Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, including colorectal cancer. In addition, β-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of β-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of β-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β-catenin stability. Analyses of β-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the β-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of β-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of β-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.

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The effect of cone voltage on the polymer mass distribution of non-polar poly(styrene) samples has been investigated using electrospray ionization. It was found that relatively high cone voltages (120 V) were required to ionize non-polar polymers. At lower cone voltages (30-60 V), cluster ions, e.g. [M+2catI2+ and [ZM+cat]', were observed. Singly-charged species predominated only at high cone voltages, but no fragmentation of the oligomer ions has been seen. The molecular mass distributions obtained by electmpray ionization have been compared with those obtained by size-exclusion chromatography and the agreement (generally within 10%) has been shown to be cone-voltage dependent.Mass spectrometry of synthetic polymers has grown considerably in importance in recent years. Advances in soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI),' and electrospray ionization mass spectrometry (ESI-MS)' previously applied to large biological molecules, are of particular interest when applied to synthetic polymers. ESI-MS should in principle enable the detection of oligomer and polymer macromolecules with little or no fragmentation. The strength of these new soft ionization techniques lies in their ability to give accurate macromolecular masses in the absence of fragment ions, allowing determination of polymer structure, including that of polymer end groups, in addition to yielding derived number-and weight-average molecular masses.ESI-MS is a rapidly developing soft ionization technique which finds wide application in the mass analysis of hydrophilic biomolecules. Application of this technique to synthetic polymers is very much in its infancy. Work has mainly concentrated on easily ionizable, hydrophilic polymers such as poly(ethy1ene glycol) (PEG).2b Unlike most biological polymers, synthetic polymers do not always contain easily ionizable functional groups. There has been very little work on the application of ESI-MS to less hydrophilic polymers, such as poly(styrene) and poly-(methylmethacrylate). ESI-MS studies of methyl methacrylate/methacrylic acid and methyl methacrylate/ butyl acrylate oligomers have been carried out in tandem with size separation by size-exclusion chromatography (SEC) prior to analysis in the mass ~pectrometer.~ Polymethyl methacrylate homopolymer and co-polymers of methyl methacrylate with butyl methacrylate, styrene and methacrylic acid4 have also proved amenable to ESI-MS.In the electrospray experiments, a sample solution doped with a potential cation in salt form is nebulized from the solution state (in the mobile phase) to the gas phase. In the first stages of the ion source-to-vacuum interface, a beam of sample-related ions is accelerated across a potential created relative to the sampling cone. The potential applied to the cone can be varied over the range 1 to 200 V and determines the amount of collision-induced decomposition (CID) which the ions experience before entering the first mass analyser. At low cone voltages (Cv), cluster such as [2M+cat]+, [M+2cat...

Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec/Elitek), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme contains a cysteine adduct on Cys(103). In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys(35) and Cys(290)) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.