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.

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.

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

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.

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

Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

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

Bame, K.J.

1986-01-01

Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The bindingmore » of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.« less

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.

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

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.

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.

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.

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.

The Influence of pH and Sodium Hydroxide Exposure Time on Glucosamine and Acrylamide Levels in California-Style Black Ripe Olives.

PubMed

Charoenprasert, Suthawan; Zweigenbaum, Jerry A; Zhang, Gong; Mitchell, Alyson E

2017-07-01

Acrylic acid, N-acetyl-glucosamine and glucosamine were investigated for their role in the formation of acrylamide in California-style black ripe olives [CBROs]. Levels of acrylic acid and glucosamine are reported for the first time in fresh (333.50 ± 21.88 and 243.59 ± 10.06 nmol/g, respectively) and in brine-stored olives (184.50 ± 6.02 and 165.88 ± 11.51 nmol/g, respectively). Acrylamide levels significantly increased when acrylic acid (35.2%), N-acetyl-glucosamine (29.9%), and glucosamine (124.0%) were added to olives prior to sterilization. However, isotope studies indicate these compounds do not contribute carbon and/or nitrogen atoms to acrylamide. The base-catalyzed degradation of glucosamine is demonstrated in olive pulp and a strong correlation (r 2 = 0.9513) between glucosamine in olives before sterilization and acrylamide formed in processed CBROs is observed. Treatment with sodium hydroxide (pH > 12) significantly reduces acrylamide levels over 1 to 5 d without impacting olive fruit texture. © 2017 Institute of Food Technologists®.

Effects of Oral Glucosamine Hydrochloride Administration on Plasma Free Amino Acid Concentrations in Dogs

PubMed Central

Azuma, Kazuo; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Takamori, Yoshimori; Minami, Saburo

2011-01-01

We examined the effects of oral glucosamine hydrochloride (GlcN), N-acetyl-d-glucosamine (GlcNAc) and d-glucose (Glc) administration on plasma total free amino acid (PFAA) concentrations in dogs. The PFAA concentrations increased in the control group and the GlcNAc group at one hour after feeding, and each amino acid concentration increased. On the other hand, in the GlcN group and the Glc group PFAA concentrations decreased at one hour after feeding. A significant decrease in amino acid concentration was observed for glutamate, glycine and alanine. Our results suggest the existence of differences in PFAA dynamics after oral administration of GlcN and GlcNAc in dogs. PMID:21673884

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.

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.

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.

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

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

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.

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.

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

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

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.

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.

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

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

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.

Identification of Bacillus anthracis by Using Monoclonal Antibody to Cell Wall Galactose-N-Acetylglucosamine Polysaccharide

DTIC Science & Technology

1990-02-01

which appear to be directed to an epitope associated with the galactose-N-acetyl-D- glucosamine polysaccharide. Both demonstrated specificity in their...liquid composed primarily of D-galactose and N-acetyl-D-glu - R medium (28) buffered with 50 mM Tris hydrochloride , pH cosamine (12, 13) (Gal-NAG...Ascites fluid (5 ml) was dialyzed (Cel-Line Associates, Inc., Newfield, N.J.). Suspensions against 20 mM Tris hydrochloride (pH 8.0) for 18 to 20 h, were

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...

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

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

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...

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.

A Core Facility for the Study of Neurotoxins of Biological Origin

DTIC Science & Technology

1992-02-15

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

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

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

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.

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...

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

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

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

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

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

PubMed

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

2000-03-01

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

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.

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.

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

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.

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

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

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.

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.

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 ...

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

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

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.

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

Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.

PubMed

Sarasquete, C; González de Canales, M L; Arellano, J; Pérez-Prieto, S; García-Rosado, E; Borrego, J J

1998-01-01

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, sialidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabream, Sparus aurata. Variable amounts of glycoproteins containing sialic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, mannose and/or glucose residues were observed in the cuticle and mucous cells of the corporal skin, tails and fins. Germinative and epithelial cells of the epidermis contained glycogen, proteins, carboxylated groups, as well as glycoproteins with mannose and/or glucose and N-acetyl-D-galactosamine residues. Hyaline capsule of the mature lymphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1). Con A reacted with the granular cytoplasm, specially around hyaline capsule, and with the basophilic intracytoplasmic inclusions developed in mature lymphocystis-infected cells of Sparus aurata skin. These sugar residues (mannose and/or glucose), as well as N-acetyl-D-glucosamine and/or sialic acid and N-acetyl-D-galactosamine were not detected in the hyaline capsule of the lymphocystis disease.

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

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

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.

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.

Immobilization of sugars in supermacroporous cryogels for the purification of lectins by affinity chromatography.

PubMed

Gonçalves, Gabriel Ramos Ferreira; Gandolfi, Olga Reinert Ramos; Santos, Leandro Soares; Bonomo, Renata Cristina Ferreira; Veloso, Cristiane Martins; Veríssimo, Lizzy Ayra Alcântara; Fontan, Rafael da Costa Ilhéu

2017-11-15

Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

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.

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

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

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.

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

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

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.

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.

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

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

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

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.

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.

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.

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.

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

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

Structural Determinants of an Insect β-N-Acetyl-d-hexosaminidase Specialized as a Chitinolytic Enzyme*

PubMed Central

Liu, Tian; Zhang, Haitao; Liu, Fengyi; Wu, Qingyue; Shen, Xu; Yang, Qing

2011-01-01

β-N-Acetyl-d-hexosaminidase has been postulated to have a specialized function. However, the structural basis of this specialization is not yet established. OfHex1, the enzyme from the Asian corn borer Ostrinia furnacalis (one of the most destructive pests) has previously been reported to function merely in chitin degradation. Here the vital role of OfHex1 during the pupation of O. furnacalis was revealed by RNA interference, and the crystal structures of OfHex1 and OfHex1 complexed with TMG-chitotriomycin were determined at 2.1 Å. The mechanism of selective inhibition by TMG-chitotriomycin was related to the existence of the +1 subsite at the active pocket of OfHex1 and a key residue, Trp490, at this site. Mutation of Trp490 to Ala led to a 2,277-fold decrease in sensitivity toward TMG-chitotriomycin as well as an 18-fold decrease in binding affinity for the substrate (GlcNAc)2. Although the overall topology of the catalytic domain of OfHex1 shows a high similarity with the human and bacterial enzymes, OfHex1 is distinguished from these enzymes by large conformational changes linked to an “open-close” mechanism at the entrance of the active site, which is characterized by the “lid” residue, Trp448. Mutation of Trp448 to Ala or Phe resulted in a more than 1,000-fold loss in enzyme activity, due mainly to the effect on kcat. The current work has increased our understanding of the structure-function relationship of OfHex1, shedding light on the structural basis that accounts for the specialized function of β-N-acetyl-d-hexosaminidase as well as making the development of species-specific pesticides a likely reality. PMID:21106526

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.

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

N-terminal acetylation modulates Bax targeting to mitochondria.

PubMed

Alves, Sara; Neiri, Leire; Chaves, Susana Rodrigues; Vieira, Selma; Trindade, Dário; Manon, Stephen; Dominguez, Veronica; Pintado, Belen; Jonckheere, Veronique; Van Damme, Petra; Silva, Rui Duarte; Aldabe, Rafael; Côrte-Real, Manuela

2018-02-01

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25 -/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

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.

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

N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

PubMed Central

Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

2016-01-01

Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

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

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.

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

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.

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

[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.

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

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.

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.

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

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.

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

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

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.

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

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

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.

Acetylation of aromatic cysteine conjugates by recombinant human N-acetyltransferase 8.

PubMed

Deol, Reema; Josephy, P David

2017-03-01

1. The mercapturic acid (MA) pathway is a metabolic route for the processing of glutathione conjugates to MA (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a MA, which is excreted in the urine. We expressed human NAT8 in HEK293T cells and developed an HPLC-MS method for the quantitation of the S-aryl-substituted cysteine conjugates and their MA. 2. We measured the activity of the enzyme for acetylation of benzyl-, 4-nitrobenzyl-, and 1-menaphthylcysteine substrates. 3. NAT8 catalyzed the acetylation of all three cysteine conjugates with similar Michaelis-Menten kinetics.

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

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.

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

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

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase.

PubMed

Aboalroub, Adam A; Bachman, Ashleigh B; Zhang, Ziming; Keramisanou, Dimitra; Merkler, David J; Gelis, Ioannis

2017-01-01

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase

PubMed Central

Aboalroub, Adam A.; Bachman, Ashleigh B.; Zhang, Ziming; Keramisanou, Dimitra; Merkler, David J.

2017-01-01

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle. PMID:28486510

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.

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 ...

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

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

First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.

PubMed

Min, Jun Zhe; Tomiyasu, Yuki; Morotomi, Takashi; Jiang, Ying-Zi; Li, Gao; Shi, Qing; Yu, Hai-Fu; Inoue, Koichi; Todoroki, Kenichiro; Toyo'oka, Toshimasa

2015-04-15

Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic β-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP. Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS. Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01). The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes. Crown Copyright © 2015. Published by Elsevier B.V. All rights

Expression of N-acetyl-D-galactosamine associated epitope in synovium: a potential marker of glycoprotein production.

PubMed

El-Gabalawy, H; King, R; Bernstein, C; Ma, G; Mou, Y; Alguacil-Garcia, A; Fritzler, M; Wilkins, J

1997-07-01

To investigate synovial glycoprotein production in situ, a novel monoclonal antibody (Mab), A13D8, was used to evaluate the expression of an epitope containing N-acetyl-D-galactosamine (GalNAc) in normal and pathological synovium. Immunohistological and cytochemical analysis of synovial tissue samples was undertaken with single and double staining techniques using the A13D8 Mab, anti-CD68, vascular cell adhesion molecule-1 (VCAM-1), the hyaluronan associated enzyme uridine diphosphoglucose dehydrogenase (UDPGD), and the anti-Golgi Mab SSN/HR-1992. The specificity of the A13D8 Mab was established through blocking studies using carbohydrate residues, including GalNAc and N-acetylglucosamine (GlcNAc). A13D8 is expressed intensely in the cytoplasm of normal type B lining cells, which coexpress VCAM-1 and UDPGD, and is not expressed by CD68+ type A lining cells. In the lining layer of RA synovium, there is a negative correlation between A13D8 expression and the level of lymphocytic infiltration in the sublining areas (r = -0.43, p < 0.001). The endothelium of a subset of venules, typically in lymphocyte-rich aggregates, also stains intensely for A13D8. Pretreatment of the Mab with GalNAc completely eliminates the tissue staining, as well as the 110 kDa band seen on immunoblot, whereas pretreatment of A13D8 with GlcNAc and lactose has no effect. Double staining of HEp-2 cells with A13D8 and the anti-Golgi Mab SSN/HR-1992 reveals co-localization of the A13D8 epitope to the Golgi apparatus. Type B synovial lining cells and selected synovial endothelium express GalNAc containing epitope identified by Mab A13D8. Marked reduction in the expression of this epitope in the lining layer of inflamed RA synovium suggests that the synovial production of GalNAc containing glycoproteins, such as mucins, may be altered in this disorder.

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

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.

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.

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

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

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 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

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

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.

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

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

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

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.

Identification of Novel Potential β-N-Acetyl-D-Hexosaminidase Inhibitors by Virtual Screening, Molecular Dynamics Simulation and MM-PBSA Calculations

PubMed Central

Liu, Jianling; Liu, Mengmeng; Yao, Yao; Wang, Jinan; Li, Yan; Li, Guohui; Wang, Yonghua

2012-01-01

Chitinolytic β-N-acetyl-d-hexosaminidases, as a class of chitin hydrolysis enzyme in insects, are a potential species-specific target for developing environmentally-friendly pesticides. Until now, pesticides targeting chitinolytic β-N-acetyl-d-hexosaminidase have not been developed. This study demonstrates a combination of different theoretical methods for investigating the key structural features of this enzyme responsible for pesticide inhibition, thus allowing for the discovery of novel small molecule inhibitors. Firstly, based on the currently reported crystal structure of this protein (OfHex1.pdb), we conducted a pre-screening of a drug-like compound database with 8 × 106 compounds by using the expanded pesticide-likeness criteria, followed by docking-based screening, obtaining 5 top-ranked compounds with favorable docking conformation into OfHex1. Secondly, molecular docking and molecular dynamics simulations are performed for the five complexes and demonstrate that one main hydrophobic pocket formed by residues Trp424, Trp448 and Trp524, which is significant for stabilization of the ligand–receptor complex, and key residues Asp477 and Trp490, are respectively responsible for forming hydrogen-bonding and π–π stacking interactions with the ligands. Finally, the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) analysis indicates that van der Waals interactions are the main driving force for the inhibitor binding that agrees with the fact that the binding pocket of OfHex1 is mainly composed of hydrophobic residues. These results suggest that screening the ZINC database can maximize the identification of potential OfHex1 inhibitors and the computational protocol will be valuable for screening potential inhibitors of the binding mode, which is useful for the future rational design of novel, potent OfHex1-specific pesticides. PMID:22605995

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

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

Glucosamine hydrochloride for the treatment of osteoarthritis symptoms

PubMed Central

Fox, Beth Anne; Stephens, Mary M

2007-01-01

Osteoarthritis is the most common arthritis in the world. It affects millions of people with age being the greatest risk factor for developing the disease. The burden of disease will worsen with the aging of the world’s population. The disease causes pain and functional disability. The direct costs of osteoarthritis include hospital and physician visits, medications, and assistive services. The indirect costs include work absences and lost wages. Many studies have sought to find a therapy to relieve pain and reduce disability. Glucosamine hydrochloride (HCl) is one of these therapies. There are limited studies of glucosamine HCl in humans. Although some subjects do report statistically significant improvement in pain and function from products combining glucosamine HCl and other agents, glucosamine HCl by itself appears to offer little benefit to those suffering from osteoarthritis. PMID:18225460

Evaluation of the inhibitory effect of N-acetyl-L-cysteine on Babesia and Theileria parasites.

PubMed

Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Yokoyama, Naoaki; Igarashi, Ikuo

2017-08-01

N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate. Copyright © 2017 Elsevier Inc. All rights reserved.

Radiolysis of N-acetyl amino acids as model compounds for radiation degradation of polypeptides

NASA Astrophysics Data System (ADS)

Wayne Garrett, R.; Hill, David J. T.; Ho, Sook-Ying; O'Donnell, James H.; O'Sullivan, Paul W.; Pomery, Peter J.

Radiation chemical yields of (i) the volatile radiolysis products and (ii) the trapped free radicals from the y-radiolysis of the N-acetyl derivatives of glycine, L-valine, L-phenylalanine and L-tyrosine in the polycrystalline state have been determined at room temperature (303 K). Carbon dioxide was found to be the major molecular product for all these compounds with G(CO 2) varying from 0.36 for N-acetyl-L-tyrosine to 8 for N-acetyl-L-valine. There was evidence for some scission of the N-C α bond, indicated by the production of acetamide and the corresponding aliphatic acid, but the determination reaction was found to be of much lesser importance than the decarboxylation reaction. A protective effect of the aromatic ring in N-acetyl-L-phenylalanine and in N-acetyl-L-tyrosine was indicated by the lower yields of volatile products for these compounds. The yields of trapped free radicals were found to vary with the nature of the amino acid side chain, increasing with chain length and chain branching. The radical yields were decreased by incorporation of an aromatic moiety in the side chain, this effect being greater for the tyrosyl side chain than for the phenyl side chain. The G(R·) values showed a good correlation with G(CO 2) indicating that a common reaction may be involved in radical production and carbon dioxide formation.

Ion-exchange equilibrium of N-acetyl-D-neuraminic acid on a strong anionic exchanger.

PubMed

Wu, Jinglan; Ke, Xu; Zhang, Xudong; Zhuang, Wei; Zhou, Jingwei; Ying, Hanjie

2015-09-15

N-acetyl-D-neuraminic acid (Neu5Ac) is a high value-added product widely applied in the food industry. A suitable equilibrium model is required for purification of Neu5Ac based on ion-exchange chromatography. Hence, the equilibrium uptake of Neu5Ac on a strong anion exchanger, AD-1 was investigated experimentally and theoretically. The uptake of Neu5Ac by the hydroxyl form of the resin occurred primarily by a stoichiometric exchange of Neu5Ac(-) and OH(-). The experimental data showed that the selectivity coefficient for the exchange of Neu5Ac(-) with OH(-) was a non-constant quantity. Subsequently, the Saunders' model, which took into account the dissociation reactions of Neu5Ac and the condition of electroneutrality, was used to correlate the Neu5Ac sorption isotherms at various solution pHs and Neu5Ac concentrations. The model provided an excellent fit to the binary exchange data for Cl(-)/OH(-) and Neu5Ac(-)/OH(-), and an approximate prediction of equilibrium in the ternary system Cl(-)/Neu5Ac(-)/OH(-). This basic information combined with the general mass transfer model could lay the foundation for the prediction of dynamic behavior of fixed bed separation process afterwards. Copyright © 2015 Elsevier Ltd. All rights reserved.

Formation of the thioester, N-acetyl, S-lactoylcysteine, by reaction of N-acetylcysteine with pyruvaldehyde in aqueous solution. [in prebiotic evolution

NASA Technical Reports Server (NTRS)

Weber, A. L.

1982-01-01

N-acetylcysteine reacts efficiently with pyruvaldehyde (methylglyoxal) in aqueous solution (pH 7.0) in the presence of a weak base, like imidazole or phosphate, to give the thioester, N-acetyl, S-lactoylcysteine. Reactions of 100 mM N-acetylcysteine with 14 mM, 24 mM and 41 mM pyruvaldehyde yield, respectively, 86%, 76% and 59% N-acetyl, S-lactoylcysteine based on pyruvaldehyde. The decrease in the percent yield at higher pyruvaldehyde concentrations suggests that during its formation the thioester is not only consumed by hydrolysis, but also by reaction with some substance in the pyruvaldehyde preparation. Indeed, purified N-acetyl, S-lactoylcysteine disappears much more rapidly in the presence of pyruvaldehyde than in its absence. Presumably, N-acetyl, S-lactoylcysteine synthesis occurs by rearrangement of the hemithioacetal of N-acetylcysteine and pyruvaldehyde. The significance of this pathway of thioester formation to molecular evolution is discussed.

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

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.

21 CFR 172.372 - N-Acetyl-L-methionine.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false N-Acetyl-L-methionine. 172.372 Section 172.372 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional...

Interactions between N-acetyl-L-cysteine protected CdTe quantum dots and doxorubicin through spectroscopic method

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

Yang, Xiupei, E-mail: xiupeiyang@163.com; College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000; Lin, Jia

2015-06-15

Highlights: • CdTe quantum dots with the diameter of 3–5 nm were synthesized in aqueous solution. • The modified CdTe quantum dots showed well fluorescence properties. • The interaction between the CdTe quantum dots and doxorubicin (DR) was investigated. - Abstract: N-acetyl-L-cysteine protected cadmium telluride quantum dots with a diameter of 3–5 nm were synthesized in aqueous solution. The interaction between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin was investigated by ultraviolet–visible absorption and fluorescence spectroscopy at physiological conditions (pH 7.2, 37 °C). The results indicate that electron transfer has occurred between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin under light illumination.more » The quantum dots react readily with doxorubicin to form a N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex via electrostatic attraction between the −NH{sub 3}{sup +} moiety of doxorubicin and the −COO{sup −} moiety of N-acetyl-L-cysteine/cadmium telluride quantum dots. The interaction of N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex with bovine serum albumin was studied as well, showing that the complex might induce the conformation change of bovine serum due to changes in microenvironment of bovine serum.« less

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

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

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.

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

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

Mycobacterium tuberculosis Arylamine N-Acetyltransferase Acetylates and Thus Inactivates para-Aminosalicylic Acid.

PubMed

Wang, Xude; Yang, Shanshan; Gu, Jing; Deng, Jiaoyu

2016-12-01

Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) is able to acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) techniques. The antituberculosis activity of the acetylated PAS is significantly reduced. As a result, overexpression of TBNAT in M. tuberculosis results in PAS resistance, as determined by MIC tests and drug exposure experiments. Taken together, our results suggest that TBNAT from M. tuberculosis is able to inactivate PAS by acetylating the compound. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

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.

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.

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

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.

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

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.

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.

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

Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

PubMed

Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2015-01-01

Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.

Reversible Lysine Acetylation Regulates Activity of Human Glycine N-Acyltransferase-like 2 (hGLYATL2)

PubMed Central

Waluk, Dominik P.; Sucharski, Filip; Sipos, Laszlo; Silberring, Jerzy; Hunt, Mary C.

2012-01-01

Lysine acetylation is a major post-translational modification of proteins and regulates many physiological processes such as metabolism, cell migration, aging, and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006) Mol. Cell 23, 607–618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19. Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50–80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that Lys-19 is not acetylated in wild-type hGLYATL2, indicating that Lys-19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-Oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signaling molecules that regulate functions like the perception of pain and body temperature and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulates the enzyme activity, thus linking post-translational modification of proteins with the production of biological signaling molecules, the N-acyl glycines. PMID:22408254

N-acetyl -β-D-glucosaminidase activity in cow milk as an indicator of mastitis.

PubMed

Hovinen, Mari; Simojoki, Heli; Pösö, Reeta; Suolaniemi, Jenni; Kalmus, Piret; Suojala, Leena; Pyörälä, Satu

2016-05-01

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1-1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

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

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

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

1992-09-15

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

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

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.

Glucosamine and Chondroitin for Osteoarthritis

MedlinePlus

... sheet Osteoarthritis and Complementary Health Approaches . What the Science Says About Glucosamine and Chondroitin for Osteoarthritis For ... months as those who received placebo. What the Science Says About Safety and Side Effects No serious ...

Benchmark dose for cadmium exposure and elevated N-acetyl-β-D-glucosaminidase: a meta-analysis.

PubMed

Liu, CuiXia; Li, YuBiao; Zhu, ChunShui; Dong, ZhaoMin; Zhang, Kun; Zhao, YanBin; Xu, YiLu

2016-10-01

Cadmium (Cd) is a well-known nephrotoxic contaminant, and N-acetyl-β-D-glucosaminidase (NAG) is considered to be an early and sensitive marker of tubular dysfunction. The link between Cd exposure and NAG level enables us to derive the benchmark dose (BMD) of Cd. Although several reports have already documented urinary Cd (UCd)-NAG relationships and BMD estimations, high heterogeneities arise due to the sub-populations (age, gender, and ethnicity) and BMD methodologies being employed. To clarify the influences that these variables exert, firstly, a random effect meta-analysis was performed in this study to correlate the UCd and NAG based on 92 datasets collected from 30 publications. Later, this established correlation (Ln(NAG) = 0.51 × Ln(UCd) + 0.83) was applied to derive the UCd BMD 5 of 1.76 μg/g creatinine and 95 % lower confidence limit of BMD 5 (BMDL 5 ) of 1.67 μg/g creatinine. While the regressions for different age groups and genders differed slightly, it is age and not gender that significantly affects BMD estimations. Ethnic differences may require further investigation given that limited data is currently available. Based on a comprehensive and systematic literature review, this study is a new attempt to quantify the UCd-NAG link and estimate BMD.

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

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.

Infrared and Raman spectra of N-acetyl- L-amino acid methylamides with aromatic side groups

NASA Astrophysics Data System (ADS)

Matsuura, Hiroatsu; Hasegawa, Kodo; Miyazawa, Tatsuo

Infrared and Raman spectra of N-acetyl- L-phenylalanine methylamide, N-acetyl- L-tyrosine methylamide and N-acetyl- L-tryptophan methylamide, as model compounds of aromatic amino acid residues in proteins, were measured in the solid state and in methanol solutions. Vibrational assignments of the spectra were made by utilizing the deuteration effect and by comparison with the spectra of related compounds which include toluene, p-cresol and 3-methylindole. The amide I, III and IV bands were strong in Raman scattering, but other characteristic amide bands were ill-defined. In the Raman spectra of methanol solutions, only the bands due to the aromatic side group vibrations were markedly observed, but those due to the peptide backbone vibrations were very weak, suggesting the coexistence of various molecular conformations in solution.

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

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

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

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB.

PubMed

Emirian, Aurélie; Fromentin, Sophie; Eckert, Catherine; Chau, Françoise; Dubost, Lionel; Delepierre, Muriel; Gutmann, Laurent; Arthur, Michel; Mesnage, Stéphane

2009-09-17

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.

Current concepts in the therapeutic management of osteoarthritis with glucosamine.

PubMed

Reginster, Jean-Yves; Bruyere, Olivier; Fraikin, Genevieve; Henrotin, Yves

2005-01-01

Over the last 10 years, several studies have investigated the ability of glucosamine sulfate to improve the symptoms (pain and function) and to delay the structural progression of osteoarthritis. There is now a large, convergent body of evidence that glucosamine sulfate, given at a daily oral dose of 1,500 mg, is able to significantly reduce the symptoms of osteoarthritis in the lower limbs and spine. This effect is usually seen with a minimal time for the onset of significant action - around 2 weeks. A similar dose of glucosamine sulfate has also been shown, in two independent studies, to prevent the joint space narrowing observed at the femorotibial compartment in patients with mild to moderate knee osteoarthritis. This effect, which is not affected by the radiographic technique used for the assessment of joint space width, also translated into a 50% reduction in the incidence of osteoarthritis-related surgery of the lower limbs during a 5-year period following the withdrawal of the treatment. There is a high degree of consistency in the literature showing that when glucosamine sulfate is used for the treatment of osteoarthritis, an efficacious response with minimum side effects can be expected. Since some discrepancies have been described between the results of studies performed with a patent-protected formulation of glucosamine sulfate distributed as a drug and those having used glucosamine preparations purchased from global suppliers, packaged, and sold over-the-counter as nutritional supplements (not regulated as drugs and with some potential issues concerning the reliability of their content), caution should be used when extrapolating conclusive results obtained with prescription drugs to over-the-counter or food supplements.

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.

Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

PubMed

You, Di; Yao, Li-Li; Huang, Dan; Escalante-Semerena, Jorge C; Ye, Bang-Ce

2014-09-01

Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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.

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.

Separation and characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by ammonium sulfate precipitation.

PubMed

Peng, Feng; Bian, Jing; Peng, Pai; Xiao, Huan; Ren, Jun-Li; Xu, Feng; Sun, Run-Cang

2012-04-25

Delignified Arundo donax was sequentially extracted with DMSO, saturated barium hydroxide, and 1.0 M aqueous NaOH solution. The yields of the soluble fractions were 10.2, 6.7, and 10.0% (w/w), respectively, of the dry Arundo donax materials. The DMSO-, Ba(OH)(2)- and NaOH-soluble hemicellulosic fractions were further fractionated into two subfractions by gradient 50% and 80% saturation ammonium sulfate precipitation, respectively. Monosaccharide, molecular weight, FT-IR, and 1D ((1)H and (13)C) and 2D (HSQC) NMR analysis revealed the differences in structural characteristics and physicochemical properties among the subfractions. The subfractions precipitated with 50% saturation ammonium sulfate had lower arabinose/xylose and glucuronic acid/xylose ratios but had higher molecular weight than those of the subfractions precipitated by 80% saturation ammonium sulfate. FT-IR and NMR analysis revealed that the highly acetylated DMSO-soluble hemicellulosic subfraction (H(D50)) could be precipitated with a relatively lower concentration of 50% saturated ammonium sulfate, and thus the gradient ammonium sulfate precipitation technique could discriminate acetyl and non-acetyl hemicelluloses. It was found that the DMSO-soluble subfraction H(D50) precipitated by 50% saturated ammonium sulfate mainly consisted of poorly substituted O-acetyl arabino-4-O-methylglucurono xylan with terminal units of arabinose linked on position 3 of xylose, 4-O-methylglucuronic acid residues linked on position 2 of the xylan bone, and the acetyl groups (degree of acetylation, 37%) linked on position 2 or 3. The DMSO-soluble subfraction H(D80) precipitated by 80% saturated ammonium sulfate was mainly composed of highly substituted arabino-4-O-methylglucurono xylan and β-d-glucan.

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

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

Acetaminophen analog N-acetyl-m-aminophenol, but not its reactive metabolite, N-acetyl-p-benzoquinone imine induces CYP3A activity via inhibition of protein degradation.

PubMed

Santoh, Masataka; Sanoh, Seigo; Ohtsuki, Yuya; Ejiri, Yoko; Kotake, Yaichiro; Ohta, Shigeru

2017-05-06

Cytochrome P450 (CYP) 3A subfamily members are known to metabolize various types of drugs, highlighting the importance of understanding drug-drug interactions (DDI) depending on CYP3A induction or inhibition. While transcriptional regulation of CYP3A members is widely understood, post-translational regulation needs to be elucidated. We previously reported that acetaminophen (APAP) induces CYP3A activity via inhibition of protein degradation and proposed a novel DDI concept. N-Acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP formed by CYP, is known to cause adverse events related to depletion of intracellular reduced glutathione (GSH). We aimed to inspect whether NAPQI rather than APAP itself could cause the inhibitory effects on protein degradation. We found that N-acetyl-l-cysteine, the precursor of GSH, and 1-aminobenzotriazole, a nonselective CYP inhibitor, had no effect on CYP3A1/23 protein levels affected by APAP. Thus, we used APAP analogs to test CYP3A1/23 mRNA levels, protein levels, and CYP3A activity. We found N-acetyl-m-aminophenol (AMAP), a regioisomer of APAP, has the same inhibitory effects of CYP3A1/23 protein degradation, while p-acetamidobenzoic acid (PAcBA), a carboxy-substituted form of APAP, shows no inhibitory effects. AMAP and PAcBA cannot be oxidized to quinone imine forms such as NAPQI, so the inhibitory effects could depend on the specific chemical structure of APAP. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose 1

PubMed Central

Leznicki, Antoni J.; Bandurski, Robert S.

1988-01-01

The synthesis of indole-3-acetyl-1-O-β-d-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as ρ-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid, and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed. PMID:11537439

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

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.

[Studies of urinary proteins, FDP (fibrinogen degradation products), and NAG (N-acetyl-beta-D-glucosaminidase) in renal transplanted patients].

PubMed

Kunikata, S; Ikegami, M; Imanishi, M; Nishioka, T; Ishii, T; Uemura, T; Kanda, H; Matsuura, T; Akiyama, T; Kurita, T

1989-08-01

The urinary proteins, FDP (fibrinogen degradation products), and NAG (N-acetyl-beta-D-glucosaminidase) in renal transplanted patients were studied. SDS (sodium dodecyl sulphate) electrophoresis was used for the differentiation of urinary proteins according to their molecular size. In the azathioprine-treated patients with stable renal function, most of the urinary proteins were albumin. However, the low molecular weight (LMW) proteins, which were suggestive of tubular proteins, appeared in the urine of the ciclosporin-treated patients with stable renal function. During the rejection episodes of the ciclosporin-treated patients, the fraction of LMW proteins increased. The elevation of urinary FDP and NAG index (urinary NAG/urinary Cr) were detected in association with rejection episodes. Urinary NAG index increased in proportion to the elevation of serum Cr. However, the elevation of urinary NAG index was found in some ciclosporin-treated patients with normal serum Cr. The elevation of NAG index without the elevation of urinary FDP occurred in ciclosporin nephrotoxicity. The SDS electrophoresis of urinary proteins, urinary FDP, and urinary NAG index can be useful parameters for monitoring ciclosporin nephrotoxicity.

Phenanthridine synthesis through iron-catalyzed intramolecular N-arylation of O-acetyl oxime.

PubMed

Deb, Indubhusan; Yoshikai, Naohiko

2013-08-16

O-Acetyl oximes derived from 2'-arylacetophenones undergo N-O bond cleavage/intramolecular N-arylation in the presence of a catalytic amount of iron(III) acetylacetonate in acetic acid. In combination with the conventional cross-coupling or directed C-H arylation, the reaction offers a convenient route to substituted phenanthridines.

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.

Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

PubMed

Salazar-González, Raúl A; Turiján-Espinoza, Eneida; Hein, David W; Niño-Moreno, Perla C; Romano-Moreno, Silvia; Milán-Segovia, Rosa C; Portales-Pérez, Diana P

2018-02-01

Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

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.

Acetyl transfer in arylamine metabolism

PubMed Central

Booth, J.

1966-01-01

1. N-Hydroxyacetamidoaryl compounds (hydroxamic acids) are metabolites of arylamides, and an enzyme that transfers the acetyl group from these derivatives to arylamines has been found in rat tissues. The reaction products were identified by thin-layer chromatography and a spectrophotometric method, with 4-amino-azobenzene as acetyl acceptor, was used to measure enzyme activity. 2. The acetyltransferase was in the soluble fraction of rat liver, required a thiol for maximum activity and had a pH optimum between 6·0 and 7·5. 3. The soluble fractions of various rat tissues showed decreasing activity in the following order: liver, adrenal, kidney, lung, spleen, testis, heart; brain was inactive. 4. With the exception of aniline and aniline derivatives all the arylamines tested were effective as acetyl acceptors but aromatic compounds with side-chain amino groups were inactive. 5. The N-hydroxyacetamido derivatives of 2-naphthylamine, 4-amino-biphenyl and 2-aminofluorene were active acetyl donors but N-hydroxyacetanilide showed only slight activity. Acetyl-CoA was not a donor. 6. Some properties of the enzyme are compared with those of other acetyltransferases. PMID:5969287

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.

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-L-tryptophan.

PubMed

Kouno, Yousuke; Anraku, Makoto; Yamasaki, Keishi; Okayama, Yoshiro; Iohara, Daisuke; Ishima, Yu; Maruyama, Toru; Kragh-Hansen, Ulrich; Hirayama, Fumitoshi; Otagiri, Masaki

2014-09-01

Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products. Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration. N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present. The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations. N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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

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.

Adolescent Alcohol Exposure-Induced Changes in Alpha-Melanocyte Stimulating Hormone and Neuropeptide Y Pathways via Histone Acetylation in the Brain During Adulthood.

PubMed

Kokare, Dadasaheb M; Kyzar, Evan J; Zhang, Huaibo; Sakharkar, Amul J; Pandey, Subhash C

2017-09-01

Adolescent intermittent ethanol exposure causes long-lasting alterations in brain epigenetic mechanisms. Melanocortin and neuropeptide Y signaling interact and are affected by ethanol exposure in the brain. Here, the persistent effects of adolescent intermittent ethanol on alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and their regulation by histone acetylation mechanisms were investigated in adulthood. Male rats were exposed to adolescent intermittent ethanol (2 g/kg, i.p.) or volume-matched adolescent intermittent saline from postnatal days 28 to 41 and allowed to grow to postnatal day 92. Anxiety-like behaviors were measured by the elevated plus-maze test. Brain regions from adult rats were used to examine changes in alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and the histone acetylation status of their promoters. Adolescent intermittent ethanol-exposed adult rats displayed anxiety-like behaviors and showed increased pro-opiomelanocortin mRNA levels in the hypothalamus and increased melanocortin 4 receptor mRNA levels in both the amygdala and hypothalamus compared with adolescent intermittent saline-exposed adult rats. The alpha-Melanocyte stimulating hormone and melanocortin 4 receptor protein levels were increased in the central and medial nucleus of the amygdala, paraventricular nucleus, and arcuate nucleus of the hypothalamus in adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Neuropeptide Y protein levels were decreased in the central and medial nucleus of the amygdala of adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Histone H3K9/14 acetylation was decreased in the neuropeptide Y promoter in the amygdala but increased in the melanocortin 4 receptor gene promoter in the amygdala and the melanocortin 4 receptor and pro-opiomelanocortin promoters in the

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

N-Heterocyclic Carbene-Catalyzed Alcohol Acetylation: An Organic Experiment Using Organocatalysis

ERIC Educational Resources Information Center

Morgan, John P.; Shrimp, Jonathan H.

2014-01-01

Undergraduate students in the teaching laboratory have successfully used N-heterocyclic carbenes (NHCs) as organocatalysts for the acetylation of primary alcohols, despite the high water sensitivity of uncomplexed ("free") NHCs. The free NHC readily reacted with chloroform, resulting in an air- and moisture-stable adduct that liberates…

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

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.

Synthesis and x-ray crystallographic analysis of 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide.

PubMed

Rotella, Madeline; Giovine, Matthew; Dougherty, William; Boyko, Walter; Kassel, Scott; Giuliano, Robert

2016-04-29

The glycopyranosyl cyanide 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide has been synthesized from tri-O-acetyl-D-galactal by reaction with trimethylsilyl cyanide in the presence of boron trifluoride diethyl etherate followed by catalytic hydrogenation. The synthesis provides the α-anomer stereoselectively, the structure of which was assigned based on 2D NMR techniques and x-ray crystallography. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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).

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.

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.

Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver.

PubMed

Andres, H H; Kolb, H J; Schreiber, R J; Weiss, L

1983-08-16

It could be demonstrated that a sulfhydryl group is involved in the catalysis of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver (EC 2.3.1.5). From ping-pong kinetics it was concluded that there is a covalent acetyl-enzyme intermediate. The respective intermediate could be isolated and chemically characterized as a cysteinyl thioester. Electrophoretically homogeneous acetyl-CoA:acylamine N-acetyltransferase from pigeon liver was able to acetylate a broad variety of aromatic and aliphatic amines from different acetyldonors such as acetyl-CoA, p-nitroacetanilide and p-nitrophenylacetate. Apparent Km values were determined for a number of acetyl donors and acetyl acceptors. Additionally, Ki values were evaluated for CoA, 3',5'-ADP and AMP. Correlation studies of basicity of acceptor amines and acetylation rate demonstrated that there is a limit of the pKa value (about pKa = 1) where the covalently-bound acetyl-enzyme intermediate can still be saponified. Testing crude liver homogenates of several animals including turkey, duck, chicken, cow, pig, horse, sheep, carp, trout and herring the outstanding nature of the pigeon liver enzyme in acetylating very weakly basic amines could be demonstrated. It is shown that the enzyme is quite flexible concerning sterically different acceptor amines, because arylamines whose amino group was effected by large o-substituents could be quantitatively acetylated. After enzymatic acetylation of the first amino group, 1,2-phenylendiamine formed the heterocyclic compound 2-methylbenzimidazole by a spontaneous condensation reaction. There is evidence that with distinct amines formation of heterocyclic compounds may also occur in vivo.

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

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.

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.

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

Glucosamine and chondroitin use in canines for osteoarthritis: A review

PubMed Central

Bhathal, Angel; Spryszak, Meredith; Louizos, Christopher; Frankel, Grace

2017-01-01

Osteoarthritis is a slowly progressive and debilitating disease that affects canines of all breeds. Pain and decreased mobility resulting from osteoarthritis often have a negative impact on the affected canine’s quality of life, level of comfort, daily functioning, activity, behaviour, and client-pet companionship. Despite limited and conflicting evidence, the natural products glucosamine hydrochloride (HCl) and chondroitin sulfate are commonly recommended by veterinarians for treating osteoarthritis in dogs. There is a paucity of well-designed clinical veterinary studies investigating the true treatment effect of glucosamine and chondroitin. The purposes of this review article are to provide a brief background on glucosamine and chondroitin use in canine osteoarthritis and to critically review the available literature on the role of these products for improving clinical outcomes. Based on critical review, recommendations for practice are suggested and a future study design is proposed. PMID:28331832

Crystal structure of the dopamine N-acetyltransferase–acetyl-CoA complex provides insights into the catalytic mechanism

PubMed Central

Cheng, Kuo-Chang; Liao, Jhen-Ni; Lyu, Ping-Chiang

2012-01-01

The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an AANAT. Until the present study, no insect Dat structure had been solved, and, consequently, the structural basis for its acetyl-transfer activity was not well understood. We report in the present paper the high-resolution crystal structure for a D. melanogaster Dat–AcCoA (acetyl-CoA) complex obtained using one-edge (selenium) single-wavelength anomalous diffraction. A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism. PMID:22716280

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

Micronutrients, N-Acetyl Cysteine, Probiotics and Prebiotics, a Review of Effectiveness in Reducing HIV Progression

PubMed Central

Hummelen, Ruben; Hemsworth, Jaimie; Reid, Gregor

2010-01-01

Low serum concentrations of micronutrients, intestinal abnormalities, and an inflammatory state have been associated with HIV progression. These may be ameliorated by micronutrients, N-acetyl cysteine, probiotics, and prebiotics. This review aims to integrate the evidence from clinical trials of these interventions on the progression of HIV. Vitamin B, C, E, and folic acid have been shown to delay the progression of HIV. Supplementation with selenium, N-acetyl cysteine, probiotics, and prebiotics has considerable potential, but the evidence needs to be further substantiated. Vitamin A, iron, and zinc have been associated with adverse effects and caution is warranted for their use. PMID:22254046

Optimization of Maillard Reaction between Glucosamine and Other Precursors by Measuring Browning with a Spectrophotometer.

PubMed

Ogutu, Benrick; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Hong, Dong-Lee; Lee, Yang-Bong

2017-09-01

The individual Maillard reactions of glucose, glucosamine, cyclohexylamine, and benzylamine were studied at a fixed temperature of 120°C under different durations by monitoring the absorbance of the final products at 425 nm. Glucosamine was the most individually reactive compound, whereas the reactions of glucose, cyclohexylamine, and benzylamine were not significantly different from each other. Maillard reactions of reaction mixtures consisting of glucosamine-cyclohexylamine, glucosamine-benzylamine, glucose-cyclohexylamine, and glucose-benzylamine were also studied using different concentration ratios under different durations at a fixed temperature of 120°C and pH 9. Maillard reactions in the pairs involving glucosamine were observed to be more intense than those of the pairs involving glucose. Finally, with respect to the concentration ratios, it was observed that in most instances, optimal activity was realized, when the reaction mixtures were in the ratio of 1:1.

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.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

Computational Study of Environmental Effects on Torsional Free Energy Surface of N-Acetyl-N'-methyl-L-alanylamide Dipeptide

ERIC Educational Resources Information Center

Carlotto, Silvia; Zerbetto, Mirco

2014-01-01

We propose an articulated computational experiment in which both quantum mechanics (QM) and molecular mechanics (MM) methods are employed to investigate environment effects on the free energy surface for the backbone dihedral angles rotation of the small dipeptide N-Acetyl-N'-methyl-L-alanylamide. This computation exercise is appropriate for an…

Optimization of Maillard Reaction between Glucosamine and Other Precursors by Measuring Browning with a Spectrophotometer

PubMed Central

Ogutu, Benrick; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Hong, Dong-Lee; Lee, Yang-Bong

2017-01-01

The individual Maillard reactions of glucose, glucosamine, cyclohexylamine, and benzylamine were studied at a fixed temperature of 120°C under different durations by monitoring the absorbance of the final products at 425 nm. Glucosamine was the most individually reactive compound, whereas the reactions of glucose, cyclohexylamine, and benzylamine were not significantly different from each other. Maillard reactions of reaction mixtures consisting of glucosamine-cyclohexylamine, glucosamine-benzylamine, glucose-cyclohexylamine, and glucose-benzylamine were also studied using different concentration ratios under different durations at a fixed temperature of 120°C and pH 9. Maillard reactions in the pairs involving glucosamine were observed to be more intense than those of the pairs involving glucose. Finally, with respect to the concentration ratios, it was observed that in most instances, optimal activity was realized, when the reaction mixtures were in the ratio of 1:1. PMID:29043219

S-Nitroso-N-acetyl-L-cysteine ethyl ester (SNACET) and N-acetyl-L-cysteine ethyl ester (NACET)-Cysteine-based drug candidates with unique pharmacological profiles for oral use as NO, H2S and GSH suppliers and as antioxidants: Results and overview.

PubMed

Tsikas, Dimitrios; Schwedhelm, Kathrin S; Surdacki, Andrzej; Giustarini, Daniela; Rossi, Ranieri; Kukoc-Modun, Lea; Kedia, George; Ückert, Stefan

2018-02-01

S -Nitrosothiols or thionitrites with the general formula RSNO are formally composed of the nitrosyl cation (NO + ) and a thiolate (RS - ), the base of the corresponding acids RSH. The smallest S -nitrosothiol is HSNO and derives from hydrogen sulfide (HSH, H 2 S). The most common physiological S -nitrosothiols are derived from the amino acid L-cysteine (CysSH). Thus, the simplest S -nitrosothiol is S -nitroso-L-cysteine (CysSNO). CysSNO is a spontaneous potent donor of nitric oxide (NO) which activates soluble guanylyl cyclase to form cyclic guanosine monophosphate (cGMP). This activation is associated with multiple biological actions that include relaxation of smooth muscle cells and inhibition of platelet aggregation. Like NO, CysSNO is a short-lived species and occurs physiologically at concentrations around 1 nM in human blood. CysSNO can be formed from CysSH and higher oxides of NO including nitrous acid (HONO) and its anhydride (N 2 O 3 ). The most characteristic feature of RSNO is the S-transnitrosation reaction by which the NO + group is reversibly transferred to another thiolate. By this way numerous RSNO can be formed such as the low-molecular-mass S -nitroso- N -acetyl-L-cysteine (SNAC) and S -nitroso-glutathione (GSNO), and the high-molecular-mass S -nitrosol-L-cysteine hemoglobin (HbCysSNO) present in erythrocytes and S -nitrosol-L-cysteine albumin (AlbCysSNO) present in plasma at concentrations of the order of 200 nM. All above mentioned RSNO exert NO-related biological activity, but they must be administered intravenously. This important drawback can be overcome by lipophilic charge-free RSNO. Thus, we prepared the ethyl ester of SNAC, the S -nitroso- N -acetyl-L-cysteine ethyl ester (SNACET), from synthetic N -acetyl-L-cysteine ethyl ester (NACET). Both NACET and SNACET have improved pharmacological features over N -acetyl-L-cysteine (NAC) and S -nitroso- N -acetyl-L-cysteine (SNAC), respectively, including higher oral bioavailability. SNACET

[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.

Bone resorption and remodeling in murine collagenase-induced osteoarthritis after administration of glucosamine

PubMed Central

2011-01-01

Introduction Glucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling. Methods The effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry. Results The administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels. Conclusions

A randomised, double blind, placebo-controlled trial of a fixed dose of N-acetyl cysteine in children with autistic disorder.

PubMed

Dean, Olivia M; Gray, Kylie M; Villagonzalo, Kristi-Ann; Dodd, Seetal; Mohebbi, Mohammadreza; Vick, Tanya; Tonge, Bruce J; Berk, Michael

2017-03-01

Oxidative stress, inflammation and heavy metals have been implicated in the aetiology of autistic disorder. N-acetyl cysteine has been shown to modulate these pathways, providing a rationale to trial N-acetyl cysteine for autistic disorder. There are now two published pilot studies suggesting efficacy, particularly in symptoms of irritability. This study aimed to explore if N-acetyl cysteine is a useful treatment for autistic disorder. This was a placebo-controlled, randomised clinical trial of 500 mg/day oral N-acetyl cysteine over 6 months, in addition to treatment as usual, in children with a Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision diagnosis of autistic disorder. The study was conducted in Victoria, Australia. The primary outcome measures were the Social Responsiveness Scale, Children's Communication Checklist-Second Edition and the Repetitive Behavior Scale-Revised. Additionally, demographic data, the parent-completed Vineland Adaptive Behavior Scales, Social Communication Questionnaire and clinician-administered Autism Diagnostic Observation Schedule were completed. A total of 102 children were randomised into the study, and 98 (79 male, 19 female; age range: 3.1-9.9 years) attended the baseline appointment with their parent/guardian, forming the Intention to Treat sample. There were no differences between N-acetyl cysteine and placebo-treated groups on any of the outcome measures for either primary or secondary endpoints. There was no significant difference in the number and severity of adverse events between groups. This study failed to demonstrate any benefit of adjunctive N-acetyl cysteine in treating autistic disorder. While this may reflect a true null result, methodological issues particularly the lower dose utilised in this study may be confounders.

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

Crystalline glucosamine sulfate in the management of knee osteoarthritis: efficacy, safety, and pharmacokinetic properties

PubMed Central

Girolami, Federica; Persiani, Stefano

2012-01-01

Glucosamine is an amino monosaccharide and a natural constituent of glycosaminoglycans in articular cartilage. When administered exogenously, it is used for the treatment of osteoarthritis as a prescription drug or a dietary supplement. The latter use is mainly supported by its perception as a cartilage building block, but it actually exerts specific pharmacologic effects, mainly decreasing interleukin 1-induced gene expression by inhibiting the cytokine intracellular signaling cascade in general and nuclear factor-kappa B (NF-kB) activation in particular. As a whole, the use of glucosamine in the management of osteoarthritis is supported by the clinical trials performed with the original prescription product, that is, crystalline glucosamine sulfate. This is the stabilized form of glucosamine sulfate, while other formulations or different glucosamine salts (e.g. hydrochloride) have never been shown to be effective. In particular, long-term pivotal trials of crystalline glucosamine sulfate 1500 mg once daily have shown significant and clinically relevant improvement of pain and function limitation (symptom-modifying effect) in knee osteoarthritis. Continuous administration for up to 3 years resulted in significant reduction in the progression of joint structure changes compared with placebo as assessed by measuring radiologic joint space narrowing (structure-modifying effect). The two effects combined may suggest a disease-modifying effect that was postulated based on an observed decrease in the risk of undergoing total joint replacement in the follow up of patients receiving the product for at least 12 months in the pivotal trials. The safety of the drug was good in clinical trials and in the postmarketing surveillance. Crystalline glucosamine sulfate 1500 mg once daily is therefore recommended in the majority of clinical practice guidelines and was found to be cost effective in pharmacoeconomic analyses. Compared with other glucosamine formulations, salts, or

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.

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.

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

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

PubMed

Anfelt, Josefine; Kaczmarzyk, Danuta; Shabestary, Kiyan; Renberg, Björn; Rockberg, Johan; Nielsen, Jens; Uhlén, Mathias; Hudson, Elton P

2015-10-16

There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Preparation and characterization of N-benzoyl-O-acetyl-chitosan.

PubMed

Cai, Jinping; Dang, Qifeng; Liu, Chengsheng; Fan, Bing; Yan, Jingquan; Xu, Yanyan; Li, Jingjing

2015-01-01

A novel amphipathic chitosan derivative, N-benzoyl-O-acetyl-chitosan (BACS), was prepared by using the selective partial acylation of chitosan (CS), benzoyl chloride, and acetic acid under high-intensity ultrasound. The chemical structure and physical properties of BACS were characterized by FTIR, (1)H NMR, TGA, and XRD techniques. The degrees of substitution of benzoyl and acetyl for the chitosan derivatives were 0.26 and 1.15, respectively, which were calculated from the peak areas in NMR spectra by using the combined integral methods. The foaming properties of CS and BACS were determined and the results suggested BACS had better foam capacity and stability than those of chitosan. In addition, the antimicrobial activities of CS and BACS were also investigated against two species of bacteria (Escherichia coli and Staphylococcus aureus) and a fungus (Aspergillus niger), the results indicated that the antibacterial and antifungal activities of BACS were much stronger than those of the parent chitosan. These findings suggested that BACS was preferable for use as a food additive with a dual role of both foaming agent and food preservative. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

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.

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

Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

PubMed

Tahir, I M; Iqbal, T; Saleem, S; Mehboob, H; Akhter, N; Riaz, M

2016-03-01

The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n=19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P<0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P<0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P<0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies. © The Author(s) 2015.

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.

Roles of N-methyl-d-aspartate receptors during the sensory stimulation-evoked field potential responses in mouse cerebellar cortical molecular layer.

PubMed

Xu, Yin-Hua; Zhang, Guang-Jian; Zhao, Jing-Tong; Chu, Chun-Ping; Li, Yu-Zi; Qiu, De-Lai

2017-11-01

The functions of N-methyl-d-aspartate receptors (NMDARs) in cerebellar cortex have been widely studied under in vitro condition, but their roles during the sensory stimulation-evoked responses in the cerebellar cortical molecular layer in living animals are currently unclear. We here investigated the roles of NMDARs during the air-puff stimulation on ipsilateral whisker pad-evoked field potential responses in cerebellar cortical molecular layer in urethane-anesthetized mice by electrophysiological recording and pharmacological methods. Our results showed that cerebellar surface administration of NMDA induced a dose-dependent decrease in amplitude of the facial stimulation-evoked inhibitory responses (P1) in the molecular layer, accompanied with decreases in decay time, half-width and area under curve (AUC) of P1. The IC 50 of NMDA induced inhibition in amplitude of P1 was 46.5μM. In addition, application of NMDA induced significant increases in the decay time, half-width and AUC values of the facial stimulation-evoked excitatory responses (N1) in the molecular layer. Application of an NMDAR blocker, D-APV (250μM) abolished the facial stimulation-evoked P1 in the molecular layer. These results suggested that NMDARs play a critical role during the sensory information processing in cerebellar cortical molecular layer in vivo in mice. Copyright © 2017 Elsevier B.V. All rights reserved.

The combined therapy with chondroitin sulfate plus glucosamine sulfate or chondroitin sulfate plus glucosamine hydrochloride does not improve joint damage in an experimental model of knee osteoarthritis in rabbits.

PubMed

Roman-Blas, Jorge A; Mediero, Aránzazu; Tardío, Lidia; Portal-Nuñez, Sergio; Gratal, Paula; Herrero-Beaumont, Gabriel; Largo, Raquel

2017-01-05

Osteoarthritis is the most common chronic joint disorder especially during aging. Although with controversies, glucosamine, both in its forms of sulfate and hydrochloride, and chondroitin sulfate are commonly employed to treat osteoarthritis. Due to the modest improve in the symptoms observed in patients treated with these drugs alone, a formulation combining both agents has been considered. The discrepant results achieved for pain control or structural improvement in osteoarthritis patients has been attributed to the quality of chemical formulations or different bias in clinical studies. The current study has been designed to test the effects of two different combined formulations with adequate pharmaceutical grade of these drugs in osteoarthritic joints, and to explore the underlying mechanisms modulated by both formulations in different osteoarthritis target tissues. Knee osteoarthritis was surgically induced in experimental rabbits. Some animals received the combined therapy (CT)1, (chondroitin sulfate 1200mg/day + glucosamine sulfate 1500mg/day), or the CT2 ((chondroitin sulfate 1200mg/day + glucosamine hydrochloride 1500mg/day). Neither CT1 nor CT2 significantly modified the cartilage damage or the synovial inflammation observed in osteoarthritic animals. Treatments were also unable to modify the presence of pro-inflammatory mediators, and the synthesis of metalloproteinases in the cartilage or in the synovium of osteoarthritic animals. Combined therapies did not modify the decrease in the subchondral bone mineral density observed in osteoarthritic rabbits. Therapies of chondroitin sulfate plus glucosamine sulfate or chondroitin sulfate plus glucosamine hydrochloride failed to improve structural damage or to ameliorate the inflammatory profile of joint tissues during experimental osteoarthritis. Published by Elsevier B.V.

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

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.

Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

PubMed

Xiong, Jian; Bhaskar, Ujjwal; Li, Guoyun; Fu, Li; Li, Lingyun; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

2013-09-10

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin. Copyright © 2013 Elsevier B.V. All rights reserved.

Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

PubMed Central

Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

2011-01-01

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-d-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP. PMID:21296883

Kinetics of de-N-acetylation of the chitin disaccharide in aqueous sodium hydroxide solution.

PubMed

Khong, Thang Trung; Aachmann, Finn L; Vårum, Kjell M

2012-05-01

Chitosan is prepared from chitin, a process which is carried out at highly alkaline conditions, and that can be performed either on chitin in solution (homogeneous deacetylation) or heterogeneously with the chitin as a solid throughout the reaction. We report here a study of the de-N-acetylation reaction of the chitin dimer (GlcNAc-GlcNAc) in solution. The reaction was followed by (1)H NMR spectroscopy in deuterated aqueous sodium hydroxide solution as a function of time, sodium-hydroxide concentration and temperature. The (1)H NMR spectrum of GlcNAc-GlcNAc in 2.77 M deuterated aqueous sodium hydroxide solution was assigned. The interpretation of the (1)H NMR spectra allowed us to determine the rates of de-N-acetylation of the reducing and non-reducing ends, showing that the reaction rate at the reducing end is twice the rate at the non-reducing end. The total deacetylation reaction rate was determined as a function of the hydroxide ion concentration, showing for the first time that this de-N-acetylation reaction is second order with respect to hydroxide ion concentration. No significant difference in the deacetylation rates in deuterated water compared to water was observed. The activation energy for the reaction (26-54 °C) was determined to 114.4 and 98.6 kJ/mol at 2.77 and 5.5 M in deuterated aqueous sodium hydroxide solution, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Potential involvement of N-terminal acetylation in the quantitative regulation of the ε subunit of chloroplast ATP synthase under drought stress.

PubMed

Hoshiyasu, Saki; Kohzuma, Kaori; Yoshida, Kazuo; Fujiwara, Masayuki; Fukao, Yoichiro; Yokota, Akiho; Akashi, Kinya

2013-01-01

In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation

PubMed Central

Moffett, John R.; Arun, Peethambaran; Ariyannur, Prasanth S.; Namboodiri, Aryan M. A.

2013-01-01

N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury. PMID:24421768

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

Susceptibility of N-acetyltransferase 2 slow acetylators to antituberculosis drug-induced liver injury: a meta-analysis.

PubMed

Shi, Jing; Xie, Min; Wang, Jianmiao; Xu, Yongjian; Liu, Xiansheng

2015-12-01

This study aimed to evaluate the association between N-acetyltransferase 2 (NAT2) gene polymorphisms and the risk of antituberculosis drug-induced liver injury (ATLI). A meta-analysis was performed including 27 studies with 1289 cases and 5462 controls. Odds ratio with 95% CI was used to evaluate the strength of association. Our meta-analysis found that NAT2 slow acetylators were associated with increased risk of ATLI compared with fast and intermediate acetylators when standard dose of isoniazid was administrated (odds ratio: 3.08; 95% CI: 2.29-4.15). Individuals with NAT2 slow acetylators may have increased risk of ATLI when standard dose of isoniazid was used. Detection of NAT2 genotype may benefit to the prevention of ATLI.

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 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.

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.

N-Acetylaspartate Metabolism Outside the Brain: Lipogenesis, Histone Acetylation, and Cancer

PubMed Central

Bogner-Strauss, Juliane G.

2017-01-01

N-acetylaspartate (NAA) is a highly abundant brain metabolite. Aberrant NAA concentrations have been detected in many pathological conditions and although the function of NAA has been extensively investigated in the brain it is still controversial. Only recently, a role of NAA has been reported outside the brain. In brown adipocytes, which show high expression of the NAA-producing and the NAA-cleaving enzyme, the metabolism of NAA has been implicated in lipid synthesis and histone acetylation. Increased expression of N-acetyltransferase 8-like (Nat8l, the gene encoding the NAA synthesizing enzyme) induces de novo lipogenesis and the brown adipocyte phenotype. Accordingly silencing of aspartoacylase, the NAA-cleaving enzyme, reduced brown adipocyte differentiation mechanistically by decreasing histone acetylation and gene transcription. Notably, the expression of Nat8l and the amount of NAA were also shown to be increased in several tumors and inversely correlate with patients’ survival. Additionally, Nat8l silencing reduced cell proliferation in tumor and non-tumor cells, while NAA supplementation could rescue it. However, the mechanism behind has not yet been clarified. It remains to be addressed whether NAA per se and/or its catabolism to acetate and aspartate, metabolites that have both been implicated in tumor growth, are valuable targets for future therapies. PMID:28979238

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

Central N-acetyl aspartylglutamate deficit: a possible pathogenesis of schizophrenia.

PubMed

Tsai, Shih-Jen

2005-09-01

The "glutamate hypothesis" of schizophrenia has emerged from the finding that phencyclidine (PCP) induces psychotic-like behaviors in rodents, possibly by blocking the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, thereby causing increased glutamate release. N-acetyl aspartylglutamate (NAAG), an endogenous peptide abundant in mammalian nervous systems, is localized in certain brain cells, including cortical and hippocampal pyramidal neurons. NAAG is synthesized from N-acetylaspartate (NAA) and glutamate, and NAA availability may limit the rate of NAAG synthesis. Although NAAG is known to have some neurotransmitter-like functions, NAA does not. NAAG is a highly selective agonist of the type 3 metabotropic glutamate receptor (mGluR3, a presynaptic autoreceptor) and can inhibit glutamate release. In addition, at low levels, NAAG is an NMDA receptor antagonist, and blocking of NMDA receptors may increase glutamate release. Taken together, low central NAAG levels may antagonize the effect of glutamate at NMDA receptors and decrease its agonistic effect on presynaptic mGluR3; both activities could increase glutamate release, similar to the increase demonstrated in the PCP model of schizophrenia. In this report, it is suggested that the central NAAG deficit, possibly through decreased synthesis or increased degradation of NAAG, may play a role in the pathogenesis of schizophrenia. Evidence is presented and discussed from magnetic resonance, postmortem, animal model, schizophrenia treatment, and genetic studies. The central NAAG deficit model of schizophrenia could explain the disease process, from the perspectives of both neurodevelopment and neurodegeneration, and may point to potential treatments for schizophrenia.

A Method to Determine Lysine Acetylation Stoichiometries

DOE PAGES

Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; ...

2014-01-01

Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

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

Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groups

NASA Astrophysics Data System (ADS)

Armstrong, Geoffrey S.; Cano, Kristin E.; Mandelshtam, Vladimir A.; Shaka, A. J.; Bendiak, Brad

2004-09-01

Rapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl "isotags" was made possible with high resolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimal correlation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupled with the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highly resolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).

Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groups.

PubMed

Armstrong, Geoffrey S; Cano, Kristin E; Mandelshtam, Vladimir A; Shaka, A J; Bendiak, Brad

2004-09-01

Rapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl "isotags" was made possible with high resolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimal correlation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupled with the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highly resolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).

Synthesis of C-glycosyl-bis-1,2,3-triazole derivatives from 3,4,6-tri-O-acetyl-D-glucal.

PubMed

Shamim, Anwar; Souza, Frederico B; Trossini, Gustavo H G; Gatti, Fernando M; Stefani, Hélio A

2015-08-01

We have developed an efficient, CuI-catalyzed, microwave-assisted method for the synthesis of bis-1,2,3-triazole derivatives starting from a 3,4,6-tri-O-acetyl-D-glucal-derived mesylate. This mesylate was obtained from 3,4,6-tri-O-acetyl-D-glucal through C-glycosidation, deprotection of acetate groups to alcohols, and selective mesylation of the primary alcohol. This mesylate moiety was then converted to an azide through a microwave-assisted method with good yield. The azide, once synthesized, was then treated with different terminal alkynes in the presence of CuI to synthesize various bis-triazoles in high yields and short reaction times.

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

Associations Between Glucosamine and Chondroitin Supplement Use and Biomarkers of Systemic Inflammation

PubMed Central

Lampe, Johanna W.; Navarro, Sandi L.; Song, Xiaoling; Milne, Ginger L.; White, Emily

2014-01-01

Abstract Objectives: Glucosamine and chondroitin supplements have been shown to have anti-inflammatory properties in both in vitro studies and animal models; however, little is known about these relationships in humans. The VITamins and Lifestyle (VITAL) biomarker study evaluated the associations between use of these supplements and a panel of circulating inflammatory biomarkers. Design: Study participants included 217 men and women age 50–75 years living in the Seattle metropolitan area. Use of glucosamine and chondroitin supplements was ascertained by home interview/supplement inventory. Inflammation was assessed by using blood and urine collected at the time of home interview. Measures of systemic inflammation included plasma high-sensitivity C-reactive protein (hsCRP), interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, soluble TNF receptors I and II, and urinary prostaglandin E2-metabolite (PGE-M). Multivariate-adjusted linear regression was used to evaluate the associations between supplement use and biomarkers of inflammation. Results: High users (14 or more pills/week) of chondroitin had 36% lower hsCRP (ratio, 0.64; 95% confidence interval [CI], 0.39–1.04; p for trend=.03) and 27% lower PGE-M (ratio, 0.73; 95% CI, 0.5–0.98; p for trend=.07) than nonusers. Compared with nonusers, high users of glucosamine had 28% lower hsCRP (ratio, 0.72; 95% CI, 0.47–1.08; p for trend=.09) and 24% lower PGE-M (ratio, 0.76; 95% CI, 0.59–0.97; p for trend=0.10). Use of glucosamine and chondroitin supplements was not associated with the other markers of inflammation. Conclusions: These results support prior research suggesting that use of glucosamine and chondroitin is associated with reduced hsCRP and PGE2, but further work is needed to more definitively evaluate the anti-inflammatory potential of these supplements. PMID:24738579

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

Studies on N-Acetyltransferase (NAT2) Genotype Relationships in Emiratis: Confirmation of the Existence of Phenotype Variation among Slow Acetylators.

PubMed

Al-Ahmad, Mohammad M; Amir, Naheed; Dhanasekaran, Subramanian; John, Anne; Abdulrazzaq, Yousef M; Ali, Bassam R; Bastaki, Salim

2017-09-01

Individuals with slow N-acetylation phenotype often experience toxicity from drugs such as isoniazid, sulfonamides, procainamide, and hydralazine, whereas rapid acetylators may not respond to these medications. The highly polymorphic N-acetyltransferase 2 enzyme encoded by the NAT2 gene is one of the N-acetylators in humans with a clear impact on the metabolism of a significant number of important drugs. However, there are limited studies on N-acetylation phenotypes and NAT2 genotypes among Emiratis, and thus this study was carried out to fill this gap. Five hundred seventy-six Emirati subjects were asked to consume a soft drink containing caffeine (a nontoxic and reliable probe for predicting the acetylation phenotype) and then provide a buccal swab along with a spot urine sample. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotype of each individual. Phenotyping was carried out by analyzing the caffeine metabolites using high-performance liquid chromatography (HPLC) analysis. We found that 78.5%, 19.1%, and 2.4% of the Emirati subjects were slow, intermediate, and rapid acetylators, respectively. In addition, we found that 77.4% of the subjects were homozygous or heterozygous for two nonreference alleles, whereas 18.4% and 4.2% were heterozygous or homozygous for the reference allele (NAT2*4), respectively. The most common genotypes found were NAT2*5B/*7B, NAT2*5B/*6A, NAT2*7B/*14B, and NAT2*4/*5B, with frequencies of 0.255, 0.135, 0.105, and 0.09, respectively. The degree of phenotype/genotype concordance was 96.2%. The NAT2*6A/*6A, NAT2*6A/*7B, NAT2*7B/*7B, and NAT2*5A/*5B genotypes were found to be associated with the lowest 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine (AFMU/1X) ratios. There is a high percentage of slow acetylators among Emiratis, which correlates with the presence of nonreference alleles for the NAT2 gene. Individuals who carried NAT2*6A/*6A, NAT2*6A/*7B, NAT2*7B/*7B

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

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.

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

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

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...

From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2

PubMed Central

Laurieri, Nicola; Dairou, Julien; Egleton, James E.; Stanley, Lesley A.; Russell, Angela J.; Dupret, Jean-Marie; Sim, Edith; Rodrigues-Lima, Fernando

2014-01-01

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH 3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH 3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of

Effect of N-acetyl cysteine on orthodontic primers cytotoxicity.

PubMed

D'Antò, Vincenzo; Spagnuolo, Gianrico; Schweikl, Helmut; Rengo, Sandro; Ambrosio, Luigi; Martina, Roberto; Valletta, Rosa

2011-02-01

The aims of this study were to evaluate the cytotoxicity of four orthodontic primers, including two hydrophilic and two hydrophobic materials, and to investigate the role of the reactive oxygen species (ROS) in induced cell damage. Moreover, the effects of the anti-oxidant N-acetyl cysteine (NAC) on primers toxicity was analyzed. Human gingival fibroblasts (HGF) were exposed to different concentrations of primers (0-0.25 mg/ml) in the presence or absence of NAC, and the cytotoxicity was assessed by the MTT assay, while cell death was quantified by flow cytometry after propidium iodide staining. The increase in the induced ROS levels was detected by flow cytometry measuring the fluorescence of the oxidation-sensitive dye 2',7'-dichlorofluorescein diacetate (DCFH-DA). All materials decreased cell viability in a dose-related manner after a 24 h exposure period. Cytotoxicity of orthodontic primers based on concentrations which caused a 50% decrease in cell viability (TC₅₀) in HGF was ranked as follows (median values): Eagle Fluorsure (0.078 mg/ml)>Transbond XT (0.081 mg/ml)>Transbond MIP (0.128 mg/ml)>Ortho solo (0.130 mg/ml). Moreover, in HGF cells, all materials induced a dose-dependent increase in ROS levels compared to untreated cells. Incubation of HGF with NAC significantly reduced ROS production and decreased the cell damage and cytotoxicity caused by all materials tested (p<0.001). Our results suggested that hydrophilic primers were less cytotoxic than hydrophobic materials. Moreover, we demonstrated a major role of ROS in the induction of cell death since the antioxidant N-acetyl cysteine was able to prevent cell damage induced by all materials tested. Copyright © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

NASA Technical Reports Server (NTRS)

Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

PubMed

Takenaka, Shinji; Ozeki, Takahiro; Tanaka, Kosei; Yoshida, Ken-Ichi

2017-11-01

To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126 Leu, 132 Leu, and 135 Lys were implicated in the binding of phosphopantothenic acid, and 100 Tyr and 131 Lys in that of adenosyl biphosphate. The data supported the participation of 83 Glu and 133 Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

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

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.

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.

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.

The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue.

PubMed

Vanpouille, Christophe; Deligny, Audrey; Delehedde, Maryse; Denys, Agnès; Melchior, Aurélie; Liénard, Xavier; Lyon, Malcolm; Mazurier, Joël; Fernig, David G; Allain, Fabrice

2007-08-17

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

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.

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…

Early life socioeconomic status, chronic physiological stress and hippocampal N-acetyl aspartate concentrations.

PubMed

McLean, John; Krishnadas, Rajeev; Batty, G David; Burns, Harry; Deans, Kevin A; Ford, Ian; McConnachie, Alex; McGinty, Agnes; McLean, Jennifer S; Millar, Keith; Sattar, Naveed; Shiels, Paul G; Tannahill, Carol; Velupillai, Yoga N; Packard, Chris J; Condon, Barrie R; Hadley, Donald M; Cavanagh, Jonathan

2012-12-01

Early life socioeconomic deprivation has been associated with cognitive and behavioural changes that persist through towards adulthood. In this study, we investigated whether early life socioeconomic status is associated with changes in the hippocampus N-acetyl aspartate (NAA), using the non-invasive technique of magnetic resonance spectroscopy (MRS). We performed proton magnetic resonance spectroscopy ((1)H-MRS) of the hippocampus at 3T in 30 adult males, selected from the PSOBID cohort. We conducted multiple regression analysis to examine the relationship between early socioeconomic status (SES) and concentration of N-acetyl-aspartate in the hippocampus. We also examined whether the relationship between these variables was mediated by markers of chronic physiological stress. Greater socioeconomic deprivation was associated with lower hippocampal NAA concentrations bilaterally. The relationship between early life SES and hippocampal NAA concentrations was mediated by allostatic load index - a marker of chronic physiological stress. Greater early life socioeconomic deprivation was associated with lower concentrations of NAA reflecting lesser neuronal integrity. This relationship was mediated by greater physiological stress. Further work, to better understand the biological processes underlying the effects of poverty, physiological stress on hippocampal metabolites is necessary. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

Effects of glucosamine in patients with osteoarthritis of the knee: a systematic review and meta-analysis.

PubMed

Ogata, Toru; Ideno, Yuki; Akai, Masami; Seichi, Atsushi; Hagino, Hiroshi; Iwaya, Tsutomu; Doi, Toru; Yamada, Keiko; Chen, Ai-Zhen; Li, Yingzi; Hayashi, Kunihiko

2018-04-30

Osteoarthritis (OA) of the knee is one of the main causes of mobility decline in the elderly. Non-surgical treatments such as administration of supplements to strengthen the joint cartilage matrix have become popular not only for pain relief but also for joint preservation. Glucosamine has been used in many countries based on the increasing evidence of its effectiveness for OA. Although there are many previous studies and systematic reviews, the findings vary and different conclusions have been drawn. We aimed to review recent randomized controlled trials on glucosamine for knee OA to reveal up-to-date findings about this supplement. We also performed a meta-analysis of some of the outcomes to overcome the unsolved bias in each study. Eighteen articles written between 2003 and 2016 were analyzed. Many used visual analogue scale (VAS) pain scores and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), which were assessed in our meta-analysis. We found a marginally favorable effect of glucosamine on VAS pain scores. The effect on knee function, as measured by the WOMAC, was small and not significant. A newly established knee OA scale, the Japanese Knee Osteoarthritis Measure (JKOM), is commonly used in Japan. Although the number of subjects was small, the JKOM meta-analysis indicated that glucosamine is superior to a placebo in alleviating knee OA symptoms. Given this, we concluded that glucosamine has the potential to alleviate knee OA pain. Further studies are needed to evaluate the effect of glucosamine on knee function and joint preservation, as well as to evaluate the combined effect with other components, such as chondroitin.

Glucosamine for Osteoarthritis: Biological Effects, Clinical Efficacy, and Safety on Glucose Metabolism

PubMed Central

Bello, Luis; Añez, Roberto; Bermúdez, Valmore

2014-01-01

Osteoarthritis is a chronic degenerative disorder that currently represents one of the main causes of disability within the elderly population and an important presenting complaint overall. The pathophysiologic basis of osteoarthritis entails a complex group of interactions among biochemical and mechanical factors that have been better characterized in light of a recent spike in research on the subject. This has led to an ongoing search for ideal therapeutic management schemes for these patients, where glucosamine is one of the most frequently used alternatives worldwide due to their chondroprotective properties and their long-term effects. Its use in the treatment of osteoarthritis is well established; yet despite being considered effective by many research groups, controversy surrounds their true effectiveness. This situation stems from several methodological aspects which hinder appropriate data analysis and comparison in this context, particularly regarding objectives and target variables. Similar difficulties surround the assessment of the potential ability of glucosamine formulations to alter glucose metabolism. Nevertheless, evidence supporting diabetogenesis by glucosamine remains scarce in humans, and to date, this association should be considered only a theoretical possibility. PMID:24678419

Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with A T-helper Cell Epitope as Antitumor Vaccine Candidates.

PubMed

Chang, Tsung-Che; Manabe, Yoshiyuki; Fujimoto, Yukari; Ohshima, Shino; Kametani, Yoshie; Kabayama, Kazuya; Nimura, Yuka; Lin, Chun-Cheng; Fukase, Koichi

2018-05-16

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NASA Technical Reports Server (NTRS)

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

1988-01-01

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

In vitro stimulatory effect of N-acetyl tryptophan-glucopyranoside against gamma radiation induced immunosuppression.

PubMed

Malhotra, Poonam; Singh, Darshana; Kumar, Raj

2018-03-01

Radiation-induced manifestations like free radical burst, oxidative damage and apoptosis leading to cell death. In present study, N-acetyl tryptophan glucopyranoside (NATG) was assessed for its immune-radioprotective activities using J774A.1 cells. Clonogenic cell survival, cell cycle progression and cytokines i.e. IFN-γ, TNF-α, IL-2, IL-10, IL-12, IL-13 and IL-17A expression were evaluated in irradiated and NATG pretreated cells using clonogenic formation ability, flow cytometry and ELISA assay. Results indicated that 0.25μg/ml NATG exhibited maximum radioprotection against gamma-radiation (2Gy) without intervening in cell cycle progression. NATG pretreated (-2 h) plus irradiated cells showed significant elevation in IFN-γ (∼38.2%), IL-17A (∼53.7%) and IL-12 (∼58.8%) expression as compared to only irradiated cells. Conversely, significant decrease in TNF-α (∼21.6%), IL-10 (∼31.2%), IL-2 (∼23.7%) and IL-13 expression (∼17.8%) were observed in NATG pretreated plus irradiated cells as compared to irradiated cells. Conclusively, NATG pretreatment to irradiated J774A.1 cells, stimulate Th 1 while diminish Th 2 cytokines that contributes to radioprotection. © 2017 Wiley Periodicals, Inc.

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…

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.

Design, Synthesis, and Antifouling Activity of Glucosamine-Based Isocyanides

PubMed Central

Hasegawa, Yuki; Novita, Ira S.; Suzuki, Junya; Morozumi, Tatsuya; Nogata, Yasuyuki; Yoshimura, Erina; Matsuda, Fuyuhiko

2017-01-01

Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite. Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC50 values below 1 μg/mL, without detectable toxicity even at a high concentration of 10 μg/mL. Two isocyanides had EC50 values of 0.23 and 0.25 μg/mL, comparable to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 μg/mL). PMID:28661419

Design, Synthesis, and Antifouling Activity of Glucosamine-Based Isocyanides.

PubMed

Umezawa, Taiki; Hasegawa, Yuki; Novita, Ira S; Suzuki, Junya; Morozumi, Tatsuya; Nogata, Yasuyuki; Yoshimura, Erina; Matsuda, Fuyuhiko

2017-06-29

Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite . Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC 50 values below 1 μg/mL, without detectable toxicity even at a high concentration of 10 μg/mL. Two isocyanides had EC 50 values of 0.23 and 0.25 μg/mL, comparable to that of CuSO₄, which is used as a fouling inhibitor (EC 50 = 0.27 μg/mL).

A bioinformatics-based overview of protein Lys-Ne-acetylation

USDA-ARS?s Scientific Manuscript database

Among posttranslational modifications, there are some conceptual similarities between Lys-N'-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. T...

N-terminal acetylation -an Essential Protein Modification Emerges as an Important Regulator of Stress Responses.

PubMed

Linster, Eric; Wirtz, Markus

2018-06-26

N-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. The majority of proteins is acetylated at their N-terminus in a co-translational manner by ribosome-associated N-terminal acetyltransferases (NAT). However, the recent discovery of Golgi-membrane localized NATs in metazoan, and plastid-localized NATs in plants challenged the dogma of static, co-translational imprinting of the proteome by NTA. Indeed, NTA by the cytosolic NatA is highly dynamic and under hormonal control in plants. Such active control has not been evidenced yet in other eukaryotes and might be an adaptation to the sessile lifestyle of plants forcing them to cope with diverse environmental challenges. The function of NTA for individual proteins is distinct and yet unpredictable. In yeast and humans, NTA has been shown to affect protein-protein interactions, subcellular localization, folding, aggregation, or degradation of a handful of proteins. In particular, the impact of NTA on the protein-turnover is documented by diverse examples in yeast. Consequently, NTA has recently dicovered to be a degradation signal in a distinct branch of the N-end rule pathway ubiquitin-mediated proteolysis. In this review, we summarize the current knowledge on the NAT machinery in higher plants and discuss the potential function of NTA during biotic and abiotic stresses.

Glucosamine enhances paracetamol bioavailability by reducing its metabolism.

PubMed

Qinna, Nidal A; Shubbar, Maryam H; Matalka, Khalid Z; Al-Jbour, Nawzat; Ghattas, Mohammad A; Badwan, Adnan A

2015-01-01

Paracetamol has an extensive first-pass metabolism that highly affects its bioavailability (BA); thus, dose may be repeated several times a day in order to have longer efficacy. However, hepatotoxicity may arise because of paracetamol metabolism. Therefore, this project aimed to increase paracetamol BA in rats by glucosamine (GlcN). At GlcN-paracetamol racemic mixture ratio of 4:1 and paracetamol dose of 10 mg/kg, paracetamol area under the curve (AUC) and maximum concentration (Cmax ) were significantly increased by 99% and 66%, respectively (p < 0.05). Furthermore, paracetamol AUC and Cmax levels were increased by 165% and 88% in rats prefed with GlcN for 2 days (p < 0.001). Moreover, GlcN significantly reduced phase Ι and phase I/ΙΙ metabolic reactions in liver homogenate by 48% and 54%, respectively. Furthermore, GlcN molecule was found to possess a good in silico binding mode into the CYP2E1 active site-forming bidentate hydrogen bonding with the Thr303 side chain. Finally, serum ALT and AST levels of rats-administered high doses of paracetamol were significantly reduced when rats were prefed with GlcN (p < 0.01). In conclusion, GlcN can increase the relative BA of paracetamol through reducing its metabolism. This phenomenon is associated with reduction in hepatocytes injury following ingestion of high doses of paracetamol. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

PubMed Central

Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

2011-01-01

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

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.

Effectiveness and safety of Glucosamine, chondroitin, the two in combination, or celecoxib in the treatment of osteoarthritis of the knee

PubMed Central

Zeng, Chao; Wei, Jie; Li, Hui; Wang, Yi-lun; Xie, Dong-xing; Yang, Tuo; Gao, Shu-guang; Li, Yu-sheng; Luo, Wei; Lei, Guang-hua

2015-01-01

This study aimed to investigate the effectiveness and safety of glucosamine, chondroitin, the two in combination, or celecoxib in the treatment of knee osteoarthritis (OA). PubMed, Embase and Cochrane Library were searched through from inception to February 2015. A total of 54 studies covering 16427 patients were included. Glucosamine plus chondroitin, glucosamine alone, and celecoxib were all more effective than placebo in pain relief and function improvement. Specifically, celecoxib is most likely to be the best treatment option, followed by the combination group. All treatment options showed clinically significant improvement from baseline pain, but only glucosamine plus chondroitin showed clinically significant improvement from baseline function. In terms of the structure-modifying effect, both glucosamine alone and chondroitin alone achieved a statistically significant reduction in joint space narrowing. Although no significant difference was observed among the five options with respect to the three major adverse effects (withdrawal due to adverse events, serious adverse events and the number of patients with adverse events), the additional classical meta-analysis showed that celecoxib exhibited a higher rate of gastrointestinal adverse effect comparing with the placebo group. The present study provided evidence for the symptomatic efficacy of glucosamine plus chondroitin in the treatment of knee OA. PMID:26576862

Identification and analysis of o-acetylated sialoglycoproteins.

PubMed

Mandal, Chandan; Mandal, Chitra

2013-01-01

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell-cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7-C8-C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice.

PubMed

Itzhak, Yossef; Anderson, Karen L; Kelley, Jonathan B; Petkov, Martin

2012-05-01

Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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

Optimization of Maillard Reaction in Model System of Glucosamine and Cysteine Using Response Surface Methodology

PubMed Central

Arachchi, Shanika Jeewantha Thewarapperuma; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Lee, Yang-Bong

2017-01-01

Sulfur-containing amino acids play important roles in good flavor generation in Maillard reaction of non-enzymatic browning, so aqueous model systems of glucosamine and cysteine were studied to investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and cysteine. Response surface methodology was applied to optimize the independent reaction parameters of cysteine and glucosamine in Maillard reaction. Box-Behnken factorial design was used with 30 runs of 16 factorial levels, 8 axial levels and 6 central levels. The degree of Maillard reaction was determined by reading absorption at 425 nm in a spectrophotometer and Hunter’s L, a, and b values. ΔE was consequently set as the fifth response factor. In the statistical analyses, determination coefficients (R2) for their absorbance, Hunter’s L, a, b values, and ΔE were 0.94, 0.79, 0.73, 0.96, and 0.79, respectively, showing that the absorbance and Hunter’s b value were good dependent variables for this model system. The optimum processing parameters were determined to yield glucosamine-cysteine Maillard reaction product with higher absorbance and higher colour change. The optimum estimated absorbance was achieved at the condition of initial pH 8.0, 111°C reaction temperature, 2.47 h reaction time, and 1.30 concentration ratio. The optimum condition for colour change measured by Hunter’s b value was 2.41 h reaction time, 114°C reaction temperature, initial pH 8.3, and 1.26 concentration ratio. These results can provide the basic information for Maillard reaction of aqueous model system between glucosamine and cysteine. PMID:28401086

Optimization of Maillard Reaction in Model System of Glucosamine and Cysteine Using Response Surface Methodology.

PubMed

Arachchi, Shanika Jeewantha Thewarapperuma; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Lee, Yang-Bong

2017-03-01

Sulfur-containing amino acids play important roles in good flavor generation in Maillard reaction of non-enzymatic browning, so aqueous model systems of glucosamine and cysteine were studied to investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and cysteine. Response surface methodology was applied to optimize the independent reaction parameters of cysteine and glucosamine in Maillard reaction. Box-Behnken factorial design was used with 30 runs of 16 factorial levels, 8 axial levels and 6 central levels. The degree of Maillard reaction was determined by reading absorption at 425 nm in a spectrophotometer and Hunter's L, a, and b values. ΔE was consequently set as the fifth response factor. In the statistical analyses, determination coefficients (R 2 ) for their absorbance, Hunter's L, a, b values, and ΔE were 0.94, 0.79, 0.73, 0.96, and 0.79, respectively, showing that the absorbance and Hunter's b value were good dependent variables for this model system. The optimum processing parameters were determined to yield glucosamine-cysteine Maillard reaction product with higher absorbance and higher colour change. The optimum estimated absorbance was achieved at the condition of initial pH 8.0, 111°C reaction temperature, 2.47 h reaction time, and 1.30 concentration ratio. The optimum condition for colour change measured by Hunter's b value was 2.41 h reaction time, 114°C reaction temperature, initial pH 8.3, and 1.26 concentration ratio. These results can provide the basic information for Maillard reaction of aqueous model system between glucosamine and cysteine.

Iron-Catalyzed Intramolecular C(sp(2))-N Cyclization of 1-(N-Arylpyrrol-2-yl)ethanone O-Acetyl Oximes toward Pyrrolo[1,2-a]quinoxaline Derivatives.

PubMed

Zhang, Zhiguo; Li, Junlong; Zhang, Guisheng; Ma, Nana; Liu, Qingfeng; Liu, Tongxin

2015-07-02

An efficient and convenient iron-catalyzed protocol has been developed for the synthesis of substituted pyrrolo[1,2-a]quinoxalines from 1-(N-arylpyrrol-2-yl)ethanone O-acetyl oximes through N-O bond cleavage and intramolecular directed C-H arylation reactions in acetic acid.

Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

PubMed Central

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-01-01

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

PubMed

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-10-21

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

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.

Glucosamine Activates Autophagy In Vitro and In Vivo

PubMed Central

Caramés, Beatriz; Kiosses, William B.; Akasaki, Yukio; Brinson, Diana C.; Eap, William; Koziol, James; Lotz, Martin K.

2013-01-01

Objectives Aging-associated changes in articular cartilage represent a main Osteoarthritis (OA) risk factor. Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimental defects in autophagy contribute to organismal and tissue specific aging while enhancement of autophagy may protect against certain aging related pathologies such as OA. The objective of this study was to determine whether glucosamine (GlcN) could activate autophagy. Methods Chondrocytes from normal human articular cartilage were treated with GlcN (0.1-10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3 and ribosomal protein S6 (prbS6) were determined by Western blotting. Autophagosome formation was analyzed by microscopy. Transgenic reporter mice with green fluorescent protein fused to LC3 (GFP-LC3 mice) were used to test changes in autophagy in response to starvation and GlcN administration. Results GlcN treatment of chondrocytes activated autophagy as indicated by increased of LC3-II levels, formation of LC3 puncta and increased LC3 turnover. This was associated with GlcN-mediated inhibition of Akt, FoxO3 and mTOR pathway. Administration of GlcN to GFP-LC3 mice markedly activated autophagy in articular cartilage. Conclusions GlcN modulates molecular targets of the autophagy pathway in vitro and in vivo and the enhancement of autophagy was mainly dependent on the Akt/FoxO and mTOR pathway. These findings suggest that GlcN is an effective autophagy activator and motivate future studies on its efficacy in modifying aging-related cellular changes and supporting joint health. PMID:23606170

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.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

PubMed

Renguet, Edith; Ginion, Audrey; Gélinas, Roselle; Bultot, Laurent; Auquier, Julien; Robillard Frayne, Isabelle; Daneault, Caroline; Vanoverschelde, Jean-Louis; Des Rosiers, Christine; Hue, Louis; Horman, Sandrine; Beauloye, Christophe; Bertrand, Luc

2017-08-01

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [ 13 C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart. NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation

A step-by-step approach to study the influence of N-acetylation on the adjuvanticity of N,N,N-trimethyl chitosan (TMC) in an intranasal nanoparticulate influenza virus vaccine.

PubMed

Verheul, Rolf J; Hagenaars, Niels; van Es, Thomas; van Gaal, Ethlinn V B; de Jong, Pascal H J L F; Bruijns, Sven; Mastrobattista, Enrico; Slütter, Bram; Que, Ivo; Heldens, Jacco G M; van den Bosch, Han; Glansbeek, Harrie L; Hennink, Wim E; Jiskoot, Wim

2012-03-12

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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

The Codacs™ direct acoustic cochlear implant actuator: exploring alternative stimulation sites and their stimulation efficiency.

PubMed

Grossöhmichen, Martin; Salcher, Rolf; Kreipe, Hans-Heinrich; Lenarz, Thomas; Maier, Hannes

2015-01-01

This work assesses the efficiency of the Codacs system actuator (Cochlear Ltd., Sydney Australia) in different inner ear stimulation modalities. Originally the actuator was intended for direct perilymph stimulation after stapedotomy using a piston prosthesis. A possible alternative application is the stimulation of middle ear structures or the round window (RW). Here the perilymph stimulation with a K-piston through a stapes footplate (SFP) fenestration (N = 10) as well as stimulation of the stapes head (SH) with a Bell prosthesis (N = 9), SFP stimulation with an Omega/Aerial prosthesis (N = 8) and reverse RW stimulation (N = 10) were performed in cadaveric human temporal bones (TBs). Codacs actuator output is expressed as equivalent sound pressure level (eq. SPL) using RW and SFP displacement responses, measured by Laser Doppler velocimetry as reference. The axial actuator coupling force in stimulation of stapes and RW was adjusted to ~5 mN. The Bell prosthesis and Omega/Aerial prosthesis stimulation generated similar mean eq. SPLs (Bell: 127.5-141.8 eq. dB SPL; Omega/Aerial: 123.6-143.9 eq. dB SPL), being significantly more efficient than K-piston perilymph stimulation (108.6-131.6 eq. dB SPL) and RW stimulation (108.3-128.2 eq. dB SPL). Our results demonstrate that SH, SFP and RW are adequate alternative stimulation sites for the Codacs actuator using coupling prostheses and an axial coupling force of ~5 mN. Based on the eq. SPLs, all investigated methods were adequate for in vivo hearing aid applications, provided that experimental conditions including constant coupling force will be implemented.

N-Acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in RAT and human plasma after disulfiram administration

PubMed Central

Winefield, Robert D.; Heemskerk, Anthonius A.M.; Kaul, Swetha; Williams, Todd D.; Caspers, Michael J.; Prisinzano, Thomas E.; McCance-Katz, Elinore F.; Lunte, Craig E.

2015-01-01

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291 > 128) is easily separable from DETC-NAC (MIM: 263 > 100) on C18 RP media with a methanol gradient. The method's linear range is 0.5–500 nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24 h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5 mg/d and 250mg/d dosing, CARB concentration peaks at 0.3 and 4 nM at 3 h followed by DETC-NAC peaks of 11 and 70 nM 2 h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80 nM at 6 h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95–105) is discussed. PMID:25720821

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

Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

PubMed

Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

2007-05-01

The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

SENIEUR status of the originating cell donor negates certain 'anti-immunosenescence' effects of ebselen and N-acetyl cysteine in human T cell clone cultures.

PubMed

Marthandan, Shiva; Freeburn, Robin; Steinbrecht, Susanne; Pawelec, Graham; Barnett, Yvonne

2014-01-01

Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined. T cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined. In this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor. The results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit.

Survey of the human acetylator polymorphism in spontaneous disorders.

PubMed Central

Evans, D A

1984-01-01

There is ample evidence that the human acetylator phenotypes are associated with drug induced phenomena. It is principally the slow acetylators who exhibit toxic adverse effects because of their relative inability to detoxify the original drug compounds. In rare instances, however, it is the rapid acetylators who are at a disadvantage. In the matter of association of spontaneous disease with either acetylator phenotype, there are two groups of disorders to consider. First, disorders in which carcinogenic amines are known to be an aetiological factor. This is because these amines are substrates for the polymorphic N-acetyltransferase activity and hence there is a possible rational basis for searching for an association. Secondly, other disorders where searches for associations are based more on hunches. In the first group there is a definite statistical association between cancer of the bladder and the slow acetylator phenotype. In prevalence studies the slow phenotype is 39% more associated with bladder cancer than is the rapid phenotype. On the basis of the evidence now available it is not possible to say whether this association is because slow acetylators develop the disease more frequently or whether they survive longer. In the second group the relevant studies show (1) a greatly increased prevalence of slow acetylators in Gilbert's disease; (2) a confirmed association between the rapid acetylator phenotype and diabetes; (3) a possible association between the rapid acetylator phenotype and breast cancer; (4) a possible association between the slow acetylator phenotype and leprosy in Chinese patients; (5) an earlier age of onset of thyrotoxicosis (Graves' disease) in slow acetylators than in rapid acetylators; (6) no evidence of an association between either phenotype and spontaneous systemic lupus erythematosus. PMID:6387123

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.

Co-Administration of Metformin and N-Acetyl Cysteine Fails to Improve Clinical Manifestations in PCOS Individual Undergoing ICSI

PubMed Central

Cheraghi, Ebrahim; Soleimani Mehranjani, Malek; Shariatzadeh, Mohammad Ali; Nasr Esfahani, Mohammad Hossein; Ebrahimi, Zahra

2014-01-01

Background Studies have demonstrated the efficacy of metformin (MTF ) in reducing insulin resistance and N-acetyl cysteine (NAC) in inhibiting oxidative stress which are involved in the pathogenesis of polycystic ovarian syndrome (PCOS). We aimed to compare the effects of MTF and NAC combination on serum metabolite and hormonal levels during the course of ovulation induction in PCOS individual candidates of intracytoplasmic sperm injection (ICSI). Materials and Methods In this prospective randomized clinical trial, placebo con- trolled pilot study, 80 patients of polycystic ovarian syndrome at the age of 25-35 years were divided into 4 groups (n=20): i. NAC=treated with N-acetyl cysteine (600 mg three times daily), ii. MTF=treated with metformin (500 mg three times daily), iii. MTF+NAC=treated with N-acetyl cysteine plus metformin (the offered doses) and iv. placebo (PLA). A total number of 20 patients (6 from MTF group, 4 from NAC group, 6 from MTF+NAC group and 4 from PLA group) were dropped of the study. The drugs were administrated from day 3 of menses of previous cycle until ovum pick-up. Results Serum levels of luteinizing hormone (LH), total testosterone, cholester- ol and triglyceride, insulin and leptin significantly reduced in the MTF and NAC groups compared to the placebo (p<0.01). But levels of LH, total testosterone, cholesterol and triglyceride had no significant reduction in the MTF+NAC groups compared to the placebo. The serum levels of malonyldialdehyde (MDA), insulin and leptin reduced significantly after treatment in the MTF+NAC group compared to the placebo (p<0.05). Conclusion Considering the adverse effect of combination therapy, we proposed the conadministration might have no beneficial effect for PCOS patient during course of ovulation induction of ICSI (Registration Number: IRCT201204159476N1). PMID:25083175

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

PubMed

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

2018-05-17

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

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

Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

PubMed Central

Wallace, H M; Nuttall, M E; Robinson, F C

1988-01-01

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine. PMID:3421945

[PHI regulates histone methylation and acetylation in Burkitt lymphoma Daudi cell line].

PubMed

Hong, Ling-Ling; Ma, Xu-Dong; Huang, Yi-Qun

2011-02-01

This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.

The Codacs™ Direct Acoustic Cochlear Implant Actuator: Exploring Alternative Stimulation Sites and Their Stimulation Efficiency

PubMed Central

Grossöhmichen, Martin; Salcher, Rolf; Kreipe, Hans-Heinrich; Lenarz, Thomas; Maier, Hannes

2015-01-01

This work assesses the efficiency of the Codacs system actuator (Cochlear Ltd., Sydney Australia) in different inner ear stimulation modalities. Originally the actuator was intended for direct perilymph stimulation after stapedotomy using a piston prosthesis. A possible alternative application is the stimulation of middle ear structures or the round window (RW). Here the perilymph stimulation with a K-piston through a stapes footplate (SFP) fenestration (N = 10) as well as stimulation of the stapes head (SH) with a Bell prosthesis (N = 9), SFP stimulation with an Omega/Aerial prosthesis (N = 8) and reverse RW stimulation (N = 10) were performed in cadaveric human temporal bones (TBs). Codacs actuator output is expressed as equivalent sound pressure level (eq. SPL) using RW and SFP displacement responses, measured by Laser Doppler velocimetry as reference. The axial actuator coupling force in stimulation of stapes and RW was adjusted to ~ 5 mN. The Bell prosthesis and Omega/Aerial prosthesis stimulation generated similar mean eq. SPLs (Bell: 127.5–141.8 eq. dB SPL; Omega/Aerial: 123.6–143.9 eq. dB SPL), being significantly more efficient than K-piston perilymph stimulation (108.6–131.6 eq. dB SPL) and RW stimulation (108.3–128.2 eq. dB SPL). Our results demonstrate that SH, SFP and RW are adequate alternative stimulation sites for the Codacs actuator using coupling prostheses and an axial coupling force of ~ 5 mN. Based on the eq. SPLs, all investigated methods were adequate for in vivo hearing aid applications, provided that experimental conditions including constant coupling force will be implemented. PMID:25785860

A pharmacoproteomic study confirms the synergistic effect of chondroitin sulfate and glucosamine

PubMed Central

Calamia, Valentina; Mateos, Jesús; Fernández-Puente, Patricia; Lourido, Lucía; Rocha, Beatriz; Fernández-Costa, Carolina; Montell, Eulalia; Vergés, Josep; Ruiz-Romero, Cristina; Blanco, Francisco J.

2014-01-01

Osteoarthritis (OA) is the most common age-related rheumatic disease. Chondrocytes play a primary role in mediating cartilage destruction and extracellular matrix (ECM) breakdown, which are main features of the OA joint. Quantitative proteomics technologies are demonstrating a very interesting power for studying the molecular effects of some drugs currently used to treat OA patients, such as chondroitin sulfate (CS) and glucosamine (GlcN). In this work, we employed the iTRAQ (isobaric tags for relative and absolute quantitation) technique to assess the effect of CS and GlcN, both alone and in combination, in modifying cartilage ECM metabolism by the analysis of OA chondrocytes secretome. 186 different proteins secreted by the treated OA chondrocytes were identified. 36 of them presented statistically significant differences (p ≤ 0.05) between untreated and treated samples: 32 were increased and 4 decreased. The synergistic chondroprotective effect of CS and GlcN, firstly reported by our group at the intracellular level, is now demonstrated also at the extracellular level. PMID:24912619

Evidence for lysine acetylation in the coat protein of a polerovirus.

PubMed

Cilia, Michelle; Johnson, Richard; Sweeney, Michelle; DeBlasio, Stacy L; Bruce, James E; MacCoss, Michael J; Gray, Stewart M

2014-10-01

Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

Reactions of the melatonin metabolite N(1)-acetyl-5-methoxykynuramine with carbamoyl phosphate and related compounds.

PubMed

Kuesel, Jana T; Hardeland, Rüdiger; Pfoertner, Henrike; Aeckerle, Nelia

2010-01-01

N-[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.

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

PubMed

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

1992-09-01

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

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.

Improved expression, purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice (Oryza sativa)

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

Miura-Ohnuma, Jun; Nonaka, Tsuyoshi; Katoh, Shizue

2005-12-01

Crystals of OsAGPR were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å. N-Acetyl-γ-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-γ-glutamyl phosphate to N-acetylglutamate-γ-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His){sub 6}] extension. OsAGPR(Ala50–Pro366) was purified and crystals weremore » obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å.« less

Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N- and O-Glycans without Direct Incorporation*

PubMed Central

Barthel, Steven R.; Antonopoulos, Aristotelis; Cedeno-Laurent, Filiberto; Schaffer, Lana; Hernandez, Gilberto; Patil, Shilpa A.; North, Simon J.; Dell, Anne; Matta, Khushi L.; Neelamegham, Sriram; Haslam, Stuart M.; Dimitroff, Charles J.

2011-01-01

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis. PMID:21493714

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.

Acetyl-coenzyme A deacylase activity in liver is not an artifact. Subcellular distribution and substrate specificity of acetyl-coenzyme A deacylase activities in rat liver

PubMed Central

Grigat, Klaus-P.; Koppe, Klaus; Seufert, Claus-D.; Söling, Hans-D

1979-01-01

Whole liver and isolated liver mitochondria are able to release free acetate, especially under conditions of increased fatty acid oxidation. In the present paper it is shown that rat liver contains acetyl-CoA deacylase (EC 3.1.2.1) activity (0.72μmol/min per g wet wt. of liver at 30°C and 0.5mm-acetyl-CoA). At 0.5mm-acetyl-CoA 73% of total enzyme activity was found in the mitochondria, 8% in the lysosomal fraction and 19% in the postmicrosomal supernatant. Mitochondrial subfractionation shows that mitochondrial acetyl-CoA deacylase activity is restricted to the matrix space. Mitochondrial acetyl-CoA deacylase showed almost no activity with either butyryl- or hexanoyl-CoA. Acetyl-CoA hydrolase activity from purified rat liver lysosomes exhibited a very low affinity for acetyl-CoA (apparent Km>15mm compared with an apparent Km value of 0.5mm for the mitochondrial enzyme) and reacted at about the same rate with acetyl-, n-butyryl- and hexanoyl-CoA. We could not confirm the findings of Costa & Snoswell [(1975) Biochem. J. 152, 167–172] according to which mitochondrial acetyl-CoA deacylase was considered to be an artifact resulting from the combined actions of acetyl-CoA–l-carnitine acetyltransferase (EC 2.3.1.7) and acetylcarnitine hydrolase. The results are in line with the concept that free acetate released by the liver under physiological conditions stems from the intramitochondrial deacylation of acetyl-CoA. PMID:34392

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

PubMed Central

Leung, Yiu Tak; Shi, Lihua; Maurer, Kelly; Song, Li; Zhang, Zhe; Petri, Michelle; Sullivan, Kathleen E

2015-01-01

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1. PMID

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.

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

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.

Slow acetylator mutations in the human polymorphic N-acetyltransferase gene in 786 Asians, blacks, Hispanics, and whites: Application to metabolic epidemiology

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

Lin, H.J.; Chunya Han; Lin, B.K.

1993-04-01

The aim was to determine the population frequencies of the major slow acetylator alleles of the polymorphic N-acetyltransferase (NA T2) gene, whose locus maps to chromosome 8. The authors used allele-specific PCR amplification on 786 dried blood spots obtained from Hong Kong Chinese, US Koreans, US blacks, US Hispanics, Germans, and US whites. Their results show that four slow acetylator alleles can be detected as mutations at positions 481, 590, and 857 in the NA T2 gene. Recognized base substitutions at positions 341 and 803 need not be determined, because they were almost always associated with the 481T mutation. Themore » known mutation at position 282 was strongly associated with the 590A mutation. The 481T, 590A, and 857A mutations accounted for virtually all of the slow acetylator alleles in Asian and white populations. The 857A mutation proved to be an Asiatic allele. The results will be useful in large-scale epidemiologic studies of cancer and other conditions potentially associated with the acetylator polymorphism. 20 refs., 3 figs., 4 tabs.« less

Glucosamine-containing supplement improves locomotor functions in subjects with knee pain: a randomized, double-blind, placebo-controlled study.

PubMed

Kanzaki, Noriyuki; Ono, Yoshiko; Shibata, Hiroshi; Moritani, Toshio

2015-01-01

The aim of this study was to investigate the ability of a glucosamine-containing supplement to improve locomotor functions in subjects with knee pain. A randomized, double-blind, placebo-controlled, parallel-group comparative study was conducted for 16 weeks in 100 Japanese subjects (age, 51.8±0.8 years) with knee pain. Subjects were randomly assigned to one of the two supplements containing 1) 1,200 mg of glucosamine hydrochloride, 60 mg of chondroitin sulfate, 45 mg of type II collagen peptides, 90 mg of quercetin glycosides, 10 mg of imidazole peptides, and 5 μg of vitamin D per day (GCQID group, n=50) or 2) a placebo (placebo group, n=50). Japanese Knee Osteoarthritis Measure, visual analog scale score, normal walking speed, and knee-extensor strength were measured to evaluate the effects of the supplement on knee-joint functions and locomotor functions. In subjects eligible for efficacy assessment, there was no significant group × time interaction, and there were improvements in knee-joint functions and locomotor functions in both groups, but there was no significant difference between the groups. In subjects with mild-to-severe knee pain at baseline, knee-extensor strength at week 8 (104.6±5.0% body weight vs 92.3±5.5% body weight, P=0.030) and the change in normal walking speed at week 16 (0.11±0.03 m/s vs 0.05±0.02 m/s, P=0.038) were significantly greater in the GCQID group than in the placebo group. Further subgroup analysis based on Kellgren-Lawrence (K-L) grade showed that normal walking speed at week 16 (1.36±0.05 m/s vs 1.21±0.02 m/s, P<0.05) was significantly greater in the GCQID group than in the placebo group in subjects with K-L grade I. No adverse effect of treatment was identified in the safety assessment. In subjects with knee pain, GCQID supplementation was effective for relieving knee pain and improving locomotor functions.

Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation

PubMed Central

Cohen, Todd J.; Constance, Brian H.; Hwang, Andrew W.; James, Michael; Yuan, Chao-Xing

2016-01-01

Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies. PMID:27383765

Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

PubMed

Cohen, Todd J; Constance, Brian H; Hwang, Andrew W; James, Michael; Yuan, Chao-Xing

2016-01-01

Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

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.

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

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Neuroprotection in rabbit retina with N-acetyl-aspartylglutamate and 2-phosphonyl-methyl pentanedioic acid

NASA Astrophysics Data System (ADS)

Hacker, Henry D.; Yourick, Debra L.; Koenig, Michael K.; Slusher, Barbara S.; Meyerhoff, James L.

1999-06-01

Retinal tissue is subject to ischemia from diabetic retinopathy and other conditions that affect the retinal vasculature such as lupus erythematosus and temporal arteritis. There is evidence in animal models of reversible ischemia that a therapeutic window exists during early recovery when agents that reduce glutamate activity at its receptor sites can rescue neurons from injury. To model ischemia, we used sodium cyanide (NaCN), to inhibit oxidative metabolism, and 2-deoxyglucose (2-DG) to inhibit glycolysis. Dissociated rabbit retina cells were studied to evaluate the potential neuroprotective effects of N-acetyl-aspartyl-glutamate (MAAG), which competes with glutamate as a low-potency agonist at the NMDA receptor complex. N-acetylated α-linked acidic dipeptidase (NAALADase; the NAAG-hydrolyzing enzyme) is responsible for the hydrolysis of NAAG into glutamate, a neurotransmitter and potent excitotoxin, and N-acetylaspartate. 2-Phosphonyl-methyl pentanedioic acid (PMPA) and β-linked NAAG (β-NAAG), inhibitors of NAALADase, were also tested, since inhibition of NAALADase could reduce synaptic glutamate and increase the concentration of NAAG. We found that metabolic inhibition with NaCN/2-DG for 1 hour caused 50% toxicity as assessed with the MTT assay. Co-treatment with NAAG resulted in dose-dependent protection of up to 55% (p<0.005). When the non-hydrolyzable, NAALADase inhibitor β-NAAG was employed dose-dependent protection of up to 37% was observed (p<0.001). PMPA also showed 48% protection (p<.05-.001) against these insults. These data suggest that NAAG may antagonize the effect of glutamate at the NMDA receptor complex in retina. Inhibition of NAALADase by PMPA and β-NAAG may increase the activity of endogenous NAAG.

Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

PubMed

Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Hellebrand, Pauline H M; Savelkoul, Paul H M; Stassen, Frank R M

2017-05-01

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-beta-D glucosaminidase is associated with type 2 diabetes in Mexican Americans.

PubMed

Lehman, Donna M; Fu, Dong-Jing; Freeman, Angela B; Hunt, Kelly J; Leach, Robin J; Johnson-Pais, Teresa; Hamlington, Jeanette; Dyer, Thomas D; Arya, Rector; Abboud, Hanna; Göring, Harald H H; Duggirala, Ravindranath; Blangero, John; Konrad, Robert J; Stern, Michael P

2005-04-01

Excess O-glycosylation of proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) may be involved in the pathogenesis of type 2 diabetes. The enzyme O-GlcNAc-selective N-acetyl-beta-d glucosaminidase (O-GlcNAcase) encoded by MGEA5 on 10q24.1-q24.3 reverses this modification by catalyzing the removal of O-GlcNAc. We have previously reported the linkage of type 2 diabetes and age at diabetes onset to an overlapping region on chromosome 10q in the San Antonio Family Diabetes Study (SAFADS). In this study, we investigated menangioma-expressed antigen-5 (MGEA5) as a positional candidate gene. Twenty-four single nucleotide polymorphisms (SNPs), identified by sequencing 44 SAFADS subjects, were genotyped in 436 individuals from 27 families whose data were used in the original linkage report. Association tests indicated significant association of a novel SNP with the traits diabetes (P = 0.0128, relative risk = 2.77) and age at diabetes onset (P = 0.0017). The associated SNP is located in intron 10, which contains an alternate stop codon and may lead to decreased expression of the 130-kDa isoform, the isoform predicted to contain the O-GlcNAcase activity. We investigated whether this variant was responsible for the original linkage signal. The variance attributed to this SNP accounted for approximately 25% of the logarithm of odds. These results suggest that this variant within the MGEA5 gene may increase diabetes risk in Mexican Americans.

Efficacy and tolerance of enzymatic hydrolysed collagen (EHC) vs. glucosamine sulphate (GS) in the treatment of knee osteoarthritis (KOA).

PubMed

Trč, Tomáš; Bohmová, Jana

2011-03-01

This was a 13-week, multicentre, randomised, parallel, double-blind study. One hundred men and women volunteers aged ≥ 40 years with knee osteoarthritis (KOA) were randomised to once daily enzymatic hydrolysed collagen (EHC) 10 g or glucosamine sulphate (GS) 1.5 g for 90 consecutive days. Follow-up took place after two weeks and after one, two and three months. Primary [visual analogue scale (VAS), Western Ontario and McMaster Universities (WOMAC Index)] and secondary outcomes variables, assessed at weeks two, four, eight and 12, were KOA pain intensity measured by quadruple visual analogue scales in the target knee, the WOMAC total score index, patient's and investigator's global assessments of disease activity, joint assessment, use of rescue medication (ibuprofen 400 mg tablets) and assessment of Quality of Life index (SF-36 Questionnaire). Safety and tolerability were also evaluated. Clear improvement was observed in both joint pain and symptoms in patients with KOA treated with EHC (Colatech®) and significant differences were observed. Mean reductions from baseline for EHC 10 g daily and GS 1.5 g, respectively, were KOA pain intensity reduction in the target knee for Colatech® (p < 0.05): WOMAC index decrease ≤ 15 points at the last visit (day 90) for Colatech® in 16 patients (34.04%) (p < 0.05) and for glucosamine in six patients (13.04%); total score index for painful joints: Colatech® 1.6 (p < 0.05) and glucosamine 1.8; total score index for swollen joints: Colatech® 0.5 (p < 0.05) and glucosamine 0.7; patient's global assessment of efficacy as the sum of improvement good + ideal: 80.8% for Colatech® and 46.6% for glucosamine (p < 0.05). EHC (Colatech®) showed superior improvement over GS in the SF-36 Questionnaire in the Physical Health Index (42.0 for Colatech and 40.0 for glucosamine). The incidence of adverse events was similar in both groups. Both EHC and GS were well tolerated.

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.

Mapping sugar beet pectin acetylation pattern.

PubMed

Ralet, Marie-Christine; Cabrera, Juan Carlos; Bonnin, Estelle; Quéméner, Bernard; Hellìn, Pilar; Thibault, Jean-François

2005-08-01

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi.

PubMed

Su, Chang; Lu, Yang; Liu, Haoping

2016-10-03

N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription.

N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

PubMed Central

Su, Chang; Lu, Yang; Liu, Haoping

2016-01-01

N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

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

Elephant’s breast milk contains large amounts of glucosamine

PubMed Central

TAKATSU, Zenta; TSUDA, Muneya; YAMADA, Akio; MATSUMOTO, Hiroshi; TAKAI, Akira; TAKEDA, Yasuhiro; TAKASE, Mitsunori

2016-01-01

Hand-reared elephant calves that are nursed with milk substitutes sometimes suffer bone fractures, probably due to problems associated with nutrition, exercise, sunshine levels and/or genetic factors. As we were expecting the birth of an Asian elephant (Elephas maximus), we analyzed elephant’s breast milk to improve the milk substitutes for elephant calves. Although there were few nutritional differences between conventional substitutes and elephant’s breast milk, we found a large unknown peak in the breast milk during high-performance liquid chromatography-based amino acid analysis and determined that it was glucosamine (GlcN) using liquid chromatography/mass spectrometry. We detected the following GlcN concentrations [mean ± SD] (mg/100 g) in milk hydrolysates produced by treating samples with 6M HCl for 24 hr at 110°C: four elephant’s breast milk samples: 516 ± 42, three cow’s milk mixtures: 4.0 ± 2.2, three mare’s milk samples: 12 ± 1.2 and two human milk samples: 38. The GlcN content of the elephant’s milk was 128, 43 and 14 times greater than those of the cow’s, mare’s and human milk, respectively. Then, we examined the degradation of GlcN during 0–24 hr hydrolyzation with HCl. We estimated that elephant’s milk contains >880 mg/100 g GlcN, which is similar to the levels of major amino acids in elephant’s milk. We concluded that a novel GlcN-containing milk substitute should be developed for elephant calves. The efficacy of GlcN supplements is disputed, and free GlcN is rare in bodily fluids; thus, the optimal molecular form of GlcN requires a further study. PMID:28049867

Engineering of N-acetylglucosamine metabolism for improved antibiotic production in Streptomyces coelicolor A3(2) and an unsuspected role of NagA in glucosamine metabolism.

PubMed

Świątek, Magdalena A; Urem, Mia; Tenconi, Elodie; Rigali, Sébastien; van Wezel, Gilles P

2012-01-01

N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

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

Postmortem Toxicology Findings of Acetyl Fentanyl, Fentanyl, and Morphine in Heroin Fatalities in Tampa, Florida

PubMed Central

Pearson, Julia; Poklis, Justin; Poklis, Alphonse; Wolf, Carl; Mainland, Mary; Hair, Laura; Devers, Kelly; Chrostowski, Leszek; Arbefeville, Elise; Merves, Michele

2017-01-01

In the last two years, an epidemic of 40 fatal heroin overdose cases has occurred in the Tampa area of Florida. Of these cases, 14 involved fentanyl and acetyl fentanyl. Victim demographics, case histories, toxicology findings, and causes and manners of death for all 40 deaths are presented. In 26 deaths in which acetyl fentanyl or fentanyl were not involved, free and total peripheral blood morphine concentrations were consistent with fatal heroin intoxications, averaging 0.16 mg/L and 0.35 mg/L, respectively. In the heroin cases with fentanyl present (n=7), the average free morphine concentration was 0.040 mg/L, the average total morphine concentration was 0.080 mg/L, and the average fentanyl concentration was 0.012 mg/L. In the cases with heroin, fentanyl, and acetyl fentanyl (n=3), the average free morphine concentration was 0.010 mg/L, the average total morphine concentration was 0.030 mg/L, the average fentanyl concentration was 0.018 mg/L, and the average acetyl fentanyl concentration was 0.008 mg/L. In the cases involving only acetyl fentanyl (without heroin or fentanyl, n=4), the average acetyl fentanyl concentration was 0.47 mg/L and the average acetyl norfentanyl concentration was 0.053 mg/L. The presented cases, with associated drug concentrations, case histories, demographics, and causes and manners of death may help provide assistance with the interpretation of the postmortem findings. Based on case circumstances, autopsy results, and toxicology results, it is evident that fentanyl and/or acetyl fentanyl, when present, contributed to the cause of death. PMID:29034049

Solid-state, triboelectrostatic and dissolution characteristics of spray-dried piroxicam-glucosamine solid dispersions.

PubMed

Adebisi, Adeola O; Kaialy, Waseem; Hussain, Tariq; Al-Hamidi, Hiba; Nokhodchi, Ali; Conway, Barbara R; Asare-Addo, Kofi

2016-10-01

This work explores the use of both spray drying and d-glucosamine HCl (GLU) as a hydrophilic carrier to improve the dissolution rate of piroxicam (PXM) whilst investigating the electrostatic charges associated with the spray drying process. Spray dried PXM:GLU solid dispersions were prepared and characterised (XRPD, DSC, SEM). Dissolution and triboelectric charging were also conducted. The results showed that the spray dried PXM alone, without GLU produced some PXM form II (DSC results) with no enhancement in solubility relative to that of the parent PXM. XRPD results also showed the spray drying process to decrease the crystallinity of GLU and solid dispersions produced. The presence of GLU improved the dissolution rate of PXM. Spray dried PXM: GLU at a ratio of 2:1 had the most improved dissolution. The spray drying process generally yielded PXM-GLU spherical particles of around 2.5μm which may have contributed to the improved dissolution. PXM showed a higher tendency for charging in comparison to the carrier GLU (-3.8 versus 0.5nC/g for untreated material and -7.5 versus 3.1nC/g for spray dried materials). Spray dried PXM and spray dried GLU demonstrated higher charge densities than untreated PXM and untreated GLU, respectively. Regardless of PXM:GLU ratio, all spray dried PXM:GLU solid dispersions showed a negligible charge density (net-CMR: 0.1-0.3nC/g). Spray drying of PXM:GLU solid dispersions can be used to produce formulation powders with practically no charge and thereby improving handling as well as dissolution behaviour of PXM. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.

PubMed

Muth, V; Nadaud, S; Grummt, I; Voit, R

2001-03-15

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

The acetylation of insulin

PubMed Central

Lindsay, D. G.; Shall, S.

1971-01-01

The acetylation of the free amino groups of insulin was studied by reaction of the hormone with N-hydroxysuccinimide acetate at pH6.9 and 8.5. The products formed were separated by chromatography on DEAE-Sephadex and were characterized by isoelectric focusing, by end-group analysis, by the incorporation of [3H]acetyl groups in the molecule, and by treatment with trypsin that had been treated with 1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one (`tosylphenylalanyl chloromethyl ketone'). Three monosubstituted products, two disubstituted products and one trisubstituted derivative were prepared. The α-amino groups of the terminal residues and the ∈-amino group of the lysine-B29 were the sites of reaction. Acetylation of any of the free amino groups did not affect the biological activity of insulin. It was demonstrated, however, that substitution at the glycine-A1 amino group by the larger residues, acetoacetyl or thiazolidinecarbonyl, produced a decrease in biological activity. Modification of the lysine-B29 or phenylalanine-B1 amino groups with these larger reagents did not affect the biological activity. Modification of the phenylalanine-B1 amino group by any of the three substituents resulted in a large decrease in the affinity of insulin for anti-insulin antibodies raised in the guinea pig. Modification of the other two amino groups did not affect the reaction with antibody. These observations are correlated with the tertiary structure of insulin. ImagesFig. 4. PMID:5113488

Use of glucosamine and chondroitin supplements in relation to risk of colorectal cancer: Results from the Nurses’ Health Study and Health Professionals Follow-up Study

PubMed Central

Kantor, Elizabeth D.; Zhang, Xuehong; Wu, Kana; Signorello, Lisa B.; Chan, Andrew T.; Fuchs, Charles S.; Giovannucci, Edward L.

2016-01-01

Recent epidemiologic evidence has emerged to suggest that use of glucosamine and chondroitin supplements may be associated with reduced risk of colorectal cancer (CRC). We therefore evaluated the association between use of these non-vitamin, non-mineral supplements and risk of CRC in two prospective cohorts, the Nurses’ Health Study and Health Professionals Follow-up Study. Regular use of glucosamine and chondroitin was first assessed in 2002 and participants were followed until 2010, over which time 672 CRC cases occurred. Cox proportional hazards regression was used to estimate relative risks (RRs) within each cohort, and results were pooled using a random effects meta-analysis. Associations were comparable across cohorts, with a RR of 0.79 (95% CI: 0.63–1.00) observed for any use of glucosamine and a RR of 0.77 (95% CI: 0.59–1.01) observed for any use of chondroitin. Use of glucosamine in the absence of chondroitin was not associated with risk of CRC, whereas use of glucosamine + chondroitin was significantly associated with risk (RR: 0.77; 95% CI: 0.58–0.999). The association between use of glucosamine + chondroitin and risk of CRC did not change markedly when accounting for change in exposure status over follow-up (RR: 0.75; 95% CI: 0.58–0.96), nor did the association significantly vary by sex, aspirin use, body mass index, or physical activity. The association was comparable for cancers of the colon and rectum. Results support a protective association between use of glucosamine and chondroitin and risk of CRC. Further study is needed to better understand the chemopreventive potential of these supplements. PMID:27357024

Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess

PubMed Central

Huat, Lim Boon; Garcia, Alfonso Olivos; Ning, Tan Zi; Kin, Wong Weng; Noordin, Rahmah; Azham, Siti Shafiqah Anaqi; Jie, Lee Zhi; Ching, Guee Cher; Chong, Foo Phiaw; Dam, Pim Chau

2014-01-01

Objective To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Methods Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. Results A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. Conclusions This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations. PMID:25182945

Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

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

Yamagata, Kazutsune, E-mail: kyamagat@ncc.go.jp; Kitabayashi, Issay

2009-12-25

Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest thatmore » Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.« less

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

Unexpected Diversity of Escherichia coli Sialate O-Acetyl Esterase NanS

PubMed Central

Rangel, Ariel; Steenbergen, Susan M.

2016-01-01

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

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

PubMed

Lewis, Amanda L; Cao, Hongzhi; Patel, Silpa K; Diaz, Sandra; Ryan, Wesley; Carlin, Aaron F; Thon, Vireak; Lewis, Warren G; Varki, Ajit; Chen, Xi; Nizet, Victor

2007-09-21

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

PubMed

Cova, Marta; López-Gutiérrez, Borja; Artigas-Jerónimo, Sara; González-Díaz, Aida; Bandini, Giulia; Maere, Steven; Carretero-Paulet, Lorenzo; Izquierdo, Luis

2018-03-05

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

Role of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline in the Antifibrotic and Anti-Inflammatory Effects of the Angiotensin-Converting Enzyme Inhibitor Captopril in Hypertension

PubMed Central

Peng, Hongmei; Carretero, Oscar A.; Liao, Tang-Dong; Peterson, Edward L.; Rhaleb, Nour-Eddine

2012-01-01

Angiotensin-converting enzyme inhibitors (ACEis) are known to have antifibrotic effects on the heart and kidney in both animal models and humans. N-acetyl-seryl-aspartyl-lysyl-proline is a natural inhibitor of proliferation of hematopoietic stem cells and a natural substrate of ACEi that was reported to prevent cardiac and renal fibrosis in vivo. However, it is not clear whether N-acetyl-seryl-aspartyl-lysyl-proline participates in the antifibrotic effects of ACEi. To clarify this issue, we used a model of aldosterone-salt–induced hypertension in rats treated with the ACEi captopril either alone or combined with an anti-N-acetyl-seryl-aspartyl-lysyl-proline monoclonal antibody. These hypertensive rats had the following: (1) left ventricular and renal hypertrophy, as well as increased collagen deposition in the left ventricular and the kidney; (2) glomerular matrix expansion; and (3) increased ED1-positive cells and enhanced phosphorylated-p42/44 mitogen-activated protein kinase in the left ventricle and kidney. The ACEi alone significantly lowered systolic blood pressure (P=0.008) with no effect on organ hypertrophy; it significantly lowered left ventricular collagen content, and this effect was blocked by the monoclonal antibody as confirmed by the histological data. As expected, the ACEi significantly decreased renal collagen deposition and glomerular matrix expansion, and these effects were attenuated by the monoclonal antibody. Likewise, the ACEi significantly decreased ED1-positive cells and inhibited p42/44 mitogen-activated protein kinase phosphorylation in the left ventricle and kidney, and these effects were blocked by the monoclonal antibody. We concluded that in aldosterone-salt–induced hypertension, the antifibrotic effect of ACEi on the heart and kidney, is partially mediated by N-acetyl-seryl-aspartyl-lysyl-proline, resulting in decreased inflammatory cell infiltration and p42/44 mitogen-activated protein kinase activation. PMID:17283252

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

Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose 1

PubMed Central

Leznicki, Antoni J.; Bandurski, Robert S.

1988-01-01

The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-β-d-glucose from uridine-5′-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss. Images Fig. 4 PMID:11537438

Cloning, expression profiling, and acetylation identification of alpha-tubulin N-acetyltransferase 1 from Bombyx mori.

PubMed

Zhou, Huaixiang; Cheng, Xusheng; Xu, Xiaoyuan; Jiang, Tianlong; Zhou, Haimeng; Sheng, Qing; Nie, Zuoming

2018-03-22

Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself. © 2018 Wiley Periodicals, Inc.

Molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides

NASA Astrophysics Data System (ADS)

Hoon Choi, Seung; Yun Yu, Jeong; Kwang Shin, Jae; Shik Jhon, Mu

1994-07-01

The occurrence of cis imide bonds in proteins is much higher than that of cis amide bonds due to the unique properties of proline. In order to examine the relationship between the high occurrence of these cis imide bonds and the residues preceding the proline, we perform molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides (AcXaaProNHMe). We investigate the conformational energies and structures of trans- and cis-AcXaa where Xaa has 12 amino acids in the vacuum state and 5 amino acids in the solution state. It is found that the occurrence of the cis imide bonds is strongly affected by the residue preceding the proline, and the dihedral angles (φ,ψ) of the backbone in AcXaaProNHMe are influenced by the configuration of the imide bond. We also find that the equilibrium properties of XaaPro in solution simulations are more similar to the statistics of X-ray crystallographic data than are those in vacuum simulations and solvation causes a remarkable change in the conformation of the pyrrolidine ring from the endo to the exo form.

Cytochrome P450 2D6 and 3A4 enzyme inhibition by amine stimulants in dietary supplements.

PubMed

Liu, Yitong; Santillo, Michael F

2016-01-01

A number of dietary supplements used for weight loss and athletic performance enhancement have been recently shown to contain a variety of stimulants, for which there is a lack of pharmacological and toxicological information. One concern for these emerging compounds is their potential to inhibit metabolic enzymes in the liver such as cytochromes P450 (CYP), which can lead to unexpected interactions among dietary supplements, drugs, and other xenobiotics. In this study, inhibition of human recombinant CYP2D6 and CYP3A4 by 27 amine stimulants associated with dietary supplements and their analogs was evaluated by luminescence assays. The strongest CYP2D6 inhibitors were coclaurine (IC50  = 0.14 ± 0.01 μM) and N-benzylphenethylamine (IC50  = 0.7 ± 0.2 μM), followed by several other relatively strong inhibitors (IC50 , 2-12 μM) including β-methylphenethylamine, N,β-dimethylphenethylamine (phenpromethamine), 1,3-dimethylamylamine (DMAA), N,α-diethylphenethylamine, higenamine (norcoclaurine) and N,N-diethylphenethylamine. Only nine compounds inhibited CYP3A4 by 20-55% at 100 μM. Results of this study illustrate that several amine stimulants associated with dietary supplements inhibit CYP2D6 and CYP3A4 in vitro, and these compounds may participate in adverse drug-dietary supplement interactions in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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

O-Acetylation of Plant Cell Wall Polysaccharides

PubMed Central

Gille, Sascha; Pauly, Markus

2011-01-01

Plant cell walls are composed of structurally diverse polymers, many of which are O-acetylated. How plants O-acetylate wall polymers and what its function is remained elusive until recently, when two protein families were identified in the model plant Arabidopsis that are involved in the O-acetylation of wall polysaccharides – the reduced wall acetylation (RWA) and the trichome birefringence-like (TBL) proteins. This review discusses the role of these two protein families in polysaccharide O-acetylation and outlines the differences and similarities of polymer acetylation mechanisms in plants, fungi, bacteria, and mammals. Members of the TBL protein family had been shown to impact pathogen resistance, freezing tolerance, and cellulose biosynthesis. The connection of TBLs to polysaccharide O-acetylation thus gives crucial leads into the biological function of wall polymer O-acetylation. From a biotechnological point understanding the O-acetylation mechanism is important as acetyl-substituents inhibit the enzymatic degradation of wall polymers and released acetate can be a potent inhibitor in microbial fermentations, thus impacting the economic viability of, e.g., lignocellulosic based biofuel production. PMID:22639638

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.

Glucosamine-containing supplement improves locomotor functions in subjects with knee pain: a randomized, double-blind, placebo-controlled study

PubMed Central

Kanzaki, Noriyuki; Ono, Yoshiko; Shibata, Hiroshi; Moritani, Toshio

2015-01-01

Background The aim of this study was to investigate the ability of a glucosamine-containing supplement to improve locomotor functions in subjects with knee pain. Methods A randomized, double-blind, placebo-controlled, parallel-group comparative study was conducted for 16 weeks in 100 Japanese subjects (age, 51.8±0.8 years) with knee pain. Subjects were randomly assigned to one of the two supplements containing 1) 1,200 mg of glucosamine hydrochloride, 60 mg of chondroitin sulfate, 45 mg of type II collagen peptides, 90 mg of quercetin glycosides, 10 mg of imidazole peptides, and 5 μg of vitamin D per day (GCQID group, n=50) or 2) a placebo (placebo group, n=50). Japanese Knee Osteoarthritis Measure, visual analog scale score, normal walking speed, and knee-extensor strength were measured to evaluate the effects of the supplement on knee-joint functions and locomotor functions. Results In subjects eligible for efficacy assessment, there was no significant group × time interaction, and there were improvements in knee-joint functions and locomotor functions in both groups, but there was no significant difference between the groups. In subjects with mild-to-severe knee pain at baseline, knee-extensor strength at week 8 (104.6±5.0% body weight vs 92.3±5.5% body weight, P=0.030) and the change in normal walking speed at week 16 (0.11±0.03 m/s vs 0.05±0.02 m/s, P=0.038) were significantly greater in the GCQID group than in the placebo group. Further subgroup analysis based on Kellgren–Lawrence (K–L) grade showed that normal walking speed at week 16 (1.36±0.05 m/s vs 1.21±0.02 m/s, P<0.05) was significantly greater in the GCQID group than in the placebo group in subjects with K–L grade I. No adverse effect of treatment was identified in the safety assessment. Conclusion In subjects with knee pain, GCQID supplementation was effective for relieving knee pain and improving locomotor functions. PMID:26604721

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

Genetic incorporation of Nε-acetyllysine reveals a novel acetylation-sumoylation switch in yeast.

PubMed

Kim, Sang-Woo; Lee, Kyung Jin; Kim, Sinil; Kim, Jihyo; Cho, Kyukwang; Ro, Hyeon-Su; Park, Hee-Sung

2017-11-01

The lysine acetylation of proteins plays a key role in regulating protein functions, thereby controlling a wide range of cellular processes. Despite the prevalence and significance of lysine acetylation in eukaryotes, however, its systematic study has been challenged by the technical limitations of conventional approaches for selective lysine acetylation in vivo. Here, we report the in vivo study of lysine acetylation via the genetic incorporation of N ε -acetyllysine in yeast. We demonstrate that a newly discovered acetylation-sumoylation switch precisely controls the localization and cellular function of the yeast septin protein, Cdc11, during the cell cycle. This approach should facilitate the comprehensive in vivo study of lysine acetylation across a wide range of proteins in eukaryotic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.