Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin

Infection with Helicobacter pylori is associated with different human gastric diseases. Biochemical studies, in vitro adherence assays, and in vivo animal models revealed that epithelial attachment of H. pylori can be mediated by the blood-group antigen-binding adhesin (BabA) targeting human Lewisb surface epitopes. Studies with transgenic mice expressing the Lewisb epitope have shown that such attachment can alter disease outcome. In the current study, the presence of the babA2 gene encoding the adhesin was investigated in clinical isolates from a German population by using PCR and reverse transcription–PCR. A positive genotype was correlated to allelic variations in the genes encoding VacA and CagA and also to the prevalence of duodenal ulcer, distal gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma, and antral gastritis. The presence of babA2 was significantly associated with duodenal ulcer (P = 0.0002) and adenocarcinoma (P = 0.033). In contrast, type 1 strains (vacAs1- and cagA-positive) were associated with only duodenal ulcer (P = 0.004) but not adenocarcinoma (P = 0.235). Genotype presence of babA2, vacAs1, and cagA (“triple-positive” strains) showed a highly significant correlation to the prevalence of ulcer (P = 0.000002) and adenocarcinoma (P = 0.014) and discriminated significantly better between disease outcome than did the current type 1 classification. These results indicate that the babA2 gene is of high clinical relevance and would be a useful marker to identify patients who are at higher risk for specific H. pylori-related diseases.

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Correlation of regional cerebral blood flow and change of plasma sodium concentration during genesis and satiation of thirst

Positron emission tomography studies were conducted during genesis of moderate thirst by rapid i.v. infusion of hypertonic saline (0.51 M) and after satiation of thirst by drinking water. The correlation of regional cerebral blood flow with the change in the plasma Na concentration showed a significant group of cerebral activations in the anterior cingulate region and also a site in the middle temporal gyrus and in the periaqueductal gray. Strongest deactivations occurred in the parahippocampal and frontal gyri. The data are consistent with an important role of the anterior cingulate in the genesis of thirst.

Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.