic, toxicity and pharmacodynamic evaluation.

PubMed

Pawar, Smita; Shevalkar, Ganesh; Vavia, Pradeep

2016-09-01

Efficacy of anticancer drug is limited due to non-selectivity and toxicities allied with the drug; therefore the heart of the present work is to formulate drug delivery systems targeted selectively towards cancer cells with minimal toxicity to normal cells. Targeted drug delivery system of doxorubicin (DOX)-loaded niosomes using synthesized N-lauryl glucosamine (NLG) as a targeting ligand. NLG-anchored DOX niosomes were developed using ethanol injection method. Developed niosomes had particle size <150 nm and high entrapment efficiency ∼90%. In vivo pharmacokinetics exhibited long circulating nature of targeted niosomes with improved bioavailability, which significantly reduced CL and Vd than DOX solution and non-targeted niosomes (35 fold and 2.5 fold, respectively). Tissue-distribution study and enzymatic assays revealed higher concentration of DOX solution in heart while no toxicity to major organs with developed targeted niosomes was observed. Solid skin melanoma tumor model in mice manifested the commendable targeting potential of targeted niosomes with significant reduction in tumor volume and high % survival rate without drop in body weight in comparison with DOX solution and non-targeted niosomes of DOX. The glucosamine-anchored DOX-loaded targeted niosomes showed its potential in cancer targeted drug therapy with reduced toxicity. Abbreviations ALT alanine transaminase CL clearance CPK creatinine phosphokinase DOX doxorubicin EDC.HCL ethyl carbidimide hydrochloride GLUT glucose transporter GSH glutathione S-transferase LDH lactate dehydrogenase LHRH luteinizing hormone-releasing hormone MDA malonaldehyde NHS N-hydroxy succinimide NLG N-lauryl glucosamine NTAR DoxNio non-targeted doxorubicin niosomes PBS phosphate buffer saline RGD argynyl glycyl aspartic acid SGOT serum glutamate oxaloacetate transaminase SGPT serum glutamate pyruvate transaminase SOD superoxide dismutase TAR DoxNio targeted doxorubicin niosomes Vd volume of distribution.

Immunochemical characterization of the "native" type III polysaccharide of group B Streptococcus

PubMed Central

1976-01-01

The type III polysaccharide of -roup B Streptococcus has been isolated and purified by a method that employs washing of intact cells at neutral pH. That the polysaccharide prepared by this procedure is the "native" type III antigen is suggested by its molecular size in excess of 10(6) daltons, its degradation by acid and heat treatment to a fragment with immunologic characteristics of the classical HCl antigen, and its type-specific serologic activity. The type III polysaccharide in native form contains sialic acid, galactose, glucose, glucosamine, heptose, and mannose. It is acidic in nature, is resistant to neuramindiase degradation, contains no O-acetyl groups, and does not share antigenic determinants with capsular type K1 antigen of Escherichia coli or Group B polysaccharide antigen of Neiserria meningitidis. PMID:55450

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

PubMed

Vincent, Florence; Yates, David; Garman, Elspeth; Davies, Gideon J; Brannigan, James A

2004-01-23

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

PubMed Central

López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

2013-01-01

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

Progressive neurologic and somatic disease in a novel mouse model of human mucopolysaccharidosis type IIIC.

PubMed

Marcó, Sara; Pujol, Anna; Roca, Carles; Motas, Sandra; Ribera, Albert; Garcia, Miguel; Molas, Maria; Villacampa, Pilar; Melia, Cristian S; Sánchez, Víctor; Sánchez, Xavier; Bertolin, Joan; Ruberte, Jesús; Haurigot, Virginia; Bosch, Fatima

2016-09-01

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging because of the unfeasibility of cross-correction. In this study, we generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing investigation into sites of endogenous expression, which was particularly prominent in the CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches. © 2016. Published by The Company of Biologists Ltd.

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.

Protein O-linked ß-N-acetylglucosamine: A novel effector of cardiomyocyte metabolism and function

PubMed Central

Darley-Usmar, Victor M.; Ball, Lauren E.; Chatham, John C.

2014-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in O-GlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing O-GlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. PMID:21878340

Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

PubMed Central

Oh, Seung-Il; Park, Jin-Kook; Park, Sang-Kyu

2015-01-01

OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV), was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors. PMID:26039957

Early Urinary Markers of Diabetic Kidney Disease: A Nested Case-Control Study From the Diabetes Control and Complications Trial (DCCT)

PubMed Central

Kern, Elizabeth O; Erhard, Penny; Sun, Wanjie; Genuth, Saul; Weiss, Miriam F

2010-01-01

Background Urinary markers were tested as predictors of macroalbuminuria or microalbuminuria in type 1 diabetes. Study Design Nested case:control of participants in the Diabetes Control and Complications Trial (DCCT) Setting & Participants Eighty-seven cases of microalbuminuria were matched to 174 controls in a 1:2 ratio, while 4 cases were matched to 4 controls in a 1:1 ratio, resulting in 91 cases and 178 controls for microalbuminuria. Fifty-five cases of macroalbuminuria were matched to 110 controls in a 1:2 ratio. Controls were free of micro/macroalbuminuria when their matching case first developed micro/macroalbuminuria. Predictors Urinary N-acetyl-β-D-glucosaminidase, pentosidine, AGE fluorescence, albumin excretion rate (AER) Outcomes Incident microalbuminuria (two consecutive annual AER > 40 but <= 300 mg/day), or macroalbuminuria (AER > 300 mg/day) Measurements Stored urine samples from DCCT entry, and 1–9 years later when macroalbuminuria or microalbuminuria occurred, were measured for the lysosomal enzyme, N-acetyl-β-D-glucosaminidase, and the advanced glycosylation end-products (AGEs) pentosidine and AGE-fluorescence. AER and adjustor variables were obtained from the DCCT. Results Sub-microalbuminuric levels of AER at baseline independently predicted microalbuminuria (adjusted OR 1.83; p<.001) and macroalbuminuria (adjusted OR 1.82; p<.001). Baseline N-acetyl-β-D-glucosaminidase independently predicted macroalbuminuria (adjusted OR 2.26; p<.001), and microalbuminuria (adjusted OR 1.86; p<.001). Baseline pentosidine predicted macroalbuminuria (adjusted OR 6.89; p=.002). Baseline AGE fluorescence predicted microalbuminuria (adjusted OR 1.68; p=.02). However, adjusted for N-acetyl-β-D-glucosaminidase, pentosidine and AGE-fluorescence lost predictive association with macroalbuminuria and microalbuminuria, respectively. Limitations Use of angiotensin converting-enzyme inhibitors was not directly ascertained, although their use was proscribed during the DCCT. Conclusions Early in type 1 diabetes, repeated measurements of AER and urinary NAG may identify individuals susceptible to future diabetic nephropathy. Combining the two markers may yield a better predictive model than either one alone. Renal tubule stress may be more severe, reflecting abnormal renal tubule processing of AGE-modified proteins, among individuals susceptible to diabetic nephropathy. PMID:20138413

Histochemical Characterization of Oocytes in the Pink Cuskeel (Genypterus blacodes).

PubMed

Cohen, Stefanía; Petcoff, Gladys; Freijo, Roberto O; Portiansky, Enrique L; Barbeito, Claudio G; Macchi, Gustavo J; Díaz, Alcira O

2015-08-01

In the present study we histochemically and lectinhistochemically characterized the growing oocytes of the pink cuskeel (Genypterus blacodes). We used histochemical methods for the localization and characterization of glycoconjugates (GCs) and lectin histochemical techniques for the identification of specific sugar residues. We analyzed presence and distribution of GCs in the different structures of the growing follicles (cortical alveoli, globules, yolk granules and zona radiata). During the initial stage of vitellogenesis, the oocytes presented small yolk granules composed of GCs that gradually increased during exogenous vitellogenesis. These GCs contained moderate quantities of α-D-mannose, D-glucose, N-acetylglucosamine and N-acetyl-neuraminic acid. The cortical alveoli contained both neutral and carboxylated GCs, and lectin techniques detected N-acetylgalactosamine, galactose and L-fucose. The zona radiata showed a strong positive reaction to PAS and it reacted weakly with more specific techniques, such as KOH/PA*S and PA/Bh/KOH/PAS. This structure showed GCs with oxidizable vicinal diols, O-acyl sugars and sialic acid residues with different substitution types and presented N-acetylgalactosamine and L-fucose specific residues. The oocytes follicular envelope evidenced neutral and acidic non-sulfated GCs and high concentrations of α-D-mannose, D-glucose, galactose and N-acetylgalactosamine. The intergranular cytoplasmic GCs were mainly rich in α-D-mannose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine and N-acetyl-neuraminic acid. These results enhance the comprehension of the structure and functionality of the pink cuskeel ovarian follicles, and provide a useful tool for the study of this tissue in other teleost species.

Rabbit N-acetyltransferase 2 genotyping method to investigate role of acetylation polymorphism on N- and O-acetylation of aromatic and heterocyclic amine carcinogens.

PubMed

Hein, David W; Doll, Mark A

2017-09-01

The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 (NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine (p = 0.0014), benzidine (p = 0.0257), 4-aminobiphenyl (p = 0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p = 0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly (p > 0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens.

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

Acetylation at lysine 346 controls the transforming activity of the HTLV-1 Tax oncoprotein in the Rat-1 fibroblast model

PubMed Central

2013-01-01

Background Transformation by the Tax oncoprotein of the human T cell leukemia virus type 1 (HTLV-1) is governed by actions on cellular regulatory signals, including modulation of specific cellular gene expression via activation of signaling pathways, acceleration of cell cycle progression via stimulation of cyclin-dependent kinase activity leading to retinoblastoma protein (pRb) hyperphosphorylation and perturbation of survival signals. These actions control early steps in T cell transformation and development of Adult T cell leukemia (ATL), an aggressive malignancy of HTLV-1 infected T lymphocytes. Post-translational modifications of Tax by phosphorylation, ubiquitination, sumoylation and acetylation have been implicated in Tax-mediated activation of the NF-κB pathway, a key function associated with Tax transforming potential. Results In this study, we demonstrate that acetylation at lysine K346 in the carboxy-terminal domain of Tax is modulated in the Tax nuclear bodies by the acetyltransferase p300 and the deacetylases HDAC5/7 and controls phosphorylation of the tumor suppressor pRb by Tax-cyclin D3-CDK4-p21CIP complexes. This property correlates with the inability of the acetylation deficient K346R mutant, but not the acetylation mimetic K346Q mutant, to promote anchorage-independent growth of Rat-1 fibroblasts. By contrast, acetylation at lysine K346 had no effects on the ability of Tax carboxy-terminal PDZ-binding domain to interact with the tumor suppressor hDLG. Conclusions The identification of the acetyltransferase p300 and the deacetylase HDAC7 as enzymes modulating Tax acetylation points to new therapeutic targets for the treatment of HTLV-1 infected patients at risk of developing ATL. PMID:23880157

A newly synthetic chromium complex-chromium (D-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transport.

PubMed

Zhao, Peng; Wang, Jingying; Ma, Heng; Xiao, Yao; He, Leilei; Tong, Chao; Wang, Zhenhua; Zheng, Qiusheng; Dolence, E Kurt; Nair, Sreejayan; Ren, Jun; Li, Ji

2009-03-15

We synthesized the chromium (phenylalanine)(3) [Cr(D-phe)(3)] by chelating chromium(III) with D-phenylalanine ligand in aqueous solution to improve the bioavailability of chromium, and reported that Cr(D-phe)(3) improved insulin sensitivity. AMP-activated protein kinase (AMPK) is a key mediator for glucose uptake and insulin sensitivity. To address the molecular mechanisms by which Cr(d-phe)(3) increases insulin sensitivity, we investigated whether Cr(D-phe)(3) stimulates glucose uptake via activation of AMPK signaling pathway. H9c2 myoblasts and isolated cardiomyocytes were treated with Cr(D-phe)(3) (25microM). Western blotting was used for signaling determination. The glucose uptake was determined by 2-deoxy-D-glucose-(3)H accumulation. HPLC measured concentrations of AMP. The mitochondrial membrane potential (Deltapsi) was detected by JC-1 fluorescence assay. Cr(D-phe)(3) stimulated the phosphorylation of alpha catalytic subunit of AMPK at Thr(172), as well the downstream targets of AMPK, acetyl-CoA carboxylase (ACC, Ser(212)) and eNOS (Ser(1177)). Moreover, Cr(D-phe)(3) significantly stimulated glucose uptake in both H9c2 cells and cardiomyocytes. AMPK inhibitor compound C (10microM) dramatically inhibited the glucose uptake stimulated by Cr(D-phe)(3), while it did not affect insulin stimulation of glucose uptake. Furthermore, in vivo studies showed that Cr(D-phe)(3) also activated cardiac AMPK signaling pathway. The increase of cardiac AMP concentration and the decrease of mitochondrial membrane potential (Deltapsi) may contribute to the activation of AMPK induced by Cr(D-phe)(3). Cr(D-phe)(3) is a novel compound that activates AMPK signaling pathway, which contributes to the regulation of glucose transport during stress conditions that may be associated the role of AMPK in increasing insulin sensitivity.

Arylamine N-acetyltransferase 2 genotype-dependent N-acetylation of isoniazid in cryopreserved human hepatocytes.

PubMed

Doll, Mark A; Salazar-González, Raúl A; Bodduluri, Srineil; Hein, David W

2017-07-01

Cryopreserved human hepatocytes were used to investigate the role of arylamine N -acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N -acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N -acetylation was measured by high performance liquid chromatography. INH N -acetylation rates in vitro exhibited a robust and highly significant ( P <0.005) NAT2 phenotype-dependent metabolism. N -acetylation rates in situ were INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly ( P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.

The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

PubMed Central

Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

2013-01-01

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

Mechanism of α-Glycerophosphate Regulation of Acetyl-Coenzyme A Carboxylase of Saccharomyces cerevisiae

PubMed Central

Rasmussen, Roberta K.; Klein, Harold P.

1968-01-01

The mechanism proposed for the activation of animal acetyl-coenzyme A (CoA) carboxylase by α-glycerophosphate, namely, the removal of inhibitory palmityl-CoA via glyceride synthesis, is not the only possible one in the yeast system because extracts exhibiting marked stimulation of acetyl-CoA carboxylase activity by α-glyerophosphate show a lack of acyl-CoA compounds. PMID:5643049

JNK1 regulates histone acetylation in trigeminal neurons following chemical stimulation

PubMed Central

Wu, Jing; Zhang, Xuan; Nauta, Haring J; Lin, Qing; Li, Junfa; Fang, Li

2008-01-01

Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure. PMID:18822271

Codominant Expression of N-Acetylation and O-Acetylation Activities Catalyzed by N-Acetyltransferase 2 in Human Hepatocytes

PubMed Central

Doll, Mark A.; Zang, Yu; Moeller, Timothy

2010-01-01

Human populations exhibit genetic polymorphism in N-acetylation capacity, catalyzed by N-acetyltransferase 2 (NAT2). We investigated the relationship between NAT2 acetylator genotype and phenotype in cryopreserved human hepatocytes. NAT2 genotypes determined in 256 human samples were assigned as rapid (two rapid alleles), intermediate (one rapid and one slow allele), or slow (two slow alleles) acetylator phenotypes based on functional characterization of the NAT2 alleles reported previously in recombinant expression systems. A robust and significant relationship was observed between deduced NAT2 phenotype (rapid, intermediate, or slow) and N-acetyltransferase activity toward sulfamethazine (p < 0.0001) and 4-aminobiphenyl (p < 0.0001) and for O-acetyltransferase-catalyzed metabolic activation of N-hydroxy-4-aminobiphenyl (p < 0.0001), N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (p < 0.01), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (p < 0.0001). NAT2-specific protein levels also significantly associated with the rapid, intermediate, and slow NAT2 acetylator phenotypes (p < 0.0001). As a negative control, p-aminobenzoic acid (an N-acetyltransferase 1-selective substrate) N-acetyltransferase activities from the same samples did not correlate with the three NAT2 acetylator phenotypes (p > 0.05). These results clearly document codominant expression of human NAT2 alleles resulting in rapid, intermediate, and slow acetylator phenotypes. The three phenotypes reflect levels of NAT2 protein catalyzing both N- and O-acetylation. Our results suggest a significant role of NAT2 acetylation polymorphism in arylamine-induced cancers and are consistent with differential cancer risk and/or drug efficacy/toxicity in intermediate compared with rapid or slow NAT2 acetylator phenotypes. PMID:20430842

Production of Nα-acetylated thymosin α1 in Escherichia coli

PubMed Central

2011-01-01

Background Thymosin α1 (Tα1), a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. Results To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase) in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols β-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. Conclusions The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides. PMID:21513520

A novel ion-exclusion chromatography-mass spectrometry method to measure concentrations and cycling rates of carbohydrates and amino sugars in freshwaters.

PubMed

Horňák, Karel; Pernthaler, Jakob

2014-10-24

The concentrations of free neutral carbohydrates and amino sugars were determined in freshwater samples of distinct matrix complexity, including meso-, eu- and dystrophic lakes and ponds, using high-performance ion-exclusion chromatography (HPIEC) coupled to mass spectrometry (MS). In contrast to other methods, our approach allowed the quantification of free neutral carbohydrates and amino sugars at low nM concentrations without derivatization, de-salting or pre-concentration. New sample preparation procedures were applied prior to injection employing syringe and hollow fiber filtration. Analytes were separated on a strong cation exchange resin under 100% aqueous conditions using 0.1% formic acid as a mobile phase. To minimize background noise in MS, analytes were detected in a multiple reaction monitoring scan mode with double ion filtering. Detection limits of carbohydrates and amino sugars ranged between 0.2 and 2nM at a signal-to-noise ratio >5. Error ranged between 1 and 12% at 0.5-500nM levels. Using a stable isotope dilution approach, both the utilization and recycling of glucose in Lake Zurich was observed. In contrast, N-acetyl-glucosamine was equally rapidly consumed but there was no visible de novo production. The simple and rapid sample preparation makes our protocol suitable for routine analyses of organic compounds in freshwater samples. Application of stable isotope tracers along with accurate measures of carbohydrate and amino sugar concentrations enables novel insights into the compound in situ dynamics. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-01-01

Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be related to aniline nor to N-acetyl-4-aminophenol in man. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

Who Uses Glucosamine and Why? A Study of 266,848 Australians Aged 45 Years and Older

PubMed Central

Sibbritt, David; Adams, Jon; Lui, Chi-Wai; Broom, Alex; Wardle, Jonathan

2012-01-01

Objectives There has been a dramatic increase in the use of complementary medicines over recent decades. Glucosamine is one of the most commonly used complementary medicines in Western societies. An understanding of glucosamine consumption is of significance for public health and future health promotion. This paper, drawing upon the largest dataset to date with regards to glucosamine use (n = 266,844), examines the use and users of glucosamine amongst a sample of older Australians. Design Analysis of the self-reported data on use of glucosamine, demographics and health status as extracted from the dataset of the 45 and Up Study, which is the largest study of healthy ageing ever undertaken in the Southern Hemisphere involving over 265,000 participants aged 45 and over. Results Analysis reveals that 58,630 (22.0%) participants reported using glucosamine in the 4 weeks prior to the survey. Use was higher for those who were female, non-smokers, residing in inner/outer regional areas, with higher income and private health insurance. Of all the health conditions examined only osteoarthritis was positively associated with use of glucosamine, while cancer, heart attack or angina and other heart disease were all negatively associated with glucosamine use. Conclusions This study suggests that a considerable proportion of the Australia population aged 45 and over consume glucosamine. There is a need for health care practitioners to enquire with their patients about their use of glucosamine and for further attention to be directed to providing good quality information for patients and providers with regards to glucosamine products. PMID:22859995

Deep aquifer prokaryotic community responses to CO2 geosequestration

NASA Astrophysics Data System (ADS)

Mu, A.; Moreau, J. W.

2015-12-01

Little is known about potential microbial responses to supercritical CO2 (scCO2) injection into deep subsurface aquifers, a currently experimental means for mitigating atmospheric CO2 pollution being trialed at several locations around the world. One such site is the Paaratte Formation of the Otway Basin (~1400 m below surface; 60°C; 2010 psi), Australia. Microbial responses to scCO2 are important to understand as species selection may result in changes to carbon and electron flow. A key aim is to determine if biofilm may form in aquifer pore spaces and reduce aquifer permeability and storage. This study aimed to determine in situ, using 16S rRNA gene, and functional metagenomic analyses, how the microbial community in the Otway Basin geosequestration site responded to experimental injection of 150 tons of scCO2. We demonstrate an in situ sampling approach for detecting deep subsurface microbial community changes associated with geosequestration. First-order level analyses revealed a distinct shift in microbial community structure following the scCO2 injection event, with proliferation of genera Comamonas and Sphingobium. Similarly, functional profiling of the formation revealed a marked increase in biofilm-associated genes (encoding for poly-β-1,6-N-acetyl-D-glucosamine). Global analysis of the functional gene profile highlights that scCO2 injection potentially degraded the metabolism of CH4 and lipids. A significant decline in carboxydotrophic gene abundance (cooS) and an anaerobic carboxydotroph OTU (Carboxydocella), was observed in post-injection samples. The potential impacts on the flow networks of carbon and electrons to heterotrophs are discussed. Our findings yield insights for other subsurface systems, such as hydrocarbon-rich reservoirs and high-CO2 natural analogue sites.

Shewanella halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile.

PubMed

Zhao, Jian-Shen; Manno, Dominic; Leggiadro, Cindy; O'Neil, David; Hawari, Jalal

2006-01-01

Indigenous bacteria found in the sediment of the Emerald Basin (depth of 215 m, Atlantic Ocean) located offshore of Halifax Harbour (Nova Scotia, Canada) were previously found to be able to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study, a novel obligately respiratory, denitrifying and RDX-mineralizing bacterium, designated strain HAW-EB4(T), was isolated from the marine sediment. This bacterium utilized peptone, yeast extract, Casamino acids, esters (Tweens 20, 40 and 80), sugars (N-acetyl-D-glucosamine, ribose), several C2 and C3 acids (acetate, pyruvate, lactate, propionate) and amino acids (serine, proline) as sole carbon and energy sources. Aerobically grown cells (in marine broth 2216 at 10 degrees C) contained C(14 : 0) (6 %), iso-C(15 : 0) (12 %), C(16 : 0) (20 %), C(16 : 1)omega7 (37 %), C(18 : 1)omega7 (7 %) and C(20 : 5)omega3 (7 %) as major membrane fatty acids, and Q7 (28.1 %) and MK-7 (60.9 %) as dominant respiratory quinones, consistent with deep-sea species of Shewanella. The novel bacterium had a DNA G+C content of 45 mol% and showed similarity to Shewanella species in terms of 16S rRNA and gyrB gene sequences (93-99 and 67.3-88.4 % similarity, respectively), with Shewanella pealeana being the most closely related species. Genomic DNA-DNA hybridization between strain HAW-EB4T and S. pealeana revealed a level of relatedness of 17.9 %, lower than the 70 % species cut-off value, indicating that strain HAW-EB4T (= NCIMB 14093T = DSM 17350T) is the type strain of a novel species of Shewanella, for which the name Shewanella halifaxensis sp. nov. is proposed.

Wheat germ agglutinin-conjugated PLGA nanoparticles for enhanced intracellular delivery of paclitaxel to colon cancer cells.

PubMed

Wang, Chunxia; Ho, Paul C; Lim, Lee Yong

2010-11-15

The purpose of this study was to investigate the potentiation of the anticancer activity and enhanced cellular retention of paclitaxel-loaded PLGA nanoparticles after surface conjugation with wheat germ agglutinin (WGA) against colon cancer cells. Glycosylation patterns of representative colon cancer cells confirmed the higher expression levels of WGA-binding glycoproteins in the Caco-2 and HT-29 cells, than in the CCD-18Co cells. Cellular uptake and in vitro cytotoxicity of WNP (final formulation) against colon cell lines was evaluated alongside control formulations. Confocal microscopy and quantitative analysis of intracellular paclitaxel were used to monitor the endocytosis and retention of nanoparticles inside the cells. WNP showed enhanced anti-proliferative activity against Caco-2 and HT-29 cells compared to corresponding nanoparticles without WGA conjugation (PNP). The greater efficacy of WNP was associated with higher cellular uptake and sustained intracellular retention of paclitaxel, which in turn was attributed to the over-expression of N-acetyl-D-glucosamine-containing glycoprotein on the colon cell membrane. WNP also demonstrated increased intracellular retention in the Caco-2 (30% of uptake) and HT-29 (40% of uptake) cells, following post-uptake incubation with fresh medium, compared to the unconjugated PNP nanoparticles (18% in Caco-2) and (27% in HT-29), respectively. Cellular trafficking study of WNP showed endocytosed WNP could successful escape from the endo-lysosome compartment and release into the cytosol with increasing incubation time. It may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer. Copyright © 2010 Elsevier B.V. All rights reserved.

Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic, Synergistic Lepidopteran-larvicidal and Nematicidal Activities

PubMed Central

Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

2015-01-01

Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849

Translational Repression of NhaR, a Novel Pathway for Multi-Tier Regulation of Biofilm Circuitry by CsrA

PubMed Central

Pannuri, Archana; Yakhnin, Helen; Vakulskas, Christopher A.; Edwards, Adrianne N.; Babitzke, Paul

2012-01-01

The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na+] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5′ untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR′-′lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems. PMID:22037401

Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

PubMed Central

Sochor, Jiri; Zitka, Ondrej; Hynek, David; Jilkova, Eva; Krejcova, Ludmila; Trnkova, Libuse; Adam, Vojtech; Hubalek, Jaromir; Kynicky, Jindrich; Vrba, Radimir; Kizek, Rene

2011-01-01

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components. PMID:22346664

Evaluation of pathogenicity of Candida albicans in germination-ready states using a silkworm infection model.

PubMed

Matsumoto, Haruhito; Nagao, Jun-ichi; Cho, Tamaki; Kodama, Jun

2013-01-01

We previously developed an N-acetyl-D-glucosamine (GlcNAc) medium which induces Candida albicans to undergo a yeast-to-hyphal transition through a cAMP-PKA pathway. Microarray analysis demonstrated that 18 genes, including ALS3 that encodes a cell wall adhesion, were upregulated by 30-min incubation of yeast cells at 37°C in the GlcNAc medium. To investigate the differences between morphological transition and morphotype in C. albicans as a consequence of infection, this study utilized a silkworm infection model as an invertebrate mini-host. We prepared 3 different conditions of C. albicans cells in vitro by changing the incubation times in the GlcNAc medium: yeast-form cells at 0 min (Y0 cells), yeast-form cells in germination-ready state at 60 min (Y60 cells), and hyphal cells at 120 min (H120 cells), and compared their pathogenicities. We performed the infection study at various temperatures to find temperature-dependent virulence factors in vivo. Y60 cells in germination-ready state in the GlcNAc medium showed higher pathogenicity in vivo compared to Y0 and H120 cells at 30°C. Y60 cells proliferated in silkworms 24 h post-injection at 30°C, whereas the other 2 cell types did not. In vitro analysis demonstrated that Y60 cells, but not Y0 cells, germinated in the silkworm hemolymph at 30°C. However, Y0 and Y60 cells showed a similar degree of germination in the silkworm hemolymph at 37°C, although no significant difference in silkworm survival after infection with each cell type was observed at 37°C. These results suggested that the germination-ready state induced by the GlcNAc medium contributed to virulence in the silkworm.

Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae.

PubMed

Hathroubi, S; Fontaine-Gosselin, S-È; Tremblay, Y D N; Labrie, J; Jacques, M

2015-09-30

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-D-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components. Copyright © 2015 Elsevier B.V. All rights reserved.

The Use of Drugs to Reduce Hearing Loss Following Acute Acoustic Trama

DTIC Science & Technology

2012-07-01

noise exposure: (1) L-N- acetylcysteine (L-NAC); (2) D-Methionine (D-MET); (3) Ebselen SPI-1005; (4) Acetyl-L-carnitine (ALCAR) and (5) Src-PTK inhibitor...exposed to a 4.0 kHz octave band of noise for 6 hours at 105 dB SPL: (1) L-N- acetylcysteine (L-NAC); (2) D-Methionine (D-MET); (3) Ebselen SPI-1005...effectively absent or severely depressed from 2 kHz and above (Figs. 14 & 15). Individual cochleograms for this group are shown in Figure 16. The

N1-acetyl substituted pyrrolidine derivative CIP-A5: a novel compound that could ameliorate liver cirrhosis through modulation of hepatic stellate cell activity.

PubMed

Wang, Xiao-Dan; Gao, Zu-Hua; Xue, Xia; Cheng, Yan-Na; Yue, Pan; Fang, Xu-Wen; Qu, Xian-Jun

2011-06-01

(2S,4R)-methyl 1-acetyl-4-(N-(4-bromophenyl)sulfamoyloxy)pyrrolidine-2-carboxylate (CIP-A5) is the N1-acetyl substituted pyrrolidine derivative which was designed against the structure of matrix metalloproteinase (MMP-2) and MMP-9. CIP-A5 has been considered as a candidate compound for treatment of liver cirrhosis. In this study, we evaluated the efficacy of CIP-A5 on the activity of hepatic stellate cells. CIP-A5 prevented the transforming growth factor β (TGF-β)-induced proliferation of hepatic stellate HSC-T6 cells as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. CIP-A5 stimulated MMPs activity as evidenced by an increase of degradation of succinylated gelatin. Gelatin zymography analysis showed that CIP-A5 stimulated the secretion and activity of MMP-2 and MMP-9 in HSC-T6 cells. This stimulatory effect on MMPs was verified by the observation of increased expression of MMP-2 and MMP-9 as evaluated by Western blot assay. At the same time, a significant decrease of the expression of tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) was observed, suggesting a modulation of the balance of MMPs/TIMPs in hepatic stellate cells. CIP-A5 treatment also resulted in suppression of the profibrogenic cytokines, such as TGF-β, tumor necrosis factor alpha (TNF-α) and connective tissue growth factor (CTGF) in HSC-T6 cells. CIP-A5 did not have cytotoxicity to human normal hepatic cells. These results implied that CIP-A5 could selectively ameliorate the process of liver cirrhosis through modulation of activated hepatic stellate cell activity, which offers hope for prevention and treatment of this devastating end-stage liver disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

PubMed

Dadashipour, Mohammad; Iwamoto, Mariko; Hossain, Mohammad Murad; Akutsu, Jun-Ichi; Zhang, Zilian; Kawarabayasi, Yutaka

2018-05-15

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways. IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially. Copyright © 2018 American Society for Microbiology.

Characterisation of neuroprotective efficacy of modified poly-arginine-9 (R9) peptides using a neuronal glutamic acid excitotoxicity model.

PubMed

Edwards, Adam B; Anderton, Ryan S; Knuckey, Neville W; Meloni, Bruno P

2017-02-01

In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1-6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.

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

PubMed

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

2016-01-01

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

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

PubMed Central

Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

2009-01-01

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

Non-enzymatic interactions of glyoxylate with lysine, arginine, and glucosamine: a study of advanced non-enzymatic glycation like compounds.

PubMed

Dutta, Udayan; Cohenford, Menashi A; Guha, Madhumita; Dain, Joel A

2007-02-01

Glyoxylate is a 2 carbon aldo acid that is formed in hepatic tissue from glycolate. Once formed, the molecule can be converted to glycine by alanine-glyoxylate aminotransferase (AGAT). In defects of AGAT, glyoxylate is transformed to oxalate, resulting in high levels of oxalate in the body. The objective of this study was 2-fold. First, it was to determine, if akin to D-glucose, D-fructose or DL-glyceraldehyde, glyoxylate was susceptible to non-enzymatic attack by amino containing molecules such as lysine, arginine or glucosamine. Second, if by virtue of its molecular structure and size, glyoxylate was as reactive a reagent in non-enzymatic reactions as DL-glyceraldehyde; i.e., a glycose that we previously demonstrated to be a more effective glycating agent than D-glucose or D-fructose. Using capillary electrophoresis (CE), high performance liquid chromatography and UV and fluorescence spectroscopy, glyoxylate was found to be a highly reactive precursor of advanced glycation like end products (AGLEs) and a more effective promoter of non-enzymatic end products than D-glucose, D-fructose or DL-glyceraldehyde.

Synthesis and biological characterization of novel charge-deficient spermine analogues.

PubMed

Weisell, Janne; Hyvönen, Mervi T; Häkkinen, Merja R; Grigorenko, Nikolay A; Pietilä, Marko; Lampinen, Anita; Kochetkov, Sergey N; Alhonen, Leena; Vepsäläinen, Jouko; Keinänen, Tuomo A; Khomutov, Alex R

2010-08-12

Biogenic polyamines, spermidine and spermine, are positively charged at physiological pH. They are present in all cells and essential for their growth and viability. Here we synthesized three novel derivatives of the isosteric charge-deficient spermine analogue 1,12-diamino-3,6,9-triazadodecane (SpmTrien, 5a) that are N(1)-Ac-SpmTrien (5c), N(12)-Ac-SpmTrien (5b), and N(1),N(12)-diethyl-1,12-diamino-3,6,9-triazadodecane (N(1),N(12)-Et(2)-SpmTrien, 5d). 5a and 5d readily accumulated in DU145 cells at the same concentration range as natural polyamines and moderately competed for the uptake with putrescine (1) but not with spermine (4a) or spermidine (2). 5a efficiently down-regulated ornithine decarboxylase and decreased polyamine levels, while 5d proved to be inefficient, compared with N(1),N(11)-diethylnorspermine (6). None of the tested analogues were substrates for human recombinant spermine oxidase, but those having free aminoterminus, including 1,8-diamino-3,6-diazaoctane (Trien, 3a), were acetylated by mouse recombinant spermidine/spermine N(1)-acetyltransferase. 5a was acetylated to 5c and 5b, and the latter was further metabolized by acetylpolyamine oxidase to 3a, a drug used to treat Wilson's disease. Thus, 5a is a bioactive precursor of 3a with enhanced bioavailability.

A simple, convenient method to synthesize cobalamins: synthesis of homocysteinylcobalamin, N -acetylcysteinylcobalamin, 2-N -acetylamino-2-carbomethoxyethanethiolatocobalamin, sulfitocobalamin and nitrocobalamin†

PubMed Central

Suarez-Moreira, Edward; Hannibal, Luciana; Smith, Clyde A.; Chavez, Roberto A.; Jacobsen, Donald W.

2009-01-01

Glutathionylcobalamin, nitrocobalamin and sulfitocobalamin are important cobalamin metabolites isolable from human tissues. Herein we demonstrate that a procedure used to synthesize and isolate γ-glutamylcysteinylcobalamin and glutathionylcobalamin in aqueous solution in high yield and purity can be used to synthesize other novel, biologically relevant thiolatocobalamins, including D,L-homocysteinylcobalamin, N-acetyl-L-cysteinylcobalamin (Na+ salt) and 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin, as well as other non-alkylcobalamins, such as sulfitocobalamin (Na+ salt) and nitrocobalamin. This uncomplicated, general procedure will assist researchers in identifying unknown cobalamin metabolites isolated from biological samples, and researchers interested in studying the uptake and intracellular cobalamin processing mechanisms utilizing non-alkylcobalamin derivatives that are not yet commercially available. The X-ray structure and XAS spectrum of N-acetyl-L-cysteinylcobalamin are also presented. PMID:17088966

Glucosamine prevents in vitro collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

PubMed Central

Tiku, Moti L; Narla, Haritha; Jain, Mohit; Yalamanchili, Praveen

2007-01-01

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine. PMID:17686167

Investigation of NAA and NAAG dynamics underlying visual stimulation using MEGA-PRESS in a functional MRS experiment

PubMed Central

Landim, Ricardo C.G.; Edden, Richard A.E.; Foerster, Bernd; Li, Li Min; Covolan, Roberto J.M.; Castellano, Gabriela

2017-01-01

N-acetyl-aspartate (NAA) is responsible for the majority of the most prominent peak in 1H-MR spectra, and has been used as diagnostic marker for several pathologies. However, ~10% of this peak can be attributed to N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide whose release may be triggered by intense neuronal activation. Separate measurement of NAA and NAAG using MRS is difficult due to large superposition of their spectra. Specifically, in functional MRS (fMRS) experiments, most work has evaluated the sum NAA + NAAG, which does not appear to change during experiments. The aim of this work was to design and perform an fMRS experiment using visual stimulation and a spectral editing sequence, MEGA-PRESS, to further evaluate the individual dynamics of NAA and NAAG during brain activation. The functional paradigm used consisted of three blocks, starting with a rest (baseline) block of 320 s, followed by a stimulus block (640 s) and a rest block (640 s). Twenty healthy subjects participated in this study. On average, subjects followed a pattern of NAA decrease and NAAG increase during stimulation, with a tendency to return to basal levels at the end of the paradigm, with a peak NAA decrease of −(21 ± 19)% and a peak NAAG increase of (64 ± 62)% (Wilcoxon test, p < 0.05). These results may relate to: 1) the only known NAAG synthesis pathway is from NAA and glutamate; 2) a relationship between NAAG and the BOLD response. PMID:26656908

Investigation of NAA and NAAG dynamics underlying visual stimulation using MEGA-PRESS in a functional MRS experiment.

PubMed

Landim, Ricardo C G; Edden, Richard A E; Foerster, Bernd; Li, Li Min; Covolan, Roberto J M; Castellano, Gabriela

2016-04-01

N-acetyl-aspartate (NAA) is responsible for the majority of the most prominent peak in (1)H-MR spectra, and has been used as diagnostic marker for several pathologies. However, ~10% of this peak can be attributed to N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide whose release may be triggered by intense neuronal activation. Separate measurement of NAA and NAAG using MRS is difficult due to large superposition of their spectra. Specifically, in functional MRS (fMRS) experiments, most work has evaluated the sum NAA+NAAG, which does not appear to change during experiments. The aim of this work was to design and perform an fMRS experiment using visual stimulation and a spectral editing sequence, MEGA-PRESS, to further evaluate the individual dynamics of NAA and NAAG during brain activation. The functional paradigm used consisted of three blocks, starting with a rest (baseline) block of 320 s, followed by a stimulus block (640 s) and a rest block (640 s). Twenty healthy subjects participated in this study. On average, subjects followed a pattern of NAA decrease and NAAG increase during stimulation, with a tendency to return to basal levels at the end of the paradigm, with a peak NAA decrease of -(21±19)% and a peak NAAG increase of (64±62)% (Wilcoxon test, p<0.05). These results may relate to: 1) the only known NAAG synthesis pathway is from NAA and glutamate; 2) a relationship between NAAG and the BOLD response. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

1980-01-01

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

The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

PubMed

Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

2008-05-01

The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

Mimicking multivalent protein-carbohydrate interactions for monitoring the glucosamine level in biological fluids and pharmaceutical tablets.

PubMed

Dey, Nilanjan; Bhattacharya, Santanu

2017-05-11

An easily synthesizable probe has been employed for dual mode sensing of glucosamine in pure water. The method was also applied for glucosamine estimation in blood serum samples and pharmaceutical tablets. Further, selective detection of glucosamine was also achieved using portable color strips.

Genetically Epilepsy-Prone Rats Have Increased Brain Regional Activity of an Enzyme Which Liberates Glutamate from N-acetyl-aspartyl-glutamate

DTIC Science & Technology

1992-01-01

DISTRIBUTION C OOt .APPROVED FOR PUPLIC RELEASE: DISTRIBUTION UNLIMITED Ii. A STRA T (Minls.m200oids N-Acetylated-a- 1 n (’ed acidic dip cpL,2ase (N...aspartate (NAA) and the excitatory amino acid , glutamate (CLU). Although there is evidence that NAAG might be a neurotransmitter, this dipoptide could...Genetics; Itippocampus: E-ctlsatlltmt pilepsy-, Glutamate: N-Acetylated-o-1 inked acidic dipeptidasc-: Enrniatic: IIrosz:NAAG: Aspartalc N-Acetylated-a

A target oriented expeditious approach towards synthesis of certain bacterial rare sugar derivatives.

PubMed

Chaudhury, Aritra; Ghosh, Rina

2017-02-07

Bacterial rare amino deoxy sugars are found in the cell surface polysaccharides of multiple pathogenic bacterial strains, but are absent in the human metabolism. This helps in the differentiation between pathogens and host cells which can be exploited for target specific drug discovery and carbohydrate based vaccine development. The principal bacterial atypical sugar derivatives include 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (AAT), 2,4-diacetamido-2,4,6-trideoxy-d-galactose (DATDG) and N-acetylfucosamine (FucNAc). Herein, a highly streamlined protocol leading to the aforesaid derivatives is presented. The highlights of the method lie in radical mediated 6-deoxygenation along with a one-pot like protection profile manipulation on suitably derivatised d-glucosamine or d-mannose motifs to obtain a vital quinovosaminoside or rhamnoside from which rare sugar derivatives were synthesized in a diversity oriented manner.

Dopamine D2High receptors stimulated by phencyclidines, lysergic acid diethylamide, salvinorin A, and modafinil.

PubMed

Seeman, Philip; Guan, Hong-Chang; Hirbec, Hélène

2009-08-01

Although it is commonly stated that phencyclidine is an antagonist at ionotropic glutamate receptors, there has been little measure of its potency on other receptors in brain tissue. Although we previously reported that phencyclidine stimulated cloned-dopamine D2Long and D2Short receptors, others reported that phencyclidine did not stimulate D2 receptors in homogenates of rat brain striatum. This study, therefore, examined whether phencyclidine and other hallucinogens and psychostimulants could stimulate the incorporation of [(35)S]GTP-gamma-S into D2 receptors in homogenates of rat brain striatum, using the same conditions as previously used to study the cloned D2 receptors. Using 10 microM dopamine to define 100% stimulation, phencyclidine elicited a maximum incorporation of 46% in rat striata, with a half-maximum concentration of 70 nM for phencyclidine, when compared with 80 nM for dopamine, 89 nM for salvinorin A (48 nM for D2Long), 105 nM for lysergic acid diethylamide (LSD), 120 nM for R-modafinil, 710 nM for dizocilpine, 1030 nM for ketamine, and >10,000 nM for S-modafinil. These compounds also inhibited the binding of the D2-selective ligand [(3)H]domperidone. The incorporation was inhibited by the presence of 200 microM guanylylimidodiphosphate and also by D2 blockade, using 10 microM S-sulpiride, but not by D1 blockade with 10 microM SCH23390. Hypertonic buffer containing 150 mM NaCl inhibited the stimulation by phencyclidine, which may explain negative results by others. It is concluded that phencyclidine and other psychostimulants and hallucinogens can stimulate dopamine D2 receptors at concentrations related to their behavioral actions.

Arylamine N-Acetyltransferases in Mycobacteria

PubMed Central

Sim, Edith; Sandy, James; Evangelopoulos, Dimitrios; Fullam, Elizabeth; Bhakta, Sanjib; Westwood, Isaac; Krylova, Anna; Lack, Nathan; Noble, Martin

2008-01-01

Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure. PMID:18680471

N-acetyl Aspartate Levels in Adolescents With Bipolar and/or Cannabis Use Disorders

PubMed Central

Bitter, Samantha M.; Weber, Wade A.; Chu, Wen-Jang; Adler, Caleb M.; Eliassen, James C.; Strakowski, Stephen M.; DelBello, Melissa P.

2014-01-01

Objective Bipolar and cannabis use disorders commonly co-occur during adolescence, and neurochemical studies may help clarify the pathophysiology underlying this co-occurrence. This study compared metabolite concentrations in the left ventral lateral prefrontal cortex among: adolescents with bipolar disorder (bipolar group; n=14), adolescents with a cannabis use disorder (cannabis use group, n=13), adolescents with cannabis use and bipolar disorders (bipolar and cannabis group, n=25), and healthy adolescents (healthy controls, n=15). We hypothesized that adolescents with bipolar disorder (with or without cannabis use disorder) would have decreased N-acetyl aspartate levels in the ventral lateral prefrontal cortex compared to the other groups, and that the bipolar and cannabis group would have the lowest N-acetyl aspartate levels of all groups. Methods N-acetyl aspartate concentrations in the left ventral lateral prefrontal cortex were obtained using Proton Magnetic Resonance Spectroscopy. Results Adolescents with bipolar disorder showed significantly lower left ventral lateral prefrontal cortex N-acetyl aspartate levels, but post-hoc analyses indicated that this was primarily due to increased N-acetyl aspartate levels in the cannabis group. The cannabis use disorder group had significantly higher N-acetyl aspartate levels compared to the bipolar disorder and the bipolar and cannabis groups (p=0.0002 and p=0.0002, respectively). Pearson correlations revealed a significant positive correlation between amount of cannabis used and N-acetyl aspartate concentrations. Conclusions Adolescents with cannabis use disorder showed higher levels of N-acetyl aspartate concentrations that were significantly positively associated with the amount of cannabis used; however, this finding was not present in adolescents with comorbid bipolar disorder. PMID:24729763

[Chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 4. Study of the effect of a mixture of di- and tetrapeptides on a prolonged rat model of UV-induced cataract].

PubMed

Avetisov, S E; Polunin, G S; Sheremet, N L; Makarov, I A; Fedorov, A A; Karpova, O E; Muranov, K O; Dizhevskaia, A K; Soustov, L V; Chelnokov, E V; Bitiurin, N M; Sapogova, N V; Nemov, V V; Bodyrev, A A; Ostrovskiĭ, M A

2008-01-01

There is a potential of therapeutic action on certain stages of caractogenesis, in particular on the aggregation of water-soluble proteins of cytoplasmic lens fiber cells, giving rise to insoluble protein complexes. The effect of a combined preparation (N-acetyl carnosine and D-patethine), acting by the chaperon-like mechanism, was studied in vivo on a prolonged rat model of UV-induced cataract. The use of the combined preparation consisting of a mixture of peptides of N-acetyl carnosine and D-patethine in a ratio of 1:1 as ocular instillations and intraperitoneal injections could slow down the development of UV-induced cataract in vivo. Pathomorphological studies suggest that the combined preparation has a protective effect on lens tissue when the rat model of UV-induced cataract is employed.

Four new triterpene saponins from the seeds of Aesculus chinensis.

PubMed

Zhao, Jing; Yang, Xiu-Wei

2003-09-01

Two pairs of new geometrically isomeric triterpenoid saponins were isolated from the seeds of Aesculus chinensis and characterized as 28-acetyl-21-tigloylprotoaescigenin 3-O-[beta-D-xylopyranosyl (1 --> 2)] [beta-D-glucopyranosyl (1 --> 4)] [beta-D-glucopyranosiduronic acid (isoescin IIa, 1) and 28-acetyl-21-angeloylprotoaescigenin 3-O-[-beta-D-xylopyranosyl (1 --> 2)] [beta-D-glucopyranosyl (1 --> 4)] beta-D-glucopyranosiduronic acid (isoescin IIb, 2); 28-acetyl-21-tigloylbarringtogenol C 3-O-[beta-D-galactopyranosyl (1 --> 2)] [beta-D-glucopyranosyl (1 --> 4)] beta-D-glucopyranosiduronic acid (isoescin IIIa, 3) and 28-acetyl-21-angeloylbarringtogenol C 3-O-[beta-D-galactopyranosyl (1 --> 2)] [beta-D-glucopyranosyl (1 --> 4)] beta-D-glucopyranosiduronic acid (isoescin IIIb, 4). Their structures were established on the basis of spectroscopic and chemical evidence.

Continuous and massive intake of chitosan affects mineral and fat-soluble vitamin status in rats fed on a high-fat diet.

PubMed

Deuchi, K; Kanauchi, O; Shizukuishi, M; Kobayashi, E

1995-07-01

We investigated the effects of continuous and massive intake of chitosan with sodium ascorbate (AsN) on the mineral and the fat-soluble vitamin status in male Sprague-Dawley rats fed on a high-fat diet. The apparent fat digestibility in the chitosan-receiving group was significantly lower than that in the cellulose- or glucosamine-receiving group. Chitosan feeding for 2 weeks caused a decrease in mineral absorption and bone mineral content, and it was necessary to administer twice the amount of Ca in the AIN-76 formula, which was supplemented with AsN, to prevent such a decrease in the bone mineral content. Moreover, the ingestion of chitosan along with AsN led to a marked and rapid decrease in the serum vitamin E level, while such a loss in vitamin E was not observed for rats given glucosamine monomer instead of chitosan.

Facile synthesis of a two-photon fluorescent probe based on pyrimidine 2-isothiocyanate and its application in bioimaging.

PubMed

Yang, Jie; Hu, Wei; Li, Huirong; Hou, Hanna; Tu, Yi; Liu, Bo

2018-04-18

Two-photon microscopy imaging has been widely applied in biological imaging, but the development of two-photon absorption probes is obviously lagging behind in the development of imaging technology. In this paper, a two-photon fluorescent probe (1) based on pyrimidine 2-isothiocyanate has been designed and synthesized through a simple method for two-photon biological imaging. Probe 1 was able to couple effectively with the amino groups on biomolecules. To verify the reactivity of the isothiocyanate group on probe 1 and the amine groups on the biomolecules, d-glucosamine was chosen as a model biomolecule to conjugate with probe 1. The result showed that probe 1 could effectively conjugate with d-glucosamine to synthesize probe 2, and the yield of probe 2 was 83%. After conjugating with d-glucosamine, linear absorption spectra, single-photon fluorescence spectra, and two-photon fluorescence spectra of probes 1 and 2 did not present significant changes. Probes 1 and 2 exhibited high fluorescence quantum yields (0.71-0.79) in toluene and chloroform. They also exhibited different photo-physical properties in solvents with different polarities. The two-photon absorption cross-section of probe 1 was 953 GM in toluene. In addition, probe 1 could be effectively conjugated with transferrin, and the conjugated probe (Tf-1) could be transported into Hep G2 cells through a receptor-mediated process for biological imaging. These results demonstrate that such probes are expected to have great potential applications in two-photon fluorescence bioimaging.

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).

A novel ultrasensitive carboxymethyl chitosan-quantum dot-based fluorescence "turn on-off" nanosensor for lysozyme detection.

PubMed

Song, Yu; Li, Yang; Liu, Ziping; Liu, Linlin; Wang, Xinyan; Su, Xingguang; Ma, Qiang

2014-11-15

In this work, we developed an ultrasensitive "turn on-off" fluorescence nanosensor for lysozyme (Lyz) detection. The novel nanosensor was constructed with the carboxymethyl chitosan modified CdTe quantum dots (CMCS-QDs). Firstly, the CMCS-QDs were fabricated via the electrostatic interaction between amino groups in CMCS polymeric chains and carboxyl groups on the surface of QDs. In the fluorescence "turn-on" step, the strong binding ability between Zn(2+) and CMCS on the surface of QDs can enhance the photoluminescence intensity (PL) of QDs. In the following fluorescence "turn-off" step, the N-acetyl-glucosamine (NAG) section along the CMCS chains was hydrolyzed by Lyz. As a result, Zn(2+) was released from the surface of QDs, and the Lyz-QDs complexes were formed to quench the QDs PL. Under the optimal conditions, there was a good linear relationship between the PL of QDs and the Lyz concentration (0.1-1.2 ng/mL) with the detection limit of 0.031 ng/mL. The developed method was ultrasensitive, highly selective and fast. It has been successfully employed in the detection of Lyz in the serum with satisfactory results. Copyright © 2014 Elsevier B.V. All rights reserved.

Chitinosans as tableting excipients for modified release delivery systems.

PubMed

Rege, P R; Shukla, D J; Block, L H

1999-04-20

The term 'chitinosans' embraces the spectrum of acetylated poly(N-glucosamines) ranging from chitin to chitosan. Chitinosans (I), at acidic pH, have protonated amines which can interact with oppositely charged drug ions and, thereby, modify drug release from drug delivery systems. Tablets were compressed from a physical mixture containing salicylic acid (II) as the model drug, I, and magnesium stearate. Five commercial I compounds, varying in degree of deacetylation and molecular weight, were selected. Tablets were compressed at 5000, 10 000, and 15 000 psig using a Carver and a single punch tablet press. The differential scanning calorimetry thermograms provided evidence of I-II interaction in the powder blend. Analysis of variance (ANOVA) indicated that the compression pressure did not significantly affect the crushing strength (CS) or the release profile of II from the I-matrix tablets (P?0.05). Furthermore, the ANOVA also indicated that the tablet press used during manufacture did not affect the above properties (P?0.05); however, the chitinosans significantly affected the CS as well as the release profile of II from I-matrix tablets (P<0.05). This study provides further evidence for the use of commercial I compounds as excipients for use in modified release drug delivery systems. Copyright.

Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

PubMed

Westermayer, Sonja A; Fritz, Georg; Gutiérrez, Joaquín; Megerle, Judith A; Weißl, Mira P S; Schnetz, Karin; Gerland, Ulrich; Rädler, Joachim O

2016-05-01

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions. © 2016 John Wiley & Sons Ltd.

Structure of human O-GlcNAc transferase and its complex with a peptide substrate

PubMed Central

Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

2010-01-01

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

Postinflammatory hyperpigmentation: etiologic and therapeutic considerations.

PubMed

Callender, Valerie D; St Surin-Lord, Sharleen; Davis, Erica C; Maclin, Marissa

2011-04-01

Postinflammatory hyperpigmentation (PIH) is a reactive hypermelanosis and sequela of a variety of inflammatory skin conditions. PIH can have a negative impact on a patient's quality of life, particularly for darker-skinned patients. Studies show that dyschromias, including PIH, are one of the most common presenting complaints of darker-skinned racial ethnic groups when visiting a dermatologist. This is likely due to an increased production or deposition of melanin into the epidermis or dermis by labile melanocytes. A variety of endogenous or exogenous inflammatory conditions can culminate in PIH and typically most epidermal lesions will appear tan, brown, or dark brown while dermal hypermelanosis has a blue-gray discoloration. Depigmenting agents target different steps in the production of melanin, most commonly inhibiting tyrosinase. These agents include hydroquinone, azelaic acid, kojic acid, arbutin, and certain licorice (glycyrrhiza) extracts. Other agents include retinoids, mequinol, ascorbic acid (vitamin C), niacinamide, N-acetyl glucosamine, and soy, and these products depigment by different mechanisms. Certain procedures can also be effective in the treatment of PIH including chemical peeling and laser therapy. It is important to note that these same therapeutic modalities may also play a role in causing PIH. Lastly, those lesions that are not amenable to medical or surgical therapy may experience some improvement with cosmetic camouflage.

Protein O-GlcNAcylation: A critical regulator of the cellular response to stress.

PubMed

Chatham, John C; Marchase, Richard B

2010-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

Plant Signaling and Metabolic Pathways Enabling Arbuscular Mycorrhizal Symbiosis.

PubMed

MacLean, Allyson M; Bravo, Armando; Harrison, Maria J

2017-10-01

Plants have lived in close association with arbuscular mycorrhizal (AM) fungi for over 400 million years. Today, this endosymbiosis occurs broadly in the plant kingdom where it has a pronounced impact on plant mineral nutrition. The symbiosis develops deep within the root cortex with minimal alterations in the external appearance of the colonized root; however, the absence of macroscopic alterations belies the extensive signaling, cellular remodeling, and metabolic alterations that occur to enable accommodation of the fungal endosymbiont. Recent research has revealed the involvement of a novel N -acetyl glucosamine transporter and an alpha/beta-fold hydrolase receptor at the earliest stages of AM symbiosis. Calcium channels required for symbiosis signaling have been identified, and connections between the symbiosis signaling pathway and key transcriptional regulators that direct AM-specific gene expression have been established. Phylogenomics has revealed the existence of genes conserved for AM symbiosis, providing clues as to how plant cells fine-tune their biology to enable symbiosis, and an exciting coalescence of genome mining, lipid profiling, and tracer studies collectively has led to the conclusion that AM fungi are fatty acid auxotrophs and that plants provide their fungal endosymbionts with fatty acids. Here, we provide an overview of the molecular program for AM symbiosis and discuss these recent advances. © 2017 American Society of Plant Biologists. All rights reserved.

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.

Pediatric oral formulation of dendrimer-N-acetyl-l-cysteine conjugates for the treatment of neuroinflammation.

PubMed

Yellepeddi, Venkata K; Mohammadpour, Raziye; Kambhampati, Siva P; Sayre, Casey; Mishra, Manoj K; Kannan, Rangaramanujam M; Ghandehari, Hamidreza

2018-04-20

N-Acetyl-l-cysteine (NAC) commonly used as an antidote in acetaminophen poisoning has shown promise in the treatment of neurological disorders such as cerebral palsy (CP). However, NAC suffers from drawbacks such as poor oral bioavailability and suboptimal blood-brain-barrier (BBB) permeability limiting its clinical success. It was previously demonstrated that intravenous administration of dendrimer-NAC (D-NAC) conjugates have shown significant promise in the targeted treatment of neuroinflammation, in multiple preclinical models. Development of an oral formulation of D-NAC may open new administrative routes for this compound. Here, we report the gastrointestinal stability, in vitro transepithelial permeability, and in vivo oral absorption and pharmacokinetics in rats of a pediatric formulation of D-NAC containing Capmul MCM (glycerol monocaprylate) as a penetration enhancer. D-NAC was stable for 6 h in all five simulated gastrointestinal fluids with no signs of chemical degradation. The apparent permeability (P app ) of D-NAC increased 9-fold in the formulation containing Capmul. The area under the curve [AUC] 0-∞ of D-NAC with Capmul increased by 47% when compared to D-NAC alone. These results indicate that an oral pediatric formulation containing D-NAC and Capmul can be an effective option for the treatment of neuroinflammation. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

PubMed

Doll, Mark A; Hein, David W

2017-07-01

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

Acetylation-mediated Siah2 stabilization enhances PHD3 degradation in Helicobacter pylori-infected gastric epithelial cancer cells.

PubMed

Kokate, Shrikant Babanrao; Dixit, Pragyesh; Das, Lopamudra; Rath, Suvasmita; Roy, Arjama Dhar; Poirah, Indrajit; Chakraborty, Debashish; Rout, Niranjan; Singh, Shivaram Prasad; Bhattacharyya, Asima

2018-04-24

Gastric epithelial cells infected with Helicobacter pylori acquire highly invasive and metastatic characteristics. The seven in absentia homolog (Siah)2, an E3 ubiquitin ligase, is one of the major proteins that induces invasiveness of infected gastric epithelial cells. We find that p300-driven acetylation of Siah2 at lysine 139 residue stabilizes the molecule in infected cells, thereby substantially increasing its efficiency to degrade prolyl hydroxylase (PHD)3 in the gastric epithelium. This enhances the accumulation of an oncogenic transcription factor hypoxia-inducible factor 1α (Hif1α) in H. pylori-infected gastric cancer cells in normoxic condition and promotes invasiveness of infected cells. Increased acetylation of Siah2, Hif1α accumulation, and the absence of PHD3 in the infected human gastric metastatic cancer biopsy samples and in invasive murine gastric cancer tissues further confirm that the acetylated Siah2 (ac-Siah2)-Hif1α axis is crucial in promoting gastric cancer invasiveness. This study establishes the importance of a previously unrecognized function of ac-Siah2 in regulating invasiveness of H. pylori-infected gastric epithelial cells.-Kokate, S. B., Dixit, P., Das, L., Rath, S., Roy, A. D., Poirah, I., Chakraborty, D., Rout, N., Singh, S. P., Bhattacharyya, A. Acetylation-mediated Siah2 stabilization enhances PHD3 degradation in Helicobacter pylori-infected gastric epithelial cancer cells.

The activity of N-acetyl-β-d-hexosaminidase A and B and β-glucuronidase in nasal polyps and hypertrophic nasal concha.

PubMed

Chojnowska, Sylwia; Minarowska, Alina; Waszkiewicz, Napoleon; Kępka, Alina; Zalewska-Szajda, Beata; Gościk, Elżbieta; Kowal, Krzysztof; Olszewska, Ewa; Konarzewska-Duchnowska, Emilia; Minarowski, Łukasz; Zwierz, Krzysztof; Ładny, Jerzy Robert; Szajda, Sławomir Dariusz

2014-01-01

Nasal polyps and hypertrophic lower nasal conchae are common disorders of nasal cavity. The majority of etiopathogenetic theories indicate inflammatory background of polyps and hypertrophic concha. N-acetyl-β-D-hexosaminidase and β-glucuronidase are lysosomal exoglycosidases revealing accelerated activity in inflammatory processes. The aim of the study was to evaluate the catabolism of glycoconjugates in nasal polyps and hypertrophic nasal concha basing on the activity of N-acetyl-β-D-hexosaminidase (HEX) and β-glucuronidase (GLU). Material consisted of nasal polyps taken from 40 patients during polypectomy in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and hypertrophic lower nasal conchae taken from 20 patients during mucotomy. The activity of HEX, HEX A, HEX B and GLU in supernatant of homogenates of nasal polyps and hypertrophic lower nasal concha tissues has been estimated using colorimetric method. Statistically significant decrease has been observed in concentration of the activity (per 1mg of tissue) of HEX (p<0.05), HEX B (p<0.001) and specific activity (per 1mg of protein) of HEX B (p<0.001) in nasal polyps tissue in comparison to hypertrophic lower nasal conchae tissue. Decrease in the activity and specific activity concentration of the majority of examined lysosomal exoglycosidases (increasing in inflammations) in comparison to hypertrophic lower nasal conchae suggests electrolytes disorders and questions the inflammatory background of nasal polyps. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

PubMed

Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

Aralkyl selenoglycosides and related selenosugars in acetylated form activate protein phosphatase-1 and -2A.

PubMed

Kónya, Zoltán; Bécsi, Bálint; Kiss, Andrea; Tamás, István; Lontay, Beáta; Szilágyi, László; Kövér, Katalin E; Erdődi, Ferenc

2018-05-01

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2-4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT1 23-38 ) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (Br-BASG). MYPT1 23-38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

Isolation, chemical characterization, and immunomodulatory activity of naturally acetylated hemicelluloses from bamboo shavings* #

PubMed Central

Huang, Ju-qing; Qi, Rui-ting; Pang, Mei-rong; Liu, Cong; Li, Guang-yu; Zhang, Ying

2017-01-01

Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing. In this study we investigated the isolation, chemical characterization, and immunostimulatory activity in vitro of the hemicelluloses from bamboo shavings. Shavings were first pretreated by steam explosion. The optimal pretreatment was found to be steam explosion at 2.2 MPa for 1 min. Following this pretreatment, the yield of hemicelluloses reached (2.05±0.22)% (based on the dry dewaxed raw materials), which was 5.7-fold higher than that of untreated samples. Bamboo-shavings hemicellulose (BSH) was then prepared by hot water extraction and ethanol precipitation from the steam-exploded shavings. Purification of BSH by anion-exchange chromatography of diethylaminoethanol (DEAE)-sepharose Fast Flow resulted in a neutral fraction (BSH-1, purity of 95.3%, yield of 1.06%) and an acidic fraction (BSH-2, purity of 92.5%, yield of 0.79%). The weight-average molecular weights (M w) of BSH-1 and BSH-2 were 12 800 and 11 300 g/mol, respectively. Chemical and structural analyses by Fourier transform infrared spectroscopy (FT-IR), 1D (1H and 13C) and 2D (heteronuclear single quantum correlation (HSQC)) nuclear magnetic resonance (NMR) spectra revealed that BSH-1 was O-acetylated-arabinoxylan and BSH-2 was O-acetylated-(4-O-methylglucurono)-arabinoxylan. BSH-1 had a higher content of acetyl groups than BSH-2. For the immunomodulatory activity in vitro, BSH and BSH-2 significantly stimulated mouse splenocyte proliferation while BSH-1 had no effect; BSH, BSH-1, and BSH-2 markedly enhanced the phagocytosis activity and nitric oxide production of the murine macrophage RAW264.7 in a dose-dependent manner. Our results suggest that the water-extractable hemicelluloses from steam-exploded bamboo shavings are naturally acetylated and have immunostimulatory activity. PMID:28124842

Isolation, chemical characterization, and immunomodulatory activity of naturally acetylated hemicelluloses from bamboo shavings.

PubMed

Huang, Ju-Qing; Qi, Rui-Ting; Pang, Mei-Rong; Liu, Cong; Li, Guang-Yu; Zhang, Ying

Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing. In this study we investigated the isolation, chemical characterization, and immunostimulatory activity in vitro of the hemicelluloses from bamboo shavings. Shavings were first pretreated by steam explosion. The optimal pretreatment was found to be steam explosion at 2.2 MPa for 1 min. Following this pretreatment, the yield of hemicelluloses reached (2.05±0.22)% (based on the dry dewaxed raw materials), which was 5.7-fold higher than that of untreated samples. Bamboo-shavings hemicellulose (BSH) was then prepared by hot water extraction and ethanol precipitation from the steam-exploded shavings. Purification of BSH by anion-exchange chromatography of diethylaminoethanol (DEAE)-sepharose Fast Flow resulted in a neutral fraction (BSH-1, purity of 95.3%, yield of 1.06%) and an acidic fraction (BSH-2, purity of 92.5%, yield of 0.79%). The weight-average molecular weights (M w ) of BSH-1 and BSH-2 were 12 800 and 11 300 g/mol, respectively. Chemical and structural analyses by Fourier transform infrared spectroscopy (FT-IR), 1D ( 1 H and 13 C) and 2D (heteronuclear single quantum correlation (HSQC)) nuclear magnetic resonance (NMR) spectra revealed that BSH-1 was O-acetylated-arabinoxylan and BSH-2 was O-acetylated-(4-O-methylglucurono)-arabinoxylan. BSH-1 had a higher content of acetyl groups than BSH-2. For the immunomodulatory activity in vitro, BSH and BSH-2 significantly stimulated mouse splenocyte proliferation while BSH-1 had no effect; BSH, BSH-1, and BSH-2 markedly enhanced the phagocytosis activity and nitric oxide production of the murine macrophage RAW264.7 in a dose-dependent manner. Our results suggest that the water-extractable hemicelluloses from steam-exploded bamboo shavings are naturally acetylated and have immunostimulatory activity.

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

PubMed Central

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

2014-01-01

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

First Functional and Mutational Analysis of Group 3 N-Acetylneuraminate Lyases from Lactobacillus antri and Lactobacillus sakei 23K

PubMed Central

García-García, María Inmaculada; Gil-Ortiz, Fernando; García-Carmona, Francisco; Sánchez-Ferrer, Álvaro

2014-01-01

N-acetyl neuraminate lyases (NALs) catalyze the reversible aldol cleavage of N-acetyl neuraminic acid (Neu5Ac) to pyruvate and N-acetyl-D-mannosamine (ManNAc). Previous phylogenetic studies divided NALs into four different groups. Groups 1 and 2 have been well characterized at both kinetic and molecular levels, but no NAL from group 3 has been studied to date. In this work, a functional characterization of two group 3 members was performed using the recombinant NALs from Lactobacillus antri and Lactobacillus sakei 23K, revealing an optimal pH of between 6.0 and 7.0, low stability at basic pHs (>8.0), low optimal temperatures and, especially, low catalytic efficiency compared with their counterparts in group 1 and 2. The mutational analysis carried out showed that a plausible molecular reason for the low activity shown by Lactobacillus antri and Lactobacillus sakei 23k NALs compared with group 1 and 2 NALs could be the relatively small sugar-binding pocket they contain. A functional divergence analysis concluding that group 3 is more closely related to group 2 than to group 1. PMID:24817128

New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

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

Andres, H.H.; Klem, A.J.; Szabo, S.M.

1985-03-01

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of (/sup 3/H)acetyl-CoA in the assaymore » using (/sup 3/H)acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-(/sup 3/H)acetylarylamine after separation from (/sup 3/H)acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.« less

N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: a cross-over trial.

PubMed

Badawy, Ahmed; State, Omnia; Abdelgawad, Soma

2007-01-01

To compare clomiphene citrate plus N-acetyl cysteine versus clomiphene citrate for inducing ovulation in patients with polycystic ovary syndrome. Prospective cross-over trial. University teaching hospital and a private practice setting. Five hundred and seventy-three patients were treated with clomiphene citrate for one menstrual cycle among which 470 patients were treated with clomiphene citrate plus N-acetyl cysteine for another cycle. All women suffered from polycystic ovary syndrome. Patients had clomiphene citrate 50-mg tablets twice daily alone or with N-acetyl cysteine 1,200 mg/day orally for 5 days starting on day 3 of the menstrual cycle. Primary outcomes were number of mature follicles, serum E2, serum progesterone, and endometrial thickness. Secondary outcome was the occurrence of pregnancy. Ovulation rate improved significantly after the addition of N-acetyl cysteine (17.9% versus 52.1%). Although the number of mature follicles was more in the N-acetyl cysteine group (2.1+/-0.88 versus 3.2+/-0.93), the difference was not statistically significant. The mean E2 levels (pg/ml) at the time of human chorionic gonadotropine injection, serum progesterone levels (ng/ml) on days 21-23 of the cycle, and the endometrial thickness were significantly improved in the N-acetyl cysteine group. The overall pregnancy rate was 11.5% in the N-acetyl cysteine group. Insulin resistance occurred in 260 patients (55.4%). There was no significant difference between the insulin resistance group (n = 260) and non-insulin resistance group (n = 210) as regards ovulation rate, number of follicles, serum E2 (pg/ml), serum progesterone (ng/ml), endometrial thickness (mm), or pregnancy rate. N-Acetyl cysteine is proved effective in inducing or augmenting ovulation in polycystic ovary patients.

The Effects of Internal Rotation and 14N Quadrupole Coupling in N-Methyldiacetamide

NASA Astrophysics Data System (ADS)

Kannengießer, Raphaela; Eibl, Konrad; Nguyen, Ha Vinh Lam; Stahl, Wolfgang

2015-06-01

Acetyl- and nitrogen containing substances play an important role in chemical, physical, and especially biological systems. This applies in particular for acetamides, which are structurally related to peptide bonds. In this work, N-methyldiacetamide, CH_3N(COCH_3)_2, was investigated by a combination of molecular beam Fourier transform microwave spectroscopy and quantum chemical calculations. In N-methyldiacetamide, at least three large amplitude motions are possible: (1) the internal rotation of the methyl group attached to the nitrogen atom and (2, 3) the internal rotations of both acetyl methyl groups. This leads to a rather complicated torsional fine structure of all rotational transitions with additional quadrupole hyperfine splittings caused by the 14N nucleus. Quantum chemical calculations were carried out at the MP2/6-311++G(d,p) level of theory to support the spectral assignment. Conformational analysis was performed by calculating a full potential energy surface depending on the orientation of the two acetyl groups. This yielded three stable conformers with a maximum energy difference of 35.2 kJ/mol. The spectrum of the lowest energy conformer was identified in the molecular beam. The quadrupole hyperfine structure as well as the internal rotation of two methyl groups could be assigned. For the N-methyl group and for one of the two acetyl methyl groups, barriers to internal rotation of 147 cm-1 and of 680 cm-1, respectively, were determined. The barrier of the last methyl group seems to be so high that no additional splittings could be resolved. Using the XIAM program, a global fit with a standard deviation on the order of our experimental accuracy could be achieved.

Nitrogen-doped mesoporous carbons for high performance supercapacitors

NASA Astrophysics Data System (ADS)

Wu, Kai; Liu, Qiming

2016-08-01

The mesoporous carbons have been synthesized by using α-D(+)-Glucose, D-Glucosamine hydrochloride or their mixture as carbon precursors and mesoporous silicas (SBA-15 or MCF) as hard templates. The as-prepared products show a large pore volume (0.59-0.97 cm3 g-1), high surface areas (352.72-1152.67 m2 g-1) and rational nitrogen content (ca. 2.5-3.9 wt.%). The results of electrochemical tests demonstrate that both heteroatom doping and suitable pore structure play a decisive role in the performance of supercapacitors. The representative sample of SBA-15 replica obtained using D-Glucosamine hydrochloride only exhibits high specific capacitance (212.8 F g-1 at 0.5 A g-1) and good cycle durability (86.1% of the initial capacitance after 2000 cycles) in 6 M KOH aqueous electrolyte, which is attributed to the contribution of double layer capacitance and pseudo-capacitance. The excellent electrochemical performance makes it a promising electrode material for supercapacitors.

A SUMO-acetyl switch in PXR biology.

PubMed

Cui, Wenqi; Sun, Mengxi; Zhang, Shupei; Shen, Xunan; Galeva, Nadezhda; Williams, Todd D; Staudinger, Jeff L

2016-09-01

Post-translational modification (PTM) of nuclear receptor superfamily members regulates various aspects of their biology to include sub-cellular localization, the repertoire of protein-binding partners, as well as their stability and mode of degradation. The nuclear receptor pregnane X receptor (PXR, NR1I2) is a master-regulator of the drug-inducible gene expression in liver and intestine. The PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues. The activation of PXR also has important implications in significant human diseases including inflammatory bowel disease and cancer. Our recent investigations reveal that PXR is modified by multiple PTMs to include phosphorylation, SUMOylation, and ubiquitination. Using both primary cultures of hepatocytes and cell-based assays, we show here that PXR is modified through acetylation on lysine residues. Further, we show that increased acetylation of PXR stimulates its increased SUMO-modification to support active transcriptional suppression. Pharmacologic inhibition of lysine de-acetylation using trichostatin A (TSA) alters the sub-cellular localization of PXR in cultured hepatocytes, and also has a profound impact upon PXR transactivation capacity. Both the acetylation and SUMOylation status of the PXR protein is affected by its ability to associate with the lysine de-acetylating enzyme histone de-acetylase (HDAC)3 in a complex with silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Taken together, our data support a model in which a SUMO-acetyl 'switch' occurs such that acetylation of PXR likely stimulates SUMO-modification of PXR to promote the active repression of PXR-target gene expression. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. Copyright © 2016 Elsevier B.V. All rights reserved.

The novel IGF-IR/Akt–dependent anticancer activities of glucosamine

PubMed Central

2014-01-01

Background Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway. Methods We used various in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism. Results We found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress. Conclusions Taken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer. PMID:24438088

Targeting Dysregulated Epigenetic Enzymes for Prostate Cancer Treatment

DTIC Science & Technology

2016-10-01

decreased protein levels of SIRT2 leads to the inability to down-regulate the hyper -acetylated form of p300. SIRT2-dependent deacetylation of p300...nature06546. 32. Thompson PR, Wang D, Wang L, Fulco M, Pediconi N, Zhang D, et al. Regulation of the p300 HAT domain via a novel activation loop . Nat Struct

4-Aminobiphenyl Downregulation of NAT2 Acetylator Genotype–Dependent N- and O-acetylation of Aromatic and Heterocyclic Amine Carcinogens in Primary Mammary Epithelial Cell Cultures from Rapid and Slow Acetylator Rats

PubMed Central

Jefferson, Felicia A.; Xiao, Gong H.; Hein, David W.

2009-01-01

Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10μM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype–dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP. PMID:18842621

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

PubMed

Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

2016-03-18

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. Copyright © 2016 Elsevier Inc. All rights reserved.

N-acetyl-L-cysteine combined with mesalamine in the treatment of ulcerative colitis: Randomized, placebo-controlled pilot study

PubMed Central

Guijarro, Luis G; Mate, Jose; Gisbert, Javier P; Perez-Calle, Jose Luis; Marín-Jimenez, Ignacio; Arriaza, Encarna; Olleros, Tomás; Delgado, Mario; Castillejo, Maria S; Prieto-Merino, David; Lara, Venancio Gonzalez; Peña, Amado Salvador

2008-01-01

AIM: To evaluate the effectiveness and safety of oral N-acetyl-L-cysteine (NAC) co-administration with mesalamine in ulcerative colitis (UC) patients. METHODS: Thirty seven patients with mild to moderate UC were randomized to receive a four-wk course of oral mesalamine (2.4 g/d) plus N-acetyl-L-cysteine (0.8 g/d) (group A) or mesalamine plus placebo (group B). Patients were monitored using the Modified Truelove-Witts Severity Index (MTWSI). The primary endpoint was clinical remission (MTWSI ≤ 2) at 4 wk. Secondary endpoints were clinical response (defined as a reduction from baseline in the MTWSI of ≥ 2 points) and drug safety. The serum TNF-α, interleukin-6, interleukin-8 and MCP-1 were evaluated at baseline and at 4 wk of treatment. RESULTS: Analysis per-protocol criteria showed clinical remission rates of 63% and 50% after 4 wk treatment with mesalamine plus N-acetyl-L-cysteine (group A) and mesalamine plus placebo (group B) respectively (OR = 1.71; 95% CI: 0.46 to 6.36; P = 0.19; NNT = 7.7). Analysis of variance (ANOVA) of data indicated a significant reduction of MTWSI in group A (P = 0.046) with respect to basal condition without significant changes in the group B (P = 0.735) during treatment. Clinical responses were 66% (group A) vs 44% (group B) after 4 wk of treatment (OR = 2.5; 95% CI: 0.64 to 9.65; P = 0.11; NNT = 4.5). Clinical improvement in group A correlated with a decrease of IL-8 and MCP-1. Rates of adverse events did not differ significantly between both groups. CONCLUSION: In group A (oral NAC combined with mesalamine) contrarily to group B (mesalamine alone), the clinical improvement correlates with a decrease of chemokines such as MCP-1 and IL-8. NAC addition not produced any side effects. PMID:18473409

Glucosamine Hydrochloride

MedlinePlus

... sulfate. People take glucosamine hydrochloride by mouth for osteoarthritis, rheumatoid arthritis, glaucoma, a jaw disorder called temporomandibular ... with chondroitin sulfate, shark cartilage, and camphor for osteoarthritis. Glucosamine hydrochloride is used parenterally and short-term ...

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

PubMed Central

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

2015-01-01

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

The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme

PubMed Central

Veyhl, Joseph; Dunn, Robert J.; Johnston, Wendy L.; Bennett, Alexa; Zhang, Lijia W.; Dennis, James W.; Schachter, Harry

2017-01-01

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted β1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development–nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-β1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations–primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors. PMID:28817611

Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

PubMed

Fei, Xiang; Holmes, Thomas; Diddle, Julianna; Hintz, Lauren; Delaney, Dan; Stock, Alex; Renner, Danielle; McDevitt, Molly; Berkowitz, David B; Soukup, Juliane K

2014-12-19

The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

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

PubMed

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

2016-09-10

Sulfisoxazole (SFX) is still used in combination with trimethoprim in cattle despite adverse drug reactions (e.g., urolithiasis). Recently, SFX is known to be a promising repositioned drug candidate for pulmonary hypertension and cancer. We developed a simultaneous determination method of SFX and its N-acetylated metabolites (N(1)-acetyl SFX, N1AS; N(4)-acetyl SFX, N4AS; diacetyl SFX, DAS) using HPLC-MS/MS for the first time, and examined the pharmacokinetics of SFX in mice. N1AS and DAS were converted rapidly to SFX and N4AS, respectively, in mouse plasma. The time courses of plasma SFX and N4AS concentrations were well-characterised following the oral administration of SFX to mice. The absorption, metabolism, and/or excretion of SFX given at >700mg/kg may be saturable, and in contrast to humans and rats, the extent of systemic exposure of mice to N4AS was much greater than that of SFX. Interestingly, the acetyl groups at both N1- and N4-positions were degraded during the ionisation required to generate precursor ions. In additional experiments the carboxyl group of N-acetyl-5-aminosalicylic acid (NA5AS) was lost instead of the acetyl group during the ionisation, and acetaminophen (AAP) appeared. As the acetyl and carboxyl groups of some substances can be degraded during ionisation in the mass spectrometer, caution is appropriate when it is sought to simultaneously quantify similar structures containing these moieties; chromatographic separation is essential. Copyright © 2016 Elsevier B.V. All rights reserved.

Paracetamol (acetaminophen) decreases hydrogen sulfide tissue concentration in brain but increases it in the heart, liver and kidney in mice.

PubMed

Wiliński, Bogdan; Wiliński, Jerzy; Somogyi, Eugeniusz; Góralska, Marta; Piotrowska, Joanna

2011-01-01

The biological action ofN-acetyl-p-aminophenol - paracetamol (acetaminophen) has been demonstrated to involve different mechanisms and is still not clear. Hydrogen sulfide (H2S) has been shown to play an important role in many physiological and pathological processes including nociception. The interaction between acetaminophen and endogenous H2S is unknown. Twenty four female CBA strain mice were administered intraperitoneal injections of N-acetyl-p-aminophenol solution: paracetemol in doses of 30 mg/kg b.w. per day (group D1, n = 8) or 100 mg/kg b.w. per day (group D2, n = 8).. The control group (n = 8) received physiological saline in portions of the same volume--0.2 ml. The measurements of tissue H2S concentration were performed with the Siegel spectrophotometric modified method. In the brain, the H2S tissue level decreased, but more significantly in the lower drug dose group. Conversely, there was a significant rise in the H2S tissue concentration in D1 and D2 groups in heart and kidney with the increase more pronounced in the group with the lower paracetamol dose. In the liver only the higher acetaminophen dose elicited a change in H2S concentration, increasing after administration of acetaminophen at 100 mg/kg. Our study demonstrates that paracetamol induces H2S tissue concentration changes in different mouse organs.

Paviosides A-H, eight new oleane type saponins from Aesculus pavia with cytotoxic activity.

PubMed

Lanzotti, Virginia; Termolino, Pasquale; Dolci, Marcello; Curir, Paolo

2012-05-15

A phytochemical analysis of Aesculus pavia has led to the isolation of eight novel triterpenoid saponins, based on oleane type skeleton and named paviosides A-H (1a, 1b-4a, 4b). On the basis of chemical, and 2D NMR and mass spectrometry data, the structures of the new compounds were elucidated as 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-d-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (1a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (1b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (2a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (2b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (3a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-d-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (3b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl protoaescigenin (4a), and 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl protoaescigenin (4b). The compounds showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines. Among them, paviosides E-H (3a, 3b and 4a, 4b) showed higher activity with values ranging from 2.1 to 3.6 μg/mL. Structure-activity relationship studies indicated the positive effect on the activity of xylose unit in the place of glucose, while a little detrimental effect is observed when glucose is substituted by galactose. The aglycone structure and the presence of a tigloyl or an angeloyl group at C-21 do not affect significantly the inhibitory activity on both tested cell lines. Copyright © 2012 Elsevier Ltd. All rights reserved.

Glucosamine Supplementation Demonstrates a Negative Effect On Intervertebral Disc Matrix in an Animal Model of Disc Degeneration

PubMed Central

Jacobs, Lloydine; Vo, Nam; Coehlo, J. Paulo; Dong, Qing; Bechara, Bernard; Woods, Barrett; Hempen, Eric; Hartman, Robert; Preuss, Harry; Balk, Judith; Kang, James; Sowa, Gwendolyn

2013-01-01

Study Design Laboratory based controlled in vivo study Objective To determine the in vivo effects of oral glucosamine sulfate on intervertebral disc degeneration Summary of Background Data Although glucosamine has demonstrated beneficial effect in articular cartilage, clinical benefit is uncertain. A CDC report from 2009 reported that many patients are using glucosamine supplementation for low back pain (LBP), without significant evidence to support its use. Because disc degeneration is a major contributor of LBP, we explored the effects of glucosamine on disc matrix homeostasis in an animal model of disc degeneration. Methods Eighteen skeletally mature New Zealand White rabbits were divided into four groups: control, annular puncture, glucosamine, and annular puncture+glucosamine. Glucosamine treated rabbits received daily oral supplementation with 107mg/day (weight based equivalent to human 1500mg/day). Annular puncture surgery involved puncturing the annulus fibrosus (AF) of 3 lumbar discs with a 16G needle to induce degeneration. Serial MRIs were obtained at 0, 4, 8, 12, and 20 weeks. Discs were harvested at 20 weeks for determination of glycosaminoglycan(GAG) content, relative gene expression measured by RT-PCR, and histological analyses. Results The MRI index and NP area of injured discs of glucosamine treated animals with annular puncture was found to be lower than that of degenerated discs from rabbits not supplemented with glucosamine. Consistent with this, decreased glycosaminoglycan was demonstrated in glucosamine fed animals, as determined by both histological and GAG content. Gene expression was consistent with a detrimental effect on matrix. Conclusions These data demonstrate that the net effect on matrix in an animal model in vivo, as measured by gene expression, MRI, histology, and total proteoglycan is anti-anabolic. This raises concern over this commonly used supplement, and future research is needed to establish the clinical relevance of these findings. PMID:23324939

Worsening Renal Function in Patients With Acute Heart Failure Undergoing Aggressive Diuresis Is Not Associated With Tubular Injury.

PubMed

Ahmad, Tariq; Jackson, Keyanna; Rao, Veena S; Tang, W H Wilson; Brisco-Bacik, Meredith A; Chen, Horng H; Felker, G Michael; Hernandez, Adrian F; O'Connor, Christopher M; Sabbisetti, Venkata S; Bonventre, Joseph V; Wilson, F Perry; Coca, Steven G; Testani, Jeffrey M

2018-05-08

Worsening renal function (WRF) in the setting of aggressive diuresis for acute heart failure treatment may reflect renal tubular injury or simply indicate a hemodynamic or functional change in glomerular filtration. Well-validated tubular injury biomarkers, N -acetyl-β-d-glucosaminidase, neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1, are now available that can quantify the degree of renal tubular injury. The ROSE-AHF trial (Renal Optimization Strategies Evaluation-Acute Heart Failure) provides an experimental platform for the study of mechanisms of WRF during aggressive diuresis for acute heart failure because the ROSE-AHF protocol dictated high-dose loop diuretic therapy in all patients. We sought to determine whether tubular injury biomarkers are associated with WRF in the setting of aggressive diuresis and its association with prognosis. Patients in the multicenter ROSE-AHF trial with baseline and 72-hour urine tubular injury biomarkers were analyzed (n=283). WRF was defined as a ≥20% decrease in glomerular filtration rate estimated with cystatin C. Consistent with protocol-driven aggressive dosing of loop diuretics, participants received a median 560 mg IV furosemide equivalents (interquartile range, 300-815 mg), which induced a urine output of 8425 mL (interquartile range, 6341-10 528 mL) over the 72-hour intervention period. Levels of N -acetyl-β-d-glucosaminidase and kidney injury molecule 1 did not change with aggressive diuresis (both P >0.59), whereas levels of neutrophil gelatinase-associated lipocalin decreased slightly (-8.7 ng/mg; interquartile range, -169 to 35 ng/mg; P <0.001). WRF occurred in 21.2% of the population and was not associated with an increase in any marker of renal tubular injury: neutrophil gelatinase-associated lipocalin ( P =0.21), N -acetyl-β-d-glucosaminidase ( P =0.46), or kidney injury molecule 1 ( P =0.22). Increases in neutrophil gelatinase-associated lipocalin, N -acetyl-β-d-glucosaminidase, and kidney injury molecule 1 were paradoxically associated with improved survival (adjusted hazard ratio, 0.80 per 10 percentile increase; 95% confidence interval, 0.69-0.91; P =0.001). Kidney tubular injury does not appear to have an association with WRF in the context of aggressive diuresis of patients with acute heart failure. These findings reinforce the notion that the small to moderate deteriorations in renal function commonly encountered with aggressive diuresis are dissimilar from traditional causes of acute kidney injury. © 2018 American Heart Association, Inc.