Clinically feasible stable isotope technique at a reasonable price: analysis of 13CO2/12CO2-abundance in breath samples with a new isotope selective-nondispersive infrared spectrometer

Up to now, stable isotope analysis of carbon dioxide in breath samples is carried out with sensitive but very expensive and complex isotope ratio mass spectrometry (IRMS). Aiming at a more widespread application of breath tests in gastroenterological diagnostic routine, we tested a newly developed isotope selective non-dispersive infrared spectrometer (NDIRS) in comparison to IRMS. 13C-urea breath tests were performed in 63 patients as the routine screening method for Helicobacter pylori infection. Breath samples at baseline and (15) 30 min after administration of the test solution containing 13C-urea were analysed both by NDIRS and conventional IRMS. The correlation between the delta values of both devices was linear and in good agreement (r = 0.96; p < 0.0001; Y = 1.01 X -0.94). Comparing the delta over baseline-values, the correlation was Y = 1.11 X -0.36 (r = 0.98; p < 0.0001). Referring to the diagnosis of Helicobacter pylori infection with IRMS we calculated a sensitivity of 95.0% and an unchanged specificity (100%) for NDIR analysis. In conclusion, NDIRS appears a promising, easy to operate, and low cost potential alternative to conventional IRMS thus encouraging further detailed investigation and more widespread application of the noninvasive stable isotope technique in breath tests for gastrointestinal function testing.     

Salivary lactobacilli explain dental caries better than salivary mutants streptococci in 4-5-year-old children

The present comparative study was undertaken to determine which of the bacteria, lactobacilli (lbc) and mutans streptococci (ms), in saliva better explains the variation of caries in 2728 South African 4-5-yr-old children. Caries was diagnosed according to WHO criteria. For lbc, the Dentocult system was used. The number of ms in stimulated saliva was counted on MSB agar plates. For correction of confounding factors, data on the frequency of intake of sweets were derived from extensive interviews. Oral hygiene was determined according to the simplified debris index of Greene & Vermillion. Simple correlation analyses between dmfs and bacterial counts were done for the total material and for three caries intervals by calculating Spearman's and Pearson's coefficients of correlation. Multivariate regression analyses were done on all intervals to correct for the confounding effects of regular intake of sweets, presence of salivary ms or lbc, and oral hygiene. Of the children, 68% had detectable lbc in the saliva, and 74% had ms. Except for children with more than 6 dmfs, the explanatory values, i.e., percentage of variation in dmfs explained, were higher for the lbc than for ms. Before correction, the values for the total material were 15 vs 6%; for children with caries, 7 vs 5%; for those with 1-6 dmfs, 5 vs 0.4%; and for those with more than 6 dmfs, 0.3 vs 2%. All r-values were reduced after correction, indicating that the confounders explain some of the correlation between dmfs and bacterial count.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.     

Biostereometric quantification of clinical denture tooth wear

In this study the wear of mandibular acrylic resin teeth opposed by porcelain maxillary teeth arranged in a lingualized occlusal scheme over a period of 3 years was measured. Six edentulous subjects received complete dentures as above and with three baseline markers of amalgam. At yearly intervals casts were made of the mandibular occlusal surfaces, including markers, and plotted by stereophotogrammetry. Volumetric loss of material was quantifiable. Ranges of 0.62-3.33 mm3/mm2 on the left side and 0.71-1.64 mm3/mm2 on the right were recorded. Friedman two-way ANOVA test indicated significant wear on teeth 35, 36, 45 and 46 but not on 34 and 44. A one-sided chewer displayed greater wear on the contralateral side, a finding difficult to explain.     

Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution

N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E. coli cells and in a wheat germ cell-free translation system, and their conformations were studied. Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates). The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone. Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules). A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure. Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein.