Suitability of Science Experiment Classes for Class Reform

departmental bulletin pape

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

Serum S-glutathionylated proteins as a potential biomarker of carotid artery stenosis

Objectives: As oxidative stress is known to be associated with the development of atherosclerosis, we investigated whether the serum S-glutathionylated proteins were increased in patients with carotid artery stenosis (CS). Design and methods: Fifty-four patients with CS and 20 age-matched non-CS patients were involved in this study. S-glutathionylated proteins in serum were examined by immunoblot analysis using an antibody against S-glutathionylated bovine serum albumin. Results: The antibody against S-glutathionylated bovine serum albumin was confirmed to specifically recognize the serum S-glutathionylated proteins in patient samples. The S-glutathionylated proteins in serum were significantly increased in the patients with CS (p<. 0.01) compared to the non-CS patients, and the increase did not depend on the stage of CS. Logistic regression analysis revealed that the serum levels of S-glutathionylated proteins were associated with the development of CS (p<. 0.01). Conclusions: Oxidative stress likely contributes to the development of CS, and serum S-glutathionylated proteins may be a potential biomarker of CS