The objective of this study was to establish conditions for the selective periodate oxidation of sialic acid, and then use these conditions to develop a series of general methods for the simultaneous visualization of "neutral sugars" (ie. hexose, 6-deoxyhexose and N-acetylhexosamine) and sialic acid and its side chain O-acyl substituted variants, or O-sulphate ester. Investigations of selective conditions for the oxidation of sialic acids demonstrated that oxidation for one hour at 4°C with 0.4 mM periodic acid in approximately 1M hydrochloric acid (PA*) oxidized all available sialic acid residues of both the sialo and sialosulphoglycoproteins of human and rat colon and the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules or vicinal diols located on the "neutral sugars" of sialo and sialosulphoglycoproteins, and did not result in the extraction of epithelial glycoproteins or in the de-O-acylation of side chain substituted sialic acid residues. Therefore, PA* can be used as a specific reagent for the selective oxidation of sialic acids. Studies of the mechanism of oxidation with PA* showed that the lack of PAS reactivity of "neutral sugars" was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is possible that the selective oxidation of sialic acids with PA* results from an increase in the oxidation rate of sialic acid residues together with a decrease in the oxidation rate of "neutral sugars". Based upon this method for the selective oxidation of sialic acid residues (PA*), five new methods have been devised for the simultaneous visualization of "neutral sugars" and either sialic acid and it side chain 0-acyl derivatives or O-sulphate ester. The first of these is the selective periodate oxidatlon-borohydride reduction-saponification-selective periodate oxidation-thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique, in which sialic acids with O-acyl substituents at C7, C8, or C9 (or which have two or three side chain O-acyl substituents) stain blue while "neutral sugars" with periodate sensitive vicinal diols stain magenta. In the second method, the saponification-selective periodate oxidation-thionin Schiff-saponification- borohydride reduction-periodic acid-Schiff (KOH/PA*/T/KOH/Bh/PAS) method all sialic acids stain blue while "neutral sugars" stain magenta. In the third method, the selective periodate oxidation-thionin Schiff-borohydride reduction-periodic acid-Schiff-saponification (PA*/T/Bh/PAS/KOH) method, sialic acids without side chain substituents or which have an O-acyl substituent at C7 stain blue while "neutral sugars" stain magenta. In the fourth method, the saponification-selectlve periodate oxidation-borohydride reduction-alcian blue PH 1.0-periodic acid-Schiff (K0H/PA*/Bh/AB1.O/PAS) technique, O-sulphate esters stain aquamarine blue while "neutral sugars" stain magenta. In all of these techniques, mixtures of the components stain in various shades of purple. In the fifth and final method, the saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (KOH/PA*/Bh/PAS) technique, selective identification of "neutral sugars" in macromolecules which also contain sialic acids can be achieved. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

No description available.

Increasing drug resistance towards the front line influenza neUraminidase inhibitor Oseltamivir (Tamiflu®, Roche) has recently been reported, emphasising the need to perform further studies to gain insight into receptor ligand interactions. Recently, influenza neuraminidase activity has been tackled using a novel class of mechanism-based inactivator, which incorporates fluorine atoms at positions C-2 and C-3 of sialic acid. These inactivators are anticipated to be less susceptible to drug induced resistance as they target essential catalytic amino acids. However, individual hydrogen-bonding interactions formed between these inactivators and the neuraminidase in both the Michaelis complex and at the transition-state remain unclear. The syntheses of the four novel monodeoxygenated 2,3-difluorosialic acid inactivators at position C-4,C-7, C-8 and C-9 deploying a Barton-McCombie protocol were accomplished. The time-dependant inactivation of wild type influenza neuraminidase N9 G70C by these monodeoxygenated 2,3-difluorosialic acid inactivators was tested in a fluorescent kinetic assay. Further biochemical evaluation (performed by our collaborators) in ICso measurements against a panel of influenza viruses including wild types (wt.) and Oseltamivir resistant mutants showed potent inhibition of influenza Band H1 N1 strains. We also wished to develop a further understanding of the effects of the monodeoxygenated 2,3-difluorosialic acid inactivators upon inactivation on a physical basis. Hence, we will discuss X-ray crystallographic structures, (obtained by our collaborators) of influenza neuraminidase N9 in complex with the monodeoxygenated 2,3-difluoro-sialic acid inactivators. 11

No description available.

Sialyltransferases (STs), which catalyze the sialylation reaction by adding sialic acids to the terminal positions of oligosaccharide of glycoproteins and glycolipids, are over-expressed in cancer cells and associated with cancer metastasis. Until now, ST inhibitors are not applicable for clinical use because of poor cell permeability, although showing potent effect in vitro. In this study, we synthesize a lithocholic acid-based ST inhibitor AL10 and test its anti-metastatic effect. Overexpression of £\-2,3-ST is found in highly metastatic A549 and CL1-5 lung cancer cells. Confocal microscopy demonstrates that AL10 is cell permeable and may attenuate total sialylation on cell surface. AL10 has no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of A549 and CL1-5 cells in vitro. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of beta1 integrin. In addition, activation of the beta1 integrin downstream signaling molecule focal adhesion kinase is also attenuated. More importantly, AL10 suppresses lung metastasis in vivo and this effect may be linked with reduced sialylation of the chemokine receptor CXCR4 which has been found to play a critical role in organ-specific metastasis. Serum biochemical assay indicates that AL10 does not affect liver and kidney functions of experimental animals. Taken together, we conclude that AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo via suppression of sialylation of beta1 integrin and CXCR4.

Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

Sialic acids are prevalent in many organisms and facilitate a range of cellular processes in both bacteria and mammals. Whilst a variety of sialic acids are present in nature, N-Acetylneuraminic acid (NANA) is the most common and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host’s immune system. NANA is synthesised in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalysed by the domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). Each NANAS monomer is comprised of two distinct domains; a catalytic domain linked to an antifreeze protein-like (AFPL) domain. This thesis outlines research into the role of the AFPL domain using a range of structural and kinetic analyses to compare variant enzymes to the natural, NmeNANAS enzyme. An investigation was also made into the evolutionary relationships between NANAS and other bacterial sialic acid synthases such as Legionaminic acid synthase and Pseudaminic acid synthase.

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity on glycoproteins. The scarce supply of sialic acid hinders its advancement in basic research, diagnostic development and therapeutic production. In this work, the recombinant E. coli BRL04 (pBRL89) producing sialic acid was studied by some batch and fed batch runs of high cell density cultivation using a 3-L fermentor. Some cultivation conditions including carbon source, induction time, dissolved oxygen were optimized and different feeding strategies were compared to enhance sialic acid production. The results may be helpful to the further scale-up of sialic acid production and the production of other recombinant proteins by high cell density cultivation of E. coli.

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity on glycoproteins. The scarce supply of sialic acid hinders its advancement in basic research, diagnostic development and therapeutic production. In this work, the recombinant E. coli BRL04 (pBRL89) producing sialic acid was studied by some batch and fed batch runs of high cell density cultivation using a 3-L fermentor. Some cultivation conditions including carbon source, induction time, dissolved oxygen were optimized and different feeding strategies were compared to enhance sialic acid production. The results may be helpful to the further scale-up of sialic acid production and the production of other recombinant proteins by high cell density cultivation of E. coli.