Isotopic study of post-anthesis foliar incorporation of sulphur and nitrogen in wheat.

Nitrogen (N) and sulphur (S) supplies have a strong influence on the quality and quantity of wheat storage proteins, which play an important role in the bread-making process. In order to relate the incorporation and distribution of foliar N and S fertilisers at the post-anthesis stage to the quality of wheat, 15N and 34S isotopes were used as tracers. The incorporation of these tracers in different plant parts (leaves, stems, ears) and in each storage protein fraction (gliadins, HMW and LMW glutenin subunits) was determined by isotopic ratio mass spectrometry coupled with an elemental analyser (EA-IRMS). By this means, the true recovery coefficient of N and S (TRCNfertiliser and TRCSfertiliser) and the N and S derived from fertilisers (Ndff and Sdff) could be determined. The TRCNfertiliser and TRCSfertiliser values of the different plant parts provide evidence of the applied N and S assimilation and translocation from wheat leaves to the seeds. The determination of Ndff and Sdff incorporated into storage proteins shows the efficiency and the influence of N and S incorporation into each storage protein fraction. Moreover, a favourable stage for fertiliser application can be determined by the TRCNfertiliser values in the grain and in the whole plant. The fertilisers enriched in stable isotope used in the culture techniques can be a means of understanding the effectiveness of fertilisers in the expression of wheat quality.

Sulphation of the salivary mucin MG1 (MUC-5B) is not correlated to the degree of its sialylation and is unaffected by cystic fibrosis.

Defective acidification of intracellular organelles, particularly the trans-Golgi network, has been proposed to explain the decreased sialylation and increased sulphation of secreted proteins in cystic fibrosis (CF). To test this hypothesis we compared expression of sulphate and sialic acid on three salivary mucins namely MG1 (MUC-5B), MG2 (MUC-7) and GL. Proteins in whole mouth saliva (WMS) from four individuals were separated by fast protein liquid chromatography (FPLC) on a Superdex 200 column and the partially purified mucins slot-blotted and assayed for sulphate content by staining with Alcian Blue. Sulphation varied with the individual and with the mucin: MG1 was the most sulphated and contributed almost the entire sulphate content of WMS. These results allowed us to test small volumes of WMS from 20 CF patients and age- and sex-matched controls for estimates of sulphate content on MG1. Wherever possible sulphate on MG1 was also visualised by staining washed SDS-PAGE gels with Alcian Blue at pH 1.0. To assess the sialic acid content of salivary mucins, electroblots of SDS-PAGE gels were probed with labelled Triticum vulgaris agglutinin. In summary, our results show MG1 to be the main sulphated protein in whole mouth saliva and there are large differences in the expression of sulphate and of sialic acid on this mucin, both in control and CF groups. CF led neither a decrease in sialylation nor an increase in sulphation and direct comparisons of sialic acid content with sulphate in MG1 failed to reveal any obvious link between the two in health or in disease. Our data thus do not support the hypothesis of defective acidification as the underlying cause of altered glycosylation in CF, but point instead to inter-individual differences in expression/functioning of terminal glycosyltransferases for published observations. We thank the European Union Biomed II programme for support.

Altered sialyl- and fucosyl-linkage on mucins in cystic fibrosis patients promotes formation of the sialyl-Lewis X determinant on salivary MUC-5B and MUC-7.

Destruction of the lungs as a consequence of recurrent infections with microorganisms such as Pseudomonas aeruginosa remains the underlying cause of most morbidity and mortality in cystic fibrosis (CF). We have hypothesized that changes in the glycosylation of key tracheal mucins such as MUC5B and MUC7 might increase the risk of pulmonary disease in CF patients. However, in preference to sputum we have examined the sugar-chains on these mucins in saliva because in the latter not only can the glycoproteins be collected from controls, but they are essentially free from modifications made following bacterial infection in disease. Proteins in ductal or whole-mouth saliva from 20 CF patients with the Delta F-508 CFTR mutation and age-and sex-matched controls were separated by SDS-PAGE and blotted onto nitrocellulose and then probed with labelled lectins of known specificity. Linkage of terminal sialic acid on the blotted mucins was determined using Sambucus nigra agglutinin (detects the 2-->6 linkage) and Maackia amurensis agglutinin (the 2-->3 linkage). Fucose was detected by Ulex europaeus agglutinin-1 (1-->2 linkage) and Aleuria aurantia agglutinin (1-->3 linkage). We found that each mucin shows a characteristic glycosylation pattern and in controls most of the sialic acid is 2-->6 linked on MG1 (MUC 5B) and 2-->3 linked on MG2 (MUC 7). CF is associated with a shift from a 2-->6 linkage to a 2-->3 linkage on MG1 with some patients showing almost no 2-->6 linkage; 2-->3 linkage on MG2 is similarly increased in disease in some individuals. The expression of fucose on these mucins is also raised in CF patients. These shift to a 2-->3 linkage of sialic acid, and with increased fucosylation this promotes the formation of sialyl-Lewis X antigen detected on CF mucins in our study. These changes will be tested for their correlation with the severity of lung disease. We gratefully acknowledge support from the European Union Biomed-II Programme.