Hildegard Kindler

Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Namely, neuraminidase-treated DCs show increased gene transcription of proinflammatory and Th1-promoting cytokines and induce greater proliferative responses of T lymphocytes. Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte-derived DCs (MDDCs) express a high content of cell surface α2,6-sialylated glycans. Mock-up vaccines contain an active ingredient for an influenza virus that has not circulated recently in human populations and thus mimics the novelty of a pandemic virus. Monocytes were cultured in complete RPMI-1640 medium supplemented with 1000 U/ml of recombinant human IL-4 (rhIL-4) and rhGM-CSF, for 6 days, to give rise to immature moDC. For purification of these recombinant proteins, the corresponding expression plasmids were transformed into BL21(DE3) pLysS, and single colonies were grown overnight in 10 ml of lysogeny broth with Cm15 Kan25. To assay for oxidoreductase activity, the purified recombinant proteins were incubated in 100-μl reactions at 37 °C overnight with 1 mg/ml 2,7-anhydro-Neu5Ac or Neu5Ac in 20 mm sodium phosphate buffer, pH 7.5, in the presence 500 μm NADH. If you have any concerns regarding in which and how to use manufacturer of sialic acid powder as Raw Material for cosmetics, you can get in touch with us at the webpage. The clarified lysate was run through an immobilized metal affinity chromatography column to elute the C-terminal His6-tagged proteins, which eluted in sharp peaks with a single 500 mm imidazole step. These were further purified by size-exclusion chromatography with elution in 50 mm KPi, 200 mm NaCl, pH 7.8, dialyzed against assay buffer (20 mm Tris, 150 mm NaCl, 2 mm tris(2-carboxyethyl)phosphine, pH 7.5), and finally concentrated to 0.5-1 mm for storage at 4 °C. After harvest, pellets were resuspended in equilibration buffer (50 mm KPi, 200 mm NaCl, 20% glycerol, 40 mm imidazole, pH 7.8) and disrupted with sonication. The structure was acquired from a crystal grown in the JCSG Plus screen (100 mm sodium citrate, pH 5.5, 20% PEG 3000). The diffraction experiment was performed on the I04 beamline at Diamond Light Source Ltd. To characterize the reaction intermediate, a sample containing 3 mm 2,7-anhydro-Neu5Ac, 100 μm NADH, 15 μm RgNanOx, and 1 mm DSS-d6 in PBS/D2O was prepared and analyzed at 293 K. A full set of 2D NMR experiments, including HSQC (hsqcetgpsi), COSY (cosygpqf), TOCSY (mlevph) and TOCSY-HSQC (hsqcdietgpsi), was acquired to fully assign 1H and 13C signals of the intermediate. All 1D NMR experiments were performed using a Bruker Advance I 500-MHz spectrometer with a 5-mm PATXI 1H/D-13C/15N Z-GRD probe at 293 K. To follow the kinetics of the reaction and assess the position of deuteration, two samples containing 2 mm 2,7-anhydro-Neu5Ac, 100 μm NADH, and 60 μm RgNanOx were used, one in deuterated PBS buffer (PBS/D2O) and one in standard PBS buffer (PBS/H2O, containing 10% D2O for locking purposes). The resulting supernatants were loaded onto an AmaZon Speed ETD (Bruker) mass spectrometer and analyzed by direct injection in negative mode. Negative controls were included for each component of the experiment individually as well as a dye-only control well. In these cases, the negative fractions (CD14− PBMCs), obtained after monocyte isolation, were maintained in culture until mixed lymphocyte cultures with autologous moDCs. This allows maximization of the level of sialic acid produced in the culture medium. In the above method, micro-organism can be culture in conditions comprising an exponential growth phase which starts with the inoculation of the fermenter and last until exhaustion of the carbon substrate (for example glucose at 17.5 g.L−1). It has not yet been possible to obtain well-diffracting crystals of any substrate analog complex of the protein. An expression cassette can be constructed for production of more than one protein. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)-12, tumour necrosis factor-α, IL-6 and IL-10 cytokines, and nuclear factor-κB activation. The relative mRNA levels were normalized against the arithmetic mean of the β-actin and GAPDH expression and calculated by the adapted formula 2−ΔCt × 1000, which infers the number of mRNA molecules of the gene of interest per 1000 molecules of the endogenous controls.31 ΔCt represents the difference between the cycle threshold of the target gene and that of the endogenous control genes. Twenty-four hours after transduction, cells were analyzed for luciferase expression with a luminometer. Cells were then cultured in RPMI-Glutamax-I™ supplemented with 5% (volume/volume) FBS, 50 μg/ml gentamicin sulphate (Cellgro, Mediatech Inc., Manassas, VA), 50 μm 2-mercaptoethanol (Invitrogen) and 10 ng/ml murine GM-CSF (R&D Systems) for 7 days.

To confirm correct sequences in plasmids constructed, the plasmids can be analyzed by standard techniques such as by restriction endonuclease digestion, and/or sequencing according to known methods. Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. Media can be rich media, e.g., Luria broth or terrific broth, or synthetic or semi-synthetic medium, e.g., M9 medium. If you're ready to find out more info regarding manufacturer of sialic acid powder for food Ingredients stop by our web site. A "culture medium" refers to any liquid, semi-solid or solid media that can be used to support the growth of a microorganism used in the methods of the invention. When more than one heterologous protein is expressed in a microorganism, the genes encoding the proteins can be expressed on a single expression cassette or on multiple expression cassettes that are compatible and can be maintained in the same cell. "silent substitutions" or "silent variations," which are one species of "conservatively modified variations." Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. In connection with the present invention, we discovered that it is possible to produce genetically engineered micro-organisms, especially non-pathogenic bacteria, by introducing several heterologous genes and inactivating several endogenous genes to obtain a tailored enzymatic pathway leading to accumulation of endogenous sialic acid. By contrast in bacteria, ManNAc is used instead of ManNAc-6-P for the condensation with phosphoenolpyruvate leading to the formation of Neu5Ac in only one step. The catabolic pathway for Neu5Ac has been identified in E. coli: a specific permease encoded by nanT transports Neu5Ac into the cytoplasm, where it is cleaved into ManNAc and pyruvate by the aldolase encoded by nanA (Vimr & Troy, 1985). ManNAc is phosphorylated by the NanK kinase into NanNAc-6-P, which is subsequently converted into GlcNAc-6-P by the NanE protein (Plumbridge & Vimr, 1999) GlcNAc-6-P is then deacetylated by NagA into GlcN-6-P to join the glycolysis pathway or to be used as a precursor for UDP-GlcNAc biosynthesis. This enzyme has been identified in various microorganisms and physiologically acts as an aldolase to enable the catabolism of Neu5Ac. According to the method proposed herein, degradation of Neu5Ac and ManNAc is prevented. In a first embodiment, the invention relates to a method for producing sialic acid and analogs thereof, comprising the step consisting of culturing a microorganism in a culture medium, wherein said microorganism comprises heterologous genes encoding a sialic acid synthase (NeuB), a UDP-GlcNAc epimerase (NeuC), said micro-organism being devoid of a gene encoding CMP-Neu5Ac synthase (NeuA) or wherein a gene encoding CMP-Neu5Ac synthase (NeuA) has been inactivated or deleted; and wherein endogenous genes coding for sialic acid aldolase (NanA), for sialic acid transporter (nanT), and optionally for ManNac kinase (nanK), have been deleted or inactivated. Alternatively, the method may comprise removing the operon including nanT, nanA, nanE genes (nanEAT-), except the nanK gene. ManNAc is phosphorylated by the NanK kinase into NanNAc-6-P, which is subsequently converted into GlcNAc-6-P by the NanE protein (Plumbridge & Vimr, 1999) GlcNAc-6-P is then deacetylated by NagA into GlcN-6-P to join the glycolysis pathway or to be used as a precursor for UDP-GlcNAc biosynthesis. The mechanism of regulation of CMP-Neu5Ac biosynthesis in bacteria has not been determined. NeuB), a UDP-GlcNAc epimerase (NeuC), said micro-organism being devoid of a gene encoding CMP-Neu5Ac synthase (NeuA) or wherein a gene encoding CMP-Neu5Ac synthase (NeuA) has been inactivated or deleted; and wherein endogenous genes coding for sialic acid aldolase (NanA), for sialic acid transporter (nanT), and optionally for ManNac kinase (nanK), have been deleted or inactivated. T, nanA, nanE genes (nanEAT-), except the nanK gene. 2.995 DNA fragment containing the sequence of the genes neuBCA was amplified by PCR using the genomic DNA of Campylobacter jejuni strain ATCC 43438 as a template. Preferably, NeuB and NeuC are isolated from bacterial strains that contain sialylated structure in their cells envelope, such as C. jejuni strain ATCC Accession No. 43438. It is also within the scope of the invention to use substantially identical sequences, and/or conservatively modified variations of said sequences as defined hereafter. After 24 h, cells were differentiated into neuron-like cells by addition of 50 ng/ml nerve growth factor (NGF; Sigma-Aldrich) diluted in 500 µl differentiating medium (RPMI 1640 medium contain 1% heat-inactivated horse serum) for 7 days before starting the experiment. Hepa-1c1c7 (Sigma-Aldrich) were cultured in alpha-minimum essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FCS), 1% l-Glutamine and 1% Penicillin/Streptomycin (all from Gibco). These genes can encode a gene product, such as a protein, necessary for the survival or growth of transformed host cells grown in a selective culture medium. Neu5Ac is a relatively small molecule which is very likely to diffuse into the extracellular medium after being produced in the cytoplasm. The genes neuC and neuB encoding UDP-GlcNAc 2-epimerase (Vann et al., 2004) and Neu5Ac synthase (Annunziato et al., 1995; Vann et al., 1997) respectively have been identified in E. coli K1 and orthologs of these genes have found in various microorganisms such as Neisseria and Campylobacter species.

The internalized bacteria were estimated by flow cytometry, by measuring the mean fluorescence intensity (MFI) of the cells. The phagocytosis was analysed by flow cytometry or confocal microscopy. Images were assembled and analysed with the Leica Confocal software LCS Lite 2.6 (Leica Microsystem). Images were acquired with a TCS SP2 AOBS confocal microscope (Leica Microsystem, Mannheim, GmbH, Deutschland) with × 40 oil immersion optics and 488 nm and 633 nm laser lines for FITC and Alexa Fluor 633 excitation, respectively. Fluorescent images were obtained with a DMRA2 fluorescent microscope (Leica Microsystem) and merged with imageJ software (National Institutes of Health, Bethesda, MD). When appropriate, the MFI values obtained at 4° were subtracted from the 37° values. Values are means from three experiments. The amounts of activated Rac1 or Cdc42 (arbitrary units) are based on the optical density values obtained after subtracting negative control values. Human T-lymphocytes were obtained during the monocyte isolation procedure (CD14− peripheral blood mononuclear cell fraction) and maintained in complete RPMI medium until autologous monocytes differentiated into MDDCs. Human MDDCs were FBS-starved for 24 hr, to set the basal state of the GTPase activation, and then treated under various conditions. Total RNA was extracted using the RNeasy Mini Kit and the RNase-Free DNase Set to eliminate genomic DNA, all from Qiagen (Manchester, UK). The supernatants were removed and used for total sialic acid quantification. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. The relative mRNA levels were normalized against the arithmetic mean of the β-actin and GAPDH expression and calculated by the adapted formula 2−ΔCt × 1000, which infers the number of mRNA molecules of the gene of interest per 1000 molecules of the endogenous controls.31 ΔCt represents the difference between the cycle threshold of the target gene and that of the endogenous control genes. The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. Expression of cytokine genes was analysed by real-time PCR. After the phagocytosis assay, MDDCs were co-cultured with T lymphocytes at a DC : T-lymphocyte ratio of 1 : 4. T-lymphocyte stimulation was assessed by determining interferon-γ (IFN-γ) gene expression after 48 hr of co-culture. Human MDDCs were adhered to cover-slip glasses, fixed and then permeabilized, blocked with 3% BSA for 15 min and then stained with rabbit anti-nuclear factor-κB (NF-κB) p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1 : 100, for 1 hr at room temperature. In some experiments, phagocytosis was conducted with human MDDCs incubated with 50 μg/ml of either SNA or MAA lectins, or, alternatively, in the presence of 10 μm cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-5-NeuAc) (Sigma). "heterologous gene", as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. It will be understood that heterologous genes that can be introduced may originates from different sources, such as E. coli, Neisseria, Campylobacter species, as well as mammals or yeasts. Methods of transforming prokaryotes other than E. coli are well known. In the context of the two glycoproteins recognized in the array, AGP and apo-transferrin, the terminal α2,3/α2,6 trisaccharide motif is linked to several other sugars before they are N-linked to the protein, and thus the length of the chain is not likely to be limiting for binding. A: schematic of the endothelial glycocalyx composed of glycoproteins (dark blue), glycolipids (green-gray), proteoglycans (dark gray), and glycosaminoglycans (light gray) covering the endothelial plasma membrane. B: schematic of an N-linked, triantennary oligosaccharide found on mature glycoproteins. The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. The endothelial glycocalyx consists of glycoproteins, glycolipids, proteoglycans and glycosaminoglycans, which coat the cell surface (18, 23) (Figure 1A). The carbohydrate network that contributes to the glycocalyx is very complex, and, to date, the structure of these complex carbohydrates and their role in endothelial barrier function are poorly understood. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Molecular structure of sialic acids. Following treatment of PAECs with neuraminidase from Vibrio cholerae-positive lectin, staining was still observed, indicating that not all α(2,3)-linked sialic acids were hydrolyzed (Fig. If you have any questions regarding wherever and how to use Supplier of sialic acid powder for Supplement Ingredients, you can make contact with us at our web page. 5E). We observed the same pattern of lectin staining when the cells were treated with neuraminidase from Clostridium perfringens.

Given the structural resemblance of RgNanOx to YjhC, it is likely that the E. coli oxidoreductase also uses the same mechanism of action for the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac. Given the accessibility of sialic acids in mucus-rich environments, their utilization offers pathogenic and commensal bacteria a competitive advantage to colonize and persist within the gut (2, 19). The ability of R. gnavus strains to produce and metabolize 2,7-anhydro-Neu5Ac provides them with a nutritional advantage by scavenging sialic acid from host mucus in a form that they have preferential access to (4, 17,-19). We previously showed that the oxidoreductase RgNanOx plays a key role in the catabolism of 2,7-anhydro-Neu5Ac inside the cells by converting it into Neu5Ac, before being catabolized into GlcNAc-6-P following the canonical pathway by the successive action of NanA (Neu5Ac aldolase), NanK (ManNAc kinase), and NanE (ManNAc-6-P epimerase). The Sialic Acid Market Report offers a thorough examination of the primary competitors in the industry, incorporating previous data, SWOT analysis, and recent worldwide advancements. Taken together, these results show that both enzymatic removal and genetic removal of sialic acid on the cell surface reduce AAV1 and AAV6 binding and transduction. These results again support the idea that sialic acid facilitates AAV1 and AAV6 transduction, in particular both α2,3 and α2,6 sialic acids. Further findings from our work support that the catabolism of 2,7-anhydro-Neu5Ac is not restricted to R. gnavus strains. To date, only R. gnavus strains have been reported to produce 2,7-anhydro-Neu5Ac from Neu5Ac terminally bound glycoconjugates in the gut (4, 5, 18). Resource sharing is an important ecological feature of microbial communities living in the gut (45). Some bacteria present in the mucus might not be primary degrader but might cross-feed on mucin glycan degradation products released by other bacteria. In Ukraine’s western Chernovetsky region, an epicenter of the outbreak, doctors have said lab tests showed at least some of the fatalties appeared to be caused by a flu dissimilar to both common flu and swine flu. E. coli can transport and catabolize the common sialic acid, Neu5Ac, as a sole source of carbon and nitrogen but also related sialic acids, N-glycolylneuraminic acid (Neu5Gc) and 3-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN), which are transported via the sialic acid transporter NanT and catabolized using the sialic acid aldolase NanA (33). Here, we showed that E. coli BW25113 strain was able to grow on 2,7-anhydro-Neu5Ac as a sole carbon source and that the two-gene NanR-regulated operon nanXY (yjhBC) encodes both the transporter and oxidoreductase enzyme required for E. coli to uptake and catabolize 2,7-anhydro-Neu5Ac. This also now completes the functional characterization of all NanR-regulated genes in E. coli (25), giving us a broader picture of the sialic acid molecules it likely encounters in its natural environment. Semi-quantitative RT-PCR was performed using 200 ng cDNA, SYBR GreenER qPCR SuperMix Universal (Thermo Fisher Scientific) and specific primers were added into a final reaction volume of 25 µl. Then, SuperScript III reverse transcriptase (Thermo Fisher Scientific) and random primer mix (Roche Diagnostics) were employed for cDNA synthesis. B: PAECs and PMVECs were treated with neuraminidase from Clostridium perfringens to cleave terminal sialic acids. 200:1 preference for cleavage of terminal α(2,3)-linked sialic acids (24), and one from Clostridium perfringens, which exhibits only a slight preference for α(2,3)-linked sialic acids over α(2,6)-linked sialic acids (3, 4). It is important to note, however, that one must be cautious in oversimplification regarding neuraminidase specificity because said specificity is dependent on both the core oligosaccharide and the protein and lipid structures that the oligosaccharides are attached to; subtle differences can dramatically influence the rate of release of different glycosidic linkages (4). Furthermore, O-acetylation is one of the most common modifications that occurs on sialic acids, and it has been demonstrated that monoacetylation of the 7, 8, or 9 position of a sialic acid largely attenuates the effectiveness of neuraminidase hydrolysis; diacetylation completely abrogates the hydrolytic ability of neuraminidases from Clostridium perfringens and Vibrio cholerae (15). Cells were treated with 1 U/ml neuraminidase for 2 and 5 h, followed by staining with fluorescently tagged lectins: FITC-tagged MAA to observe α(2,3)-linked sialic acids, and FITC-tagged SNA to observe α(2,6)-linked sialic acids. Loss of sialic acids disrupts cell-cell and cell-matrix adhesions. This ability to utilise multiple sialic acid derivatives contrasts with R. If you liked this article in addition to you desire to receive more information with regards to manufacturer of sialic acid powder for food Ingredients i implore you to visit our web site. gnavus strains, which can only grow on 2,7-anhydro-Neu5Ac but not on Neu5Ac (19) and is consistent with E. coli being able to integrate diverse sialic acids into its core catabolic pathway (33). Beyond E. coli, our bioinformatics analyses revealed RgNanOx homologues across many bacterial species that also co-occurred with predicted sialic acid transporters. AAV6 is regarded as a laboratory strain derived from recombination between AAV1 and AAV2, with its upstream sequence being identical to AAV2 and its downstream sequence similar to AAV1 (34, 46). Analysis of the capsid sequence of AAV1 and AAV6 shows only six amino acid differences in the capsid protein. To test if the decreased transduction of AAV1 and AAV6 on neuraminidase-treated cells is related to reduced virus binding, a binding assay based on dot blot hybridization was performed (Fig. (Fig.2D).2D). To test the hypothesis that other bacteria can act as "scavengers" of 2,7-anhydro-Neu5Ac, we heterologously expressed and purified the NanOx protein from Hemophilus hemoglobinophilus and showed that the recombinant protein was active against 2,7-anhydro-Neu5Ac (Fig. 6). The analysis also revealed two additional couplings of NanOx-like genes to likely 2,7-anhydro-Neu5Ac transporters, namely to transporters of the SSS family, for example in Streptococcus pneumoniae TIGR4 and a transporter of the GPH family in Lactobacillus salivarius (Fig. 8), which, together with the phylogenetically broad occurrence of the NanOx-like genes, suggests that 2,7-anhydro-Neu5Ac use is not a new trait in bacteria but the result of a symbiotic evolution of bacteria in the mammalian gastrointestinal tract.

Mucin competition was carried out by pretreating rAAV with bovine submandibular gland mucin (Sigma) for 30 min at 20°C. AAV vectors, in the presence or absence of mucin, were added to Cos-7 cells in equal volumes of DMEM for 1 h at 37°C. Cells were rinsed twice with DMEM and incubated at 37°C. Two thousand particles/cell were pretreated with mucin (concentrations up to 0.5 mg/ml), and cells were assayed for luciferase expression 24 h posttransduction. Electrophoresis was performed on ice for 45 min at 120 V in 1 × Tris-Glycine buffer. Lectin staining of HepG2 cells, Pro-5 cells, and Cos-7 cells was performed by incubation with fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA), Maackia amurensis lectin (MAA), or Sambucus nigra lectin (SNA) (Vector Laboratories Inc.), as described in reference 44. Briefly, cells growing in 24-well plates (approximately 2 × 105 cells/well) were chilled to 4°C, and then lectin (10 μg/ml) was added to the respective media. Briefly, Cos-7 cells were plated at a density of 2 × 104 cells/well in a 48-well plate. Similarly, SNA blocked AAV1 and AAV6 transduction on HepG2 cells and, to a lesser extent, on Cos-7 cells but not on Pro-5 cells. Together, these results support the requirements for α2,3 and α2,6 sialic acids which are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection. Since α2,3 sialic acid is present on ciliated cells, while α2,6 sialic acid is present on both ciliated and nonciliated cells (23), it will be of interest to determine if AAV1 and AAV6 are able to transduce both types of epithelial cells. In addition, there was a 60-fold difference between Pro-5 cells and Lec-2 cells for AAV6 transduction while only an 8-fold difference for AAV1 transduction. In addition, among all other cell lines tested, AAV6 transduction markedly drops following neuraminidase treatment (Fig. (Fig.2).2). In addition, many bacteria including E. coli K12 are able to catabolise Neu5Ac and use it as a carbon energy source. All three finally agreed to use sialic acid as the family name covering all of the more than thirty derivatives of neuraminic acid, with N-acetylneuraminic acid and N-glycolylneuraminic acid forming the core structures. More specifically, following treatment with neuraminidase from Clostridium perfringens, the PAECs appeared to lose cell-cell contacts, resulting in rather evenly dispersed individual cells. Although we did not observe the complete loss of α(2,3)-linked sialic acids following treatment of either neuraminidase, we did observe that, in many areas of gap formation, the α(2,3)-linked sialic acid staining was nearly absent (Fig. 6C). 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Finally, even at 5 h postneuraminidase treatment, both PAECs and PMVECs exhibited monolayer disruption (Fig. 6D). Collectively our observations support a prominent role for terminally linked sialic acids in maintenance of endothelial barrier integrity. Following treatment of PAECs with neuraminidase from Vibrio cholerae-positive lectin, staining was still observed, indicating that not all α(2,3)-linked sialic acids were hydrolyzed (Fig. 5E). We observed the same pattern of lectin staining when the cells were treated with neuraminidase from Clostridium perfringens. In the earlier set of experiments that utilized neuraminidase, we noted that, following neuraminidase treatment, there were typically fewer cells in the dish, indicating that cells had lost cell-cell and/or cell-matrix adhesions. First, within 2 h of either neuraminidase treatment, the α(2,6)-linked sialic acids in PAECs were hydrolyzed as evidenced by loss of FITC-tagged SNA fluorescence (Fig. 5A). Second, we observed overall disruption of the PAEC monolayer following treatment with either neuraminidase although there were characteristic differences in what the resultant disrupted monolayer looked like. Following treatment, cells were washed and imaged. Thus we stained for α(2,3)-linked sialic acids using the FITC-tagged MAA following neuraminidase treatment. However, although we clearly saw disruption of the monolayer in our visual microscopy experiment utilizing neuraminidase from Clostridium perfringens, we also noted that there were areas of intact monolayer that maintained strong cell-cell border staining of α(2,3)-linked sialic acids. However, at this time we do not know whether one linkage, i.e., α(2,3) or α(2,6), is more important than the other in determining endothelial barrier integrity nor whether further substituted (e.g., acetylated) sialic acids play a role in cell-cell and/or cell matrix adhesion. Loss of sialic acids disrupts cell-cell and cell-matrix adhesions. On the other hand, treatment with neuraminidase from Vibrio cholerae resulted in large areas where there were no cells and other areas where there were still confluent cells, suggestive of loss of cell-matrix adhesions. Although we know that the neuraminidase from Vibrio cholerae does cleave terminal sialic acids, as assessed by binding of the lectin from Arachis hypogaea (Fig. 5B), we do not know whether these trace level contaminants contribute to the distinctive pattern of endothelial barrier disruption. Similar to what we saw with the PAECs, in neuraminidase-treated PMVECs, staining for α(2,3)-linked sialic acids was still positive, revealing that PMVECs also express a population of neuraminidase-resistant α(2,3)-linked sialic acids (Fig. 6B). Because we observed positive binding of the lectin from Arachis hypogaea following neuraminidase treatment, and because the α(2,3) linkage is the predominant one on PMVECs, it strongly suggests that indeed some α(2,3)-linked sialic acids were cleaved.

Bird’s nest acid, also known as sialic acid, sialic acid, scientific identify "N-acetylneuraminic acid", is a naturally occurring carbohydrate. Don't hesitate to get in touch with us if you're desirous about wholesale sialic acid, we can't let you down. Not only wholesale sialic acid we produced have certificated the worldwide trade customary, but we can also meet your customization wants. RNA viruse are inclined to have high mutation rate-more than 10.000 times larger than that of human or viral DNA- and this is true of all of the influenza viruses. Although the very younger and elderly are usually at essentially the most threat from influenza, the influenza pandemic of 1918-1919 was unusual in that mortality was excessive in health young adults. All three influenza viruses infect man and trigger disease, however influenza A represents essentially the most critical human pathogen because it causes very massive, recurrent epidemic and even pandemic with significant mortality. Although it isn't clear whether or not a new pandemic is imminent, it can be prudent to take into account the lessons we've learned from learning completely different human and animal influenza viruses. Furthermore, reclassification of influenza A viruses signifies that H1N1 viruses circulated from not less than 1918 till 1957. Thus, it is now clear that influenza pandemics occur at unpredictable intervals. The pandemic of 1918 occurred earlier than influenza virus could possibly be isolated and it has not been attainable to study the virus within the laboratory utilizing trendy tools. 1) Cinti S; Pandemic influenza: are we ready? The rapid, international spread of pandemic influenza may be a comparatively fashionable growth related to increases in population and the growth of transportation methods needed for the global transmission of the novel virus. In distinction to measles, smallpox and poliomyelitis, influenza is brought on by viruses that endure steady antigenic change and that possess an animal reservoir. Recent phylogenetic studies of influenza A viruses have revealed species-specific lineages of viral genes and have demonstrated that the prevalence of interspecies transmission depends on the animal species. In Florence through the time of the Renaissance, astrologers linked a curious juxtaposition of stars with an outbreak of infection in the town and attributed it to the "influence" of the stars, therefore influenza. Known within the sixteenth century as "the newe Acquayntance", influenza nonetheless causes main outbreaks of acute respiratory infection. The temperature rises quickly to round 39 C. Influenza shouldn't be characterized by runny noses or sore throats at the start, as are widespread chilly infections. About 80 % of them are haemagglutinin antigen and the reminders are one other antigen, neuraminidase, and have a mushroom-like shape. Influenza A viruses have been designated on the idea of the antigenic relationships of the external spike haemagglutinin (HA) and neuraminidase (NA) proteins. Type B strains are designated on the same system, but with out H and N numbers since major modifications in these antigens have to date not been noticed. If you are you looking for more info about manufacturer of sialic acid powder as Raw Material for cosmetics stop by the web site. These mutations give rise to changes within the viral polypeptides, similar to HA which, out of a total of 250 amino acids, undergoes two or three amino-acid substitutions each year. The sequences from these three victims have been virtually equivalent and confirmed that the virus belong to strain H1N1. Virus multiplies in the epithelial cells in the nostril and sinus passages and destroys the cilia, that are an essential element in the defense of the respiratory system. There aren't any difference between Influenza A and B as regards the clinical picture. There are 4 antigens present, the haemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), the matrix (M) and the nucleocapsid proteins (NP). The haemagglutinin (HA) is a rod-formed glycoprotein with a triangular cross-section. In earlier years HA and NA antigens driving from birds and other animals have been given acceptable letters (for example Hsw for haemagglutinin of a swine -type virus or Nav for a neuraminidase of avian origin). It was first recognized by its capacity to agglutinate erythrocytes, therefore its name, however it is now apparent that it also has essential roles in the attachment and entry of virus to the cells of the host and in figuring out virulence. Myxo derives from the Greek for mucus and refers to the ability of those viruses to attach to mucoproteins on the cell surface; ortho means true or regular, as in orthodox, and distinguishes these viruses from the Paramyxoviridae (measles is a member of this family). Although laymen consult with many incapacitating respiratory infection as "flu", true influenza is brought on by the small household of the Orthomyxoviridae. They are: Influenza virus A, B and C as well as Thogoto-like virus which is a tick-borne virus of mammals. Influenza viruses A and B are closely associated, but influenza A infects a wide spectrum of birds and mammals together with humans, whereas influenza B infects only people.

Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. "Conservatively modified variations" of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. If you loved this post and you would such as to get more details pertaining to manufacturer of sialic acid powder as Raw Material for beverages kindly check out the web site. Such results allow for the first time to produce sialic acid at low commercial cost. In spite of these successive improvements the manufacturing cost of Neu5Ac is still relatively high and we have investigated the possibility of reducing this cost by producing Neu5Ac by bacterial fermentation. N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of Neu5Ac is hydroxylated. Briefly, NHS (provided by this kit or obtained from Complement Technology) was co-incubated with different concentrations of polySia avDP20 (1 h at 37 °C with 26 µM, 52 µM, 106 µM, 213 µM, 426 µM) or mono-/oligosialic acid/high molecular weight polysialic acid (1 h at 37 °C with 26 µM, 106 µM, 426 µM). The complement factor C3, factor C3b, factor H, factor B, and properdin were purchased from Complement Technology (USA). A high binding microtiter plate (Thermo Fisher Scientific) was used and 5 μg/ml of each purified complement proteins (properdin/Factor P, factor B, factor H, factor D, C3b, C3; C5, C6, C7, C8, C9, and C5b-9; Complement Technology) were immobilized on the plate. Binding of properdin and factor H to the ELISA plate was confirmed by specific monoclonal antibodies directed against factor P (Tecomedical) and factor H (clone T13) followed by secondary HRP-conjugated antibodies and the enzyme-mediated color reaction. In addition, different concentrations (7.8-1000 nM) of properdin (factor P), factor H or bovine serum albumin (BSA) were coated on the ELISA plate and 500 nM biotinylated polySia avDP20 was added. Human properdin was purchased from TECOmedical (Germany)/Complement Technology (USA). Advancement in the technology has provided today’s businesses with multifaceted advantages resulting in daily economic shifts. Afterwards, the pre-incubated sera were diluted 13-fold in the diluent provided by the manufacturer to obtain the final concentration, and the whole mixtures were transferred to the pre-washed LPS-coated micro-titer plate (provided by the manufacturer) and incubated for 1 h at 37 °C. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Sialic acid is a generic term for the N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. The term "isolated" refers to material that is substantially or essentially free from components which interfere with the activity biological molecule. We prevented the degradation of Neu5Ac and ManNAc which are formed by the activity of NeuC and NeuB. A first futile cycle can result from the combined activity of the sialic acid synthase NeuB with the sialic acid aldolase NanA. Sialic Acid or N-Acetylneuraminic Acid Sialic Acid or N-Acetylneuraminic Acid factory, Supplier, Manufacturer. With such final strain with all the above modifications, we ended with high scale production of sialic acid reaching up to about 40 g/l under optimized cultured conditions. BRIEF SUMMARY OF THE INVENTION - The present invention provides a method of producing sialic acid by fermentative growth of microorganisms. In reason of the central role of Neu5Ac in sialic acids metabolism and of its potential utilization for the synthesis of biologically active sialylated oligosaccharides, there has long been a strong interest in developing economic and efficient methods for Neu5Ac preparation. With this information, stakeholders will be more capable of developing new strategies, which focus on market opportunities that will benefit them, making their business endeavors profitable in the process.

Finally, is there a threshold of sialic acid loss that must be attained before barrier disruption occurs? More specifically, treatment of endothelial monolayers with neuraminidases leads to endothelial barrier disruption. However, the fact that there was an actual increase in resistance suggests that there is something more going on, something we do not yet understand. Two of these substrates both exhibited an α(2,3)-linked sialic acid to an underlying galactose at the end of the oligosaccharide chain, yet neuraminidase from Vibrio cholerae exhibited a more rapid hydrolysis of one substrate compared with the other. A: isolated-perfused lungs were treated with 0.5 U/ml neuraminidase from Vibrio cholerae. To determine whether the increases in endothelial permeability in the isolated lung resulted from disruption of the micro-or macrovasculature, the lungs were fixed and processed for microscopy at the completion of the experiments. A third key observation in our study is the pattern of alveolar edema observed when isolated-perfused lungs were treated with neuraminidase from Vibrio cholerae. A similar dramatic pattern of barrier disruption was observed after treatment with 1.0 U/ml neuraminidase (not shown). Another interesting, and unexplained, observation relates to the different characteristics of barrier disruption exhibited by PAECs and PMVECs exposed to the two neuraminidases. Following neuraminidase treatment, the lung became swollen and edematous indicative of severe disruption of the endothelial barrier. Treatment with neuraminidase from Vibrio cholerae caused significant fluid accumulation in the alveolar spaces, septal interstitium, and perivascular cuffs (Fig. 8C). It is important to note here that, although the formation of perivascular cuffs may be caused by protease activity, alveolar flooding is not consistent with protease activity (31). If you have any kind of concerns about where by and also how you can work with Supplier of sialic acid powder as Raw Material for pharmaceuticals, you can call us from our own web site. Strikingly, the high frequency of fluid accumulation in the alveolar spaces is consistent with neuraminidase activity as reported in clinical autopsy cases involving pathogenic viral infection (7, 29). The data indicate that significant and homogeneous disruption of the barrier occurred in microvascular endothelium, validating our observations from the in vitro experiments. Perivascular cuffing was evident around some, but not all, larger vessels (left; blue star), whereas extensive damage was observed in the microvasculature as evidenced by fluid accumulation in the alveolar spaces and septal interstitium (right; yellow arrows). The diffuse alveolar damage seen in our histopathological specimens was identically observed in 25 of 34 fatalities (73.5%) reported in the 2009 influenza pandemic (7, 29). Based on these observations, it is tempting to speculate that part of the pathological mechanism of influenza infection involves neuraminidase action directly on the microvascular endothelium. The attenuated swine influenza virus of the present invention may be a chimeric virus that expresses a heterologous sequence. In addition, the method may comprise steps of purification such as removal of cells by centrifugation, followed by crystallization and filtration. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In PAECs treated with neuraminidase from Clostridium perfringens, cells pulled apart from each other presumably through loss of cell-cell adhesions, whereas, in PMVECs treated with the same neuraminidase, the cells seemed to maintain most of the cell-cell interactions while losing cell-matrix interactions. In this study, we reveal that, although both PAECs and PMVECs express sialylated oligosaccharides, the sialic acid linkages surficially expressed differ between the two cell types. Although both AAV4 and AAV5 bind to α2,3-linked sialic acid for transduction, AAV4 binds sialic acid present on O-linked oligosaccharides, whereas AAV5 binds sialic acid present on N-linked oligosaccharides (19). To determine whether O-linked or N-linked sialic acid is used by AAV1 and AAV6 for transduction, Cos-7 cells were cultured with inhibitors of O-linked (N-benzyl GalNac) or N-linked (tunicamycin) glycosylation. The N-linked inhibitor tunicamycin inhibited both AAV1 and AAV6 transduction; however, it also inhibited AAV2 and AAV4 transduction (Fig. (Fig.7B).7B). Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Therefore, it is not surprising that AAV1 and AAV6 are almost identical in their serology (14, 16). Both AAV1 and AAV6 have been shown to transduce muscle very efficiently (2, 5), although a side-by-side comparison has not been reported. Along those same lines, compared with one substrate that possessed α(2,3)-linked sialic acids (antifreeze glycoprotein 1-5) to another substrate that possessed (2,6)-linked sialic acids (α1-acid glycoprotein), neuraminidase from Vibrio cholerae hydrolyzed the (2,6)-linked sialic acids on the α1-acid glycoprotein faster than the α(2,3)-linked sialic acids on the antifreeze glycoprotein 1-5. Thus the molecular identity and structure of the protein (or lipid) and carbohydrate chains underlying the sialic acid moieties are also important in determining the availability and rate of sialic acid hydrolysis by neuraminidase enzymes. Despite the noticeable increases in permeability, neuraminidase treatment did not affect hemodynamics or airway pressures before the measurement of the final Kf.

"Commercial scale" refers to gram scale production of a sialic acid in a single reaction. In preferred embodiments, commercial scale refers to production of greater than about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 grams. An expression cassette can be constructed for production of more than one protein. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette. For cells, saccharides, nucleic acids, and polypeptides of the invention, the term "isolated" refers to material that is substantially or essentially free from components which normally accompany the material as found in its native state. Plasmids containing one or more of the above listed components employs standard ligation techniques as described in the references cited above. At last, such genetically engineered micro-organisms are not only viable but they are able to grow in standard conditions. Other transformation methods are also suitable. A "culture medium" refers to any liquid, semi-solid or solid media that can be used to support the growth of a microorganism used in the methods of the invention. Media can be rich media, e.g., Luria broth or terrific broth, or synthetic or semi-synthetic medium, e.g., M9 medium. Alternatively, selectable markers may encode proteins that complement auxotrophic deficiencies or supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Those of skill are aware that insertion of a nucleic acid into a chromosome can occur, e.g., by homologous recombination. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by band intensity on a silver stained gel or other method for determining purity. Biol. If you loved this article and you would like to obtain extra data pertaining to Supplier of sialic acid powder as Raw Material for drinks kindly take a look at our own web page. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence. The nanT, nanA, nanK and nanE genes are part of the same operon, which is regulated by the DNA binding protein NanR and induced by Neu5Ac (Kalivoda et al., 2003). The production of Neu5Ac by the NeuB and NeuC proteins can thus induce the pathway of Neu5Ac catabolism and create two futile cycles that reduce the capacity of CMP-Neu5Ac biosynthesis of the bacteria. In a first embodiment, the invention relates to a method for producing sialic acid and analogs thereof, comprising the step consisting of culturing a microorganism in a culture medium, wherein said microorganism comprises heterologous genes encoding a sialic acid synthase (NeuB), a UDP-GlcNAc epimerase (NeuC), said micro-organism being devoid of a gene encoding CMP-Neu5Ac synthase (NeuA) or wherein a gene encoding CMP-Neu5Ac synthase (NeuA) has been inactivated or deleted; and wherein endogenous genes coding for sialic acid aldolase (NanA), for sialic acid transporter (nanT), and optionally for ManNac kinase (nanK), have been deleted or inactivated. Alternatively, the method may comprise removing the operon including nanT, nanA, nanE genes (nanEAT-), except the nanK gene. Thus, the expression of neuB and neuC genes in a bacteria results in an accumulation of Neu5Ac if the bacteria is devoid of CMP-Neu5Ac synthase activity. A "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of affecting expression of a structural gene in hosts compatible with such sequences. 1977) Nucleic Acids Res. 50 residus in length, more preferably over a region of at least about 100 residus, and most preferably the sequences are substantially identical over at least about 150 residus. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residus in length, more preferably over a region of at least about 100 residus, and most preferably the sequences are substantially identical over at least about 150 residus. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. "silent substitutions" or "silent variations," which are one species of "conservatively modified variations." Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.

The reaction was followed by acquiring 1D NMR experiments at 15-min intervals over 24 h. Gene functions were inferred from BLAST searches followed by gene linkage and cluster analysis. Deletion of yjhC resulted in loss of growth on 2,7-anhydro-Neu5Ac but not on Neu5Ac (Fig. 5C), which could be complemented in trans with yjhC (Fig. 5D), suggesting that the gene encodes an equivalent protein to RgNanOx. To test this hypothesis, the YjhC protein was recombinantly expressed and purified, and its activity against 2,7-anhydro-Neu5Ac and Neu5Ac was analyzed by ESI-MS. A, ESI-MS analysis of the enzymatic reaction between RgNanOx mutants and 2,7-anhydro-Neu5Ac (290; left) or Neu5Ac (308; left). A, ESI-MS analysis of the enzymatic reaction of RgNanOx, EcNanOx, and HhNanOx with 2,7-anhydro-Neu5Ac (left) or Neu5Ac (right). Having demonstrated that NanOx-like genes are functional in both Gram-positive and Gram-negative bacteria, functioning with different classes of transporters, we extended our analysis to likely 2,7-anhydro-Neu5Ac catabolic genes across bacterial species. The genes encoding 2,7-anhydro-Neu5Ac transporters, 2,7-anhydro-Neu5Ac oxidoreductases, and IT-sialidases are distinguished by color for emphasis. TIGR4 possesses both the conserved sialic acid "supercluster," as in strain D39, and an additional, candidate 2,7-anhydro-Neu5Ac cluster bearing the siaT-like transporter gene. The first gene in the yjhBC operon, yjhB, encodes a major facilitator superfamily (MFS) transporter protein that shows homology (35% identify, 55% similarity) to NanT, the known Neu5Ac transporter in E. coli (24, 26, 27). Deletion of nanT leads to a complete loss of growth on Neu5Ac, suggesting that YjhB cannot transport this particular sialic acid (28) (Fig. 7A). Similar to the phenotype observed with the ΔyjhC strain, the ΔyjhB mutant was also unable to grow on 2,7-anhydro-Neu5Ac but could grow on Neu5Ac (Fig. 7B). The co-expression of these two genes and the requirement of YjhB for growth on 2,7-anhydro-Neu5Ac suggest that YjhB is a novel MFS transporter for 2,7-anhydro-Neu5Ac and that these two genes together form an "accessory" operon to allow E. coli to scavenge a wider range of sialic acids that are available in the human gut. The first locus is the core nanATEKyhcH operon for Neu5Ac uptake and dissimilation into the cytoplasm (62). The two "accessory" loci contain the nanCMS operon, for Neu5Ac uptake through the outer membrane, sialic acid mutarotation, and processing of O-acetylated sialic acids in periplasm (63,-65), and the nanXY (yjhBC) operon here characterized as being required for 2,7-anhydro-Neu5Ac uptake and utilization. We propose to rename these genes nanXY, because the function of the final NanR-regulated operon has been elucidated through this work. Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function , R. Schauer, Ed. Here is more info about Supplier of sialic acid powder as Raw Material for pharmaceuticals review our own webpage. A, Neu5Ac lyase; nanK, N-acetylmannosamine kinase; nanE, N-acetylmannosamine-6-phosphate epimerase; nanC, Neu5Ac outer membrane channel; nanM, Neu5Ac mutarotase; nanS, N-acetyl-9-O-acetylneuraminate esterase; nagB, glucosamine-6-phosphate deaminase; GNAT, GCN5-related N-acetyltransferase; Reg, regulator (please note that GNAT family proteins and regulator proteins, while recurrent within clusters, may belong to different clades and thus function differently in each organism); SAT2, 2,7-anhydro-Neu5Ac transporter of the ABC superfamily; siaPQM, Neu5Ac transporter of the TRAP family; satABCD, Neu5Ac transporter of the ABC superfamily (SAT); nanUVW (SAT3), Neu5Ac transporter of the ABC superfamily (also named satABC); nanT, Neu5Ac transporter of the MFS superfamily; siaT, Neu5Ac transporter of the SSS family; nanX (yjhB), 2,7-anhydro-Neu5Ac transporter (nanT-like) of the MFS superfamily ABC; MFS, major facilitator superfamily; SSS, sodium solute symporter family; GPH, glycoside-pentoside-hexuronide:cation symporter family; SBP, solute-binding protein; TMD, transmembrane domain; NBD, nucleotide-binding domain. Both mutants were grown on 2,7-anhydro-Neu5Ac (orange), Neu5Ac (blue), glucose (red), or M9 medium alone (black) in 200-µl microtiter plates. The red line marks the trajectory of hydride transfer. All strains were grown on 2,7-anhydro-Neu5Ac (orange), Neu5Ac (blue), glucose (red), or M9 medium alone (black) in 200-µl microtiter plates. We found that this can be advantageously done by disrupting the nanA and nanK genes in the strains which will be used for Neu5Ac production. E. coli K12 the nanA nanK and nanT genes are clustered in the same region of the E. coli chromosome together with the nanE gene which encodes the ManNac kinase activity. NanK NanE NagA GlmM and GlmU catalyse the formation of UDP-GlcNAc from ManNAc. In RgNanOx, by creating a keto group, the enzyme has acidified the C3 proton; this facilitates an elimination reaction and formation of a conjugated intermediate 4-keto-DANA, which we detected by NMR. In other enzymes, including RmlB of the dTDP-l-rhamnose biosynthetic pathway (31) and the multistep enzyme GDP-mannose 3,5-epimerase (32), the creation of a keto group and the consequent acidification of the α proton(s) allow a range of chemical reactions. The creation of a keto intermediate in sugars is widespread in biology; perhaps it is best-known for the SDR enzyme UDP-glucose/galactose epimerase (29, 30). In this enzyme, the oxidation and reduction of the sugar occur so as to invert the chirality at C4.