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

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

No description available.

by Ng Ping-chung. / Thesis (M. Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / Chapter 1. --- Introduction / Chapter 1.1 --- Natural History of the Bird and the Nest --- p.1 / Chapter 1.2 --- What is More Important in Saliva: Mucin or Proteoglycan? --- p.9 / Chapter 1.3 --- Extraction and characterization of salivary glycoprotein --- p.27 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Preparation of Swiftlet's Nest Extracts --- p.38 / Chapter 2.2 --- Chemical and Biochemical Analysis of SN extracts --- p.38 / Chapter 2.2.1 --- Chemical Analysis --- p.38 / Chapter 2.2.1.1 --- Element Analysis --- p.38 / Chapter 2.2.1.2 --- Ash and Atomic Absorption Analysis --- p.39 / Chapter 2.2.2 --- Biochemical Analysis --- p.39 / Chapter 2.2.2.1 --- Protein Determination --- p.39 / Chapter 2.2.2.2 --- Hexose Determination --- p.40 / Chapter 2.2.2.3 --- Uronic Acid Determination --- p.40 / Chapter 2.2.2.4 --- Hexosamine Determination --- p.41 / Chapter 2.2.2.5 --- Sialic Acid Determination --- p.42 / Chapter 2.2.2.6 --- Sulphate Determination --- p.42 / Chapter 2.3 --- Assay of Co-mitogenic Activity in Lymphocyte Culture --- p.43 / Chapter 2.3.1 --- Preparation of Human Peripheral Blood Lymphocytes --- p.43 / Chapter 2.3.2 --- Co-mitogenic Assay --- p.43 / Chapter 2.4 --- Effect of SN pretreatment on Concanavalin A-stimulated Blastogenic Response in Mouse Splenocytes --- p.44 / Chapter 2.4.1 --- Administration of SN extracts --- p.44 / Chapter 2.4.2 --- Preparation of Mouse Splenocytes --- p.45 / Chapter 2.4.3 --- Concanavalin A-stimulated Blastogenic Response Assay --- p.45 / Chapter 2.5 --- Characterization of SN extracts by Chromatographic Methods --- p.45 / Chapter 2.5.1 --- Gel Filtration Chromatography --- p.45 / Chapter 2.5.1.1 --- Sephadex G-200 Chromatography --- p.45 / Chapter 2.5.1.2 --- Superose-Fast Protein Liquid Chromatography --- p.46 / Chapter 2.5.2 --- Ion-exchange Chromatography --- p.46 / Chapter 2.5.2.1 --- DEAE-Sepharose --- p.46 / Chapter 2.5.2.2 --- Mono-Q FPLC --- p.46 / Chapter 2.5.3 --- Immobilized Metal Affinity Chromatography --- p.47 / Chapter 2.5.4 --- Wheat-germ lectin Sepharose Chromatography --- p.47 / Chapter 2.5.5 --- Octyl-Sepharose Chromatography --- p.47 / Chapter 2.5.6 --- Limulus Polyemus Agarose Chromatography --- p.48 / Chapter 2.5.7 --- Heparin-Agarose Chromatography --- p.48 / Chapter 2.6 --- Electrophoretic Analysis of SN extract --- p.49 / Chapter 2.6.1 --- Sodium Dodecyl Sulphate/Polyacrylamide Gel Electrophoresis --- p.49 / Chapter 2.6.2 --- Isoelectrofocusing in Polyacrylamide Gel --- p.50 / Chapter 2.6.2.1 --- Preparation of Gel --- p.50 / Chapter 2.6.2.2 --- Isoelectrofocusing Procedure --- p.51 / Chapter 2.6.2.3 --- "Fixing, Staining and Destaining" --- p.51 / Chapter 2.6.3 --- Gradient Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 2.6.3.1 --- Preparation of Gradient Polyacrylamide Gel --- p.52 / Chapter 2.7 --- Enzymatic Modification of SN extracts --- p.53 / Chapter 2.7.1 --- B-glucuronidase (EC 3.2.1.31) --- p.53 / Chapter 2.7.2 --- Hyaluronidase (EC 3.2.1.35) --- p.53 / Chapter 2.7.3 --- Chondroitinase ABC (EC 4.2.2.4) --- p.54 / Chapter 2.8 --- Miscellaneous Reagents --- p.54 / Chapter 2.8.1 --- Phosphate-buffer-saline (PBS) --- p.54 / Chapter 2.8.2 --- Fetal Calf Serum (FGS) --- p.54 / Chapter 2.8.3 --- Mitogen --- p.55 / Chapter 2.3.4 --- Penicillin-Streptomycin-Fungizone Solution --- p.55 / Chapter 2.8.5 --- RPMI-1640 Medium --- p.55 / Chapter 2.8.6 --- Scintillant --- p.55 / Chapter 2.8.7 --- Trypan Blue Solution --- p.55 / Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Extraction of Biologically active fractions from swiflet's nest --- p.57 / Chapter 3.2 --- Effect of SN pretreatment on Con A-stimulated Blastogenesis response in mouse splenocytes --- p.57 / Chapter 3.3 --- Chemical and Biochemical Analysis of SN extracts --- p.57 / Chapter 3.3.1 --- Atomic absorption analysis of metal elements --- p.57 / Chapter 3.3.2 --- Element analysis --- p.61 / Chapter 3.3.3 --- Biochemical analysis --- p.61 / Chapter 3.4 --- Chromatographic characterization of SN extracts --- p.67 / Chapter 3.4.1 --- Sephadex G-200 chromatography --- p.67 / Chapter 3.4.2 --- DEAE-Sepharose chromatography --- p.67 / Chapter 3.4.3 --- Immobilized Metal Affinity Chromatography --- p.73 / Chapter 3.4.4 --- Wheat-germ lectin Sepharose Chromatography --- p.73 / Chapter 3.4.5 --- Limulus Polyemus Agarose Chromatography --- p.73 / Chapter 3.4.6 --- Octyl-Sepharose Chromatography --- p.81 / Chapter 3.4.7 --- Heparin-Agarose Chromatography --- p.81 / Chapter 3.5 --- Electrophoretic Analysis of SN extract --- p.88 / Chapter 3.5.1 --- Sodium Dodecyl Sulphate/Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 3.5.2 --- Isoelectrofocusing in Thin Layer of Polyacrylamide Gel --- p.88 / Chapter 3.5.3 --- Gradient Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 3.6 --- Enzymatic Modification of SN extracts --- p.92 / Chapter 3.6.1 --- B-glucuronidase (EC 3.2.1.31) --- p.92 / Chapter 3.6.1.1 --- Alteration in co-mitogenic activity --- p.92 / Chapter 3.6.1.2 --- Alternation in uronic acid content --- p.92 / Chapter 3.7 --- Co-mitogenic activity of glycosaminoglycans --- p.92 / Chapter 3.8 --- Effect of heparin on the co-mitogenic activity of SNp2C fraction --- p.96 / Chapter 4 --- Discussion --- p.99 / Chapter 4.1 --- Extraction of Biologically active fraction from edible bird's nest --- p.99 / Chapter 4.2 --- Chemical and Biochemical characterization of active component(s) in SN extract --- p.100 / Chapter 4.3 --- Some mechanistic considerations --- p.107 / Chapter 4.4 --- Summary and Conclusion --- p.111 / References --- p.114

Sialic acids are involved in many cellular interactions. They can serve as an adhesion ligand or act as an inhibitor to cellular adhesion by charge repulsion or by masking potential ligands. Although sialic acids are implicated in the process of blastocyst implantation, their expression and regulation in uterine epithelium of mice have not been studied. The lectin, Sambucus nigra (SNA) specifically recognizes ��2,6-linked sialic acids, which are involved in cell recognition phenomena. It was used to probe frozen uterine sections from mice during days one through six of pregnancy. SNA staining was most intense at the apical surface of uterine epithelial cells on day one of pregnancy, decreased gradually through day four, and was undetectable by day five. The role of the steroid hormones, estrogen and progesterone, in regulating the expression of ��2,6-linked sialic acids was studied in uterine sections from mice during the estrous cycle and in ovariectomized mice given hormone replacement using SNA. SNA staining of these sections during the estrous cycle showed that the expression of ��2,6-linked sialic acids was stage dependent. Staining was most intense in uterine sections from mice in estrus, and was not detected in sections from mice in diestrus. In ovariectomized mice, staining was most intense in mice injected with estradiol alone, and no staining was evident in mice injected with progesterone alone. These results suggest that the expression of ��2,6-linked sialic acids decreases during the time of implantation and that estrogen stimulates and progesterone inhibits its expression. ��-Galactoside ��2,6-Sialyltransferase (��2,6-ST) is the enzyme that links sialic acids to Gal��1-4GlcNAc termini of N-linked oligosaccharides. In order to investigate the mechanism behind the hormonal regulation of ��2,6-linked sialic acids, the expression of ��2,6-ST was followed in uterine sections from mice during early pregnancy, during the estrous cycle, and in ovariectomized mice given hormone replacement. In-situ hybridization was performed using digoxigenin labeled RNA probes to characterize ��2,6-ST mRNA levels in uterine sections. Expression of ��2,6-ST protein was also measured in uterine sections with a polyclonal antibody against ��2,6-ST. The expression of ��2,6-ST mRNA and protein correlated well with the timing of the appearance of ��2,6-linked sialic acids. These results show that the expression of ��2,6-linked sialic acids on the surface of mouse uterine epithelium decreases at the time of implantation and furthermore, that this decrease is due to the regulation of ��2,6-ST by the steroid hormones. ��2,6-linked sialic acids may serve to inhibit cellular adhesion by creating a charge repulsion, or by masking potential binding sites. Removal of this inhibition may permit blastocyst implantation. / Graduation date: 2001

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.