We describe the isolation and partial characterization of Saccharomyces cerevisiae nonconditional mutants that show defects in N-glycosylation of proteins. The selection method is based on the reduction of affinity for the ion exchanger QAE-Sephadex as a consequence of the decrease in the negative charge of the cell surface. This characteristic reflects a decrease in the incorporation of mannosylphosphate units into the N-linked oligosaccharides of the mannoproteins. The mutants exhibit low affinity for the basic dye alcian blue and for that reason we have called them Idb (low dye binding) mutants. Eight of the complementation groups seem to be new as shown by complementation studies with previously isolated mutants of similar phenotype. Four of the groups showed a significant reduction in the number and/or size of the N-linked oligosaccharides attached to secreted invertase. We have analyzed the N-linked oligosaccharides of Idb1 and Idb2, the mutants that show the most drastic reduction in the affinity for the alcian blue dye. In both cases, the purified endo H-released oligosaccharides from the mannoproteins lacked detectable amounts of phosphate groups as shown by ion exchange chromatography and the 1H NMR spectra. In addition, Ibd1 synthesizes a truncated and unbranched outer chain lacking any alpha (1,2) linked mannoses attached to the alpha (1,6) linear backbone.
Study design: A cross-sectional study. Objective: To clarify the existing controversy with regard to whether paraplegic patients suer a loss of bone mass in the upper limbs. Setting: Madrid, Spain. Methods: We evaluated bone mass by phalangeal ultrasonography in 35 male patients with paraplegia (mean age 49+12 years), and 25(OH)D3 and PTH to exclude the presence of osteomalacia and secondary hyperparathyroidism. Spasticity was evaluated according to the Ashworth scale. Patients were compared with a control group of 35 healthy male subjects (mean age 48+13 years). Results: The patients had lower 25(OH)D 3 levels and amplitude-dependent speed of sound (Ad-SOS) than controls (both P50.001), and higher PTH levels (P50.05). There was a statistically signi®cant negative association between PTH and 25(OH)D 3 levels (r=70.52, P50.0001, CI 70.73 to 70.22) and between 25(OH)D 3 and injury duration (r=0.34, P50.05, CI 70.60 to 70.01). There was no correlation between Ad-SOS values, levels of PTH or 25(OH)D 3 , and the injury duration. No signi®cant dierence in Ad-SOS values was found in patients grouped according to low-to-normal 25(OH)D 3 level or according to normal-to-high PTH level. There were no dierences in relation to muscle tone. Only alkaline phosphatase and tartrate-resistant acid phosphatase levels were higher in patients than in controls (both P50.001). Conclusion: Paraplegic patients had a loss of phalangeal bone mass that was unrelated to the levels of vitamin D or PTH, or to muscle tone, so it seems to be related to increased bone resorption rather than to de®cient bone formation.
In patients with CD, gluten-free diet and increased nutritional intake were accompanied by normal bone mass values as determined by ultrasound on phalanges.
We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernändez, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648^13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A 3 (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C 1 ) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. ß
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