- Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria
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The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid intermediate and NAD1 regeneration. The crystal structure of RgNanOx in complex with the NAD1 cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.
- Angulo, Jesus,Bell, Andrew,Juge, Nathalie,Latousakis, Dimitrios,Lee, Micah,Monaco, Serena,Naismith, James H.,Severi, Emmanuele,Thomas, Gavin H.
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- Efficient whole-cell biocatalytic synthesis of N-Acetyl-D-neuraminic acid
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N-Acetyl-D-neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32 ± 0.56 g/liter Neu5Ac were obtained from 65.61 ± 2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4 ± 0.4% purity, 80.87 ± 0.79% recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes.
- Xu, Ping,Qiu, Jian Hua,Zhang, Yi Nan,Chen, Jing,Wang, Peng George,Yan, Bing,Song, Jing,Xi, Ri Mo,Deng, Zi Xin,Ma, Cui Qing
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- Enzymatic synthesis of cytidine 5′-monophospho-N-acetylneuraminic acid
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We have established an efficient method for enzymatic production of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NeuAc) from inexpensive materials, N-acetylglucosamine (GlcNAc) and cytidine 5′-monophosphate (CMP). The Haemophilus influenzae nanE gene encoding GlcNAc 6-phosphate (GlcNAc 6-P) 2-epimerase and the Campylobacter jejuni neuB1 gene encoding N-acetylneuraminic acid (NeuAc) synthetase, both of whose products are involved in NeuAc biosynthesis, were cloned and co-expressed in Escherichia coli cells. We examined the synthesis of NeuAc from GlcNAc via GlcNAc 6-P, N-acetylmannosamine (ManNAc) 6-P, and ManNAc by the use of E. coli cells producing GlcNAc 6-P 2-epimerase and NeuAc synthetase, in expectation of biological functions of E. coli such as the supply of phosphoenolpyruvate (PEP), which is an essential substrate for NeuAc synthetase, GlcNAc phospholylation by the PEP-dependent phosphotransferase system, and dephospholylation of ManNAc 6-P. Eleven mM NeuAc was synthesized from 50 mM GlcNAc by recombinant E. coli cells with the addition of glucose as an energy source. Next we attempted to synthesize CMP-NeuAc from GlcNAc and CMP using yeast cells, recombinant E. coli cells, and H. influenzae CMP-Neu-Ac synthetase, and succeeded in efficient production of CMP-NeuAc due to a sufficient supply of PEP and efficient conversion of CMP to cytidine 5′-triphosphate by yeast cells.
- Hamamoto, Tomoki,Takeda, So,Noguchi, Toshitada
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- Quantitative Standards of 4-O-Acetyl- and 9-O-Acetyl-N-Acetylneuraminic Acid for the Analysis of Plasma and Serum
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N-Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine-carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac2, as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9-O- and 4-O-acetylated sialic acids (Neu5,9Ac2 and Neu4,5Ac2) with optimisation of previously reported synthetic routes. Neu5,9Ac2 was synthesised in 1 step in 68 % yield. Neu4,5Ac2 was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated.
- Badia, Concepcion,Cheeseman, Jack,Gardner, Richard A.,Kuhnle, Gunter,Osborn, Helen M. I.,Spencer, Daniel I. R.,Thomson, Rebecca I.
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- Three-step synthesis of sialic acids and derivatives
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(Chemical Equation Presented) Flexible yet efficient: Sialic acids such as L-N-acetylneuraminic acid (see picture) can be synthesized in only three steps by 1) vinylation of an aldose through a modified Petasis coupling reaction, 2) 1,3-dipolar cycloaddition with a nitrone to construct an isoxazolidine ring, and 3) base-catalyzed β elimination/ring opening of the isoxazolidine to generate a γ-hydroxy-α-keto acid.
- Hong, Zhangyong,Liu, Lei,Hsu, Che-Chang,Wong, Chi-Huey
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p. 7417 - 7421
(2007/10/03)
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- Randomly generated glycopeptide combinatorial libraries
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Randomly generated glycopeptide combinatorial libraries are generated by randomly glycosylating a peptide having at least one glycosylation site with at least one glycosyl donor, optionally blocking unreacted glycosylation sites on the glycopeptides and optionally selectively removing one or more protecting groups on the carbohydrate groups introduced at the first level; whereby a first level library of glycopeptides is created; and then optionally randomly glycosylating said first level library of glycopeptides, or a combination of first level libraries of glycopeptides, with at least one glycosyl donor, and optionally selectively removing one or more designated protecting groups on the carbohydrate groups introduced at the second level; whereby a second level library of glycopeptides is created. Further iterations of the process result in higher level libraries of increased diversity. The glycopeptide libraries including, e.g., carcinoma-associated mucins such as MUC1, are screened for drug-like, competitive inhibitory, immunostimulatory, antibody-like, and other biological activities.
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- Nucleic acid-coupled colorimetric analyte detectors
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The present invention relates to methods and compositions for the direct detection of analytes and membrane conformational changes through the detection of color changes in biopolymeric materials. In particular, the present invention provide for the direct colorimetric detection of analytes using nucleic acid ligands at surfaces of polydiacetylene liposomes and related molecular layer systems.
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- Sialic acid derivatives
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Sialic acid derivatives represented by the general formula (I): STR1 wherein R1 is a steroidal compound residue; R2 is H or alkyl; R3 is alkyl; STR2 wherein each of R6 and R7 is H, alkyl or the like and I is an integer of 0 to 6; or the like; X is O or S; R4 is H or acyl; and R5 is R14 O-- (R14 is H or acyl) or R15 NH--(R15 is acyl or the like); their salts, hydrates or solvates are provided. Sialic acid derivatives of the present invention are expected to be effective medicines for the prevention and therapy of senile dementia including Alzheimer's disease and the like, because they increase ChAT activity in cholinergic neurons.
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- Sialic acid derivatives
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Sialic acid derivatives represented by the general formula (I): wherein R1 is a steroidal compound residue; R2 is H or alkyl; R3 is alkyl; STR1 wherein each of R6 and R7 is H, alkyl or the like and I is an integer of 0 to 6; or the like; X is O or S; R4 is H or acyl; and R5 is R14 O--(R14 is H or acyl) or R15 NH--(R15 is acyl or the like); their salts, hydrates or solvates are provided. Sialic acid derivatives of the present invention are expected to be effective medicines for the prevention and therapy of senile dementia including Alzheimer's disease and the like, because they increase ChAT activity in cholinergic neurons.
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- Derivatives of neuraminic acid
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Provided are new derivatives of neuraminic acid of formula (I), where Ac represents an acyl residue of an aliphatic, araliphatic, aromatic, alicyclic, or heterocyclic carboxylic acid, including carboxylic amides, their 2-hydrocarbyl-glycosides, and their peracylated derivatives at the hydroxy groups of both these series of amides. These compositions are therapeutically useful in providing a protective effect against the neurotoxicity induced by excitatory amino acids, and can therefore be used in therapies of the central nervous system. STR1
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- Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
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A polypeptide or glycosylated polypeptide with at least one new carbohydrate chain produced by means of recombinant DNA technique, which has protease resistance and thermal stability and is expected to have longer lifetime in blood than those of a naturally-occurring form.
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- Method for production of sialic acid
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The present invention is directed to a method for producing crude sialic acid, comprising hydrolysis of a delipidated egg yolk and a method for producing high purity sialic acid, which comprises desalting a solution containing sialic acid obtainable by hydrolyzing a delipidated egg yolk, adsorbing sialic acid to an anion exchange resin and then eluting said sialic acid. The present invention makes it possible to produce and purify sialic acid from delipidated egg yolk on an industrial scale.
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- Blocked lectins, methods and affinity support for making same using affinity ligands, and method of killing selected cell populations having reduced nonselective cytotoxicity
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An activated affinity ligand is described comprising: a ligand having (a) a region with affinity for binding sites of a lectin; and (b) a reactive group capable of covalently linking the ligand to the lectin to thereby block one or more of the binding sites of the lectin. A blocked lectin is described comprising one or more affinity ligands covalently linked by means of a reactive group present on each of the ligands to a lectin such that one or more binding sites of the lectin is blocked. A cell-binding agent-blocked lectin conjugate is described comprising the above-described blocked lectin and a cell-binding agent covalently linked to: (a) one of the covalently linked affinity ligands; or (b) the lectin. A method of preparing a blocked lectin is described. A method of preparing the cell-binding agent-blocked lectin conjugate is described. An affinity support capable of binding to a lectin to form a blocked lectin is described comprising an activated affinity ligand covalently linked to a solid support. A method of preparing the affinity support capable of binding to a lectin to form a blocked lectin is described. A method of killing selected cell populations having reduced cytotoxicity to non-selected cell populations is described comprising contacting a cell population or tissue suspected of containing cells from said selected cell population with the above-described cell-binding agent-blocked lectin conjugate, wherein the lectin is a cytotoxic lectin. Medicaments and methods of treatment using the above-described cell-binding agent-blocked lectin conjugate also are described.
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