Periodontitis and concentrations of the cellular immune activation marker neopterin in saliva and urine.

Neopterin concentrations in diverse body fluids provide a well established indication for activation of the cell-mediated immune system. Neopterin concentrations were measured in the saliva and urine of 29 patients with varying numbers of teeth affected by periodontitis. While neopterin concentrations in urine increased slightly but not significantly in parallel with increasing numbers of affected teeth, salivary neopterin levels showed a significant and positive correlation with number of diseased teeth (linear correlation coefficient = 0.48, P = 0.012). Additionally, when the patients were grouped according to the median number of affected teeth (20), salivary specimens of subjects with one to 20 affected teeth showed significantly lower neopterin concentrations than specimens from those with more than 20 diseased teeth (P = 0.0045, Student's t-test). In contrast, urinary neopterin concentrations did not differ significantly among these two groups. This pilot study suggests that salivary neopterin concentrations may reflect local immune activation even in situations where no systemic activation can be detected.

Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling with synthetic inhibitors.

Kinetic parameters for the inactivation of the 6-phospho-beta-galactosidase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of 2,4-dinitrophenyl-2-deoxy-2-halogeno- galactoside-6-phosphates have been determined. These inhibitors function by the formation of a stabilised glycosyl-enzyme intermediate. Inactivation and reactivation studies indicate that the fluoro derivative is formed most rapidly, but is also hydrolysed fastest. The chloro derivative forms the most stable covalent intermediate. HPLC profiles of V8-protease digestion of native and inhibited protein show significant differences, whereas the inhibited 6-phospho-beta-galactosidase and a point mutant of 6-phospho-beta- galactosidase (E375Q) yield the same proteolytic fragments. The suggestion that E375 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two peptides, residues 336-375 and 376-383, are not produced, due to the absence of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of the respective V8 cleavage site due to the chemical reaction of the inhibitor at position 375. Specific modification of the glycosyl bond between the inhibitor and E375 by aminolysis with benzylamine generated a glutamatic-acid-5-benzylamide complex at that position in the peptide. The Edman derivative of the modified E375 appears to be stable and was isolated by Edman degradation of trypsin-digested V8-peptide. It was shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-galactosidase, consistent with previous findings for enzymes in this family.