http://experimentationlab.berkeley.edu/sites/default/files/AFMImages/butt_AFM.pdf

Force measurements with the atomic force microscope: Technique, interpretation and applications

Gut microbiota is an assortment of microorganisms inhabiting the length and width of the mammalian gastrointestinal tract. The composition of this microbial community is host specific, evolving throughout an individual's lifetime and susceptible to both exogenous and endogenous modifications. Recent renewed interest in the structure and function of this "organ" has illuminated its central position in health and disease. The microbiota is intimately involved in numerous aspects of normal host physiology, from nutritional status to behavior and stress response. Additionally, they can be a central or a contributing cause of many diseases, affecting both near and far organ systems. The overall balance in the composition of the gut microbial community, as well as the presence or absence of key species capable of effecting specific responses, is important in ensuring homeostasis or lack thereof at the intestinal mucosa and beyond. The mechanisms through which microbiota exerts its beneficial or detrimental influences remain largely undefined, but include elaboration of signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and mucosal immune cells. The advances in modeling and analysis of gut microbiota will further our knowledge of their role in health and disease, allowing customization of existing and future therapeutic and prophylactic modalities.

Gut Microbiota in Health and Disease

Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences

https://gut.bmj.com/content/gutjnl/45/suppl_2/II1.full.pdf

The functional gastrointestinal disorders and the Rome III process.

Mucins — large extracellular proteins that are heavily glycosylated with complex oligosaccharides — establish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. Mucins are used as diagnostic markers in cancer, and are under investigation as therapeutic targets for cancer.

Mucins in cancer: Protection and control of the cell surface

http://www.biochemsoctrans.org/content/ppbiost/15/3/391.full.pdf

Demonstration of 9-O-lactyl-N-acetylneuraminic acid in human gastric aspirates

Biochemical analysis of mucin glycoproteins in paediatric colonic mucus

The specific assay of glycosyl transfer to 0-glycosidically linked oligosaccharides on glycoproteins is limited by the complexity of glycoprotein structure. One molecule frequently contains different oligosaccharides. Conventional assay systems employ radioactive nucleotide sugars as donors (e.g. Brockhausen et al . , 1986), although the amount of transfer together with the complexity of oligosaccharide structure has often limited accurate product identification (Sadler, 1984). We have applied established radioactive labelling methods to glycoprotein substrates containing known 0-linked oligosaccharides, such that identification of the products of glycosy1 transfer can be attempted. Samples of bovine submandibular gland asialoglycoprotein, boar seminal gel asialoglycoprotein, human asialo-a, -acid glycoprotein and antarctic fish antifreeze glycoprotein were labelled in their terminal galactose or N-acetylgalactosamine residues using galactose oxidase (Morell et ul., 1966). The glycoproteins were made up as 1 % solutions in 50 mM-sodium phosphate/5 mM-NaC1, pH 7.0, and galactose oxidase added to give a final concentration of 5 units/ml. Incubation was for 25 h at 37°C. Each solution was diluted five-fold with 50 mM-sodium phosphate/ 50 rnM-NaC1, pH 7.8, and sodium boro['H]hydride ( 1 3 GBq/ mmol) added to give a concentration of 12 MBq/mg of glycoprotein (0.9 pmol/mg) and incubated for 30 min at room temperature. Non-labelled sodium borohydride (2 pmol/mg of glycoprotein) was added and incubated for a further 30min; the reactants were purified by gel filtration on Sephadex G25. The specific radioactivities of the products are shown in Table I . The nature of the labelled oligosaccharide/monosaccharide moiety was analysed after release by alkali catalysed a-elimination (Clamp et al., 1983) and fractionation on Biogel P4, by comparison with authentic standards of galactose, N-acetylgalactosaminitol and Galfil-3GalNAc-ol on gel filtration and t.1.c. Also the action of bovine testis or Escherichiu coli P-galactosidase was used to confirm the PI-3 or PI-4 nature of the terminal galactose residues. Glycosyltransfer to submandibular gland ['H]asialoglycoprotein and 'H-labelled antifreeze glycoprotein was determined in incubations containing 10 kBq of 'H-labelled acceptor, 0.75 pmol of UDP-GlcNAc, UDP-Gal or CMP-sialic acid, 0.5 pmol of ATP, 1 .Opmol of dimercaptopropan-1-01 in IoOmM-Mops, IOmM-MnCl,, 0.2% Triton X-100, pH 7.5, and proximal or distal rat colonic mucosal cell homogenate (Corfield et al., 1985) in a final volume of 750~1 . Incubation at 37°C was for 20h, and the reaction products were 8eliminated and fractionated on Biogel P4. Each nucleotide sugar donor gave a characteristic pattern of products as di-, trior tetra-saccharide with variation between normal proximal and distal colonic mucosae. Significant [j-galactosidase activity was also indicated under the conditions of the assay. Assay of 8-galactosidase preparations was carried out using [3H]asialc-a, acid glycoprotein (PI-4 linkage) and 'H-labelled antil'reeze glycoprotein (PI-3 linkage). Incubations contained P-galactosidase from E. coli (5 units at pH 7.3), Aspergillus niger (0.2 units at pH 4.0), bovine liver ( 1 unit at pH 7.3), bovine testis (0.05 unit at pH 4.3) or coffee bean a-galactosidase (0.2 unit at pH 6.5) and 'H-labelled substrate (5 kBq) at 37°C for 20 h under toluene. The reaction products were separated on Sephadex G25 superfine (25cm x 1 cm) in 0.1 M-NH,HCO, and the ['Hlgalactose fraction quantified. The final separation step allows discrimination between true P-galactosidase activity and the action of proteases releasing glycopeptides, which remains a source of error in precipitation based assays. The [3H]asialo-a, -acid glycoprotein (Dl -4) was a substrate for E. coli and A . niger preparations while the 'H-labelled antifreeze glycoprotein (PI-3) was cleaved by the bovine testis enzyme. The bovine liver preparation acted on both substrates and the a-galactosidase on neither. Specific radioactive labelling of defined glycoproteins yields substrates suitable for both glycosyltransferase and P-galactosidase assay.

Anthony P. Corfield

Papers

Demonstration of 9-O-lactyl-N-acetylneuraminic acid in human gastric aspirates

Biochemical analysis of mucin glycoproteins in paediatric colonic mucus

Radioactive substrates for the specific assay of glycoprotein glycosyl transfer and β-galactosidase activities

Individual variation in human conjunctival mucins

The expression of mucin genes by nasal polyps