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Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.

O-Linked fucose is an unusual form of glycosylation recently shown to modify the hydroxyls of serine or threonine residues at a strict consensus site within epidermal growth factor-like domains of several serum proteins. Here we demonstrate that Chinese hamster ovary cells modify numerous proteins with O-linked fucose and that the fucose is elongated on specific proteins. We have identified at least two forms of O-linked fucose elongation in Chinese hamster ovary cells: a disaccharide (Glcbeta1,3Fuc) and a larger oligosaccharide of indeterminate structure. Interestingly, it appears that the level of monosaccharide accumulates in the cells over time whereas the disaccharide does not. Analysis of the O-linked fucose-containing saccharides on individual proteins revealed that some proteins are modified with the monosaccharide only, whereas others are modified with monosaccharide and disaccharide, or monosaccharide and oligosaccharide. These results suggest that elongation of the O-linked fucose monosaccharide is a protein-specific phenomena. The presence of elongated O-linked fucose moieties suggests that a novel glycosylation pathway exists in mammalian cells with O-linked fucose as the core.

In order to assess the benefit to patients with thin malignant melanoma (< 0.76 mm) of a 5-year clinical follow-up programme, we have studied 602 patients with a minimum time from primary surgery of 5 years. Tumour recurrence occurred in 24 patients (4% of all patients) but only five surgically treatable recurrences (< 1% of all patients) occurred within the 5-year period following primary surgery. After 5 years there were four surgically treatable recurrences, but their prognosis was generally poor. The remaining cases of tumour recurrence were not surgically treatable. In the face of an increasing incidence of melanoma, and the accompanying increase in demand for surgical treatment and outpatient review, we question the need for prolonged hospital follow-up of thin melanoma.

We have found that double opposing rectangular advancement flaps in the forehead may be easily performed under either local or general anaesthesia, and are associated with high patient satisfaction and low morbidity. We claim no originality for the mode of reconstruction, but highlight its broad spectrum of application and its popularity with junior plastic surgeons passing through our unit., We have found that the term 'H-fla' provides a readily communicated alternative to 'double opposing rectangular advancement flaps'.