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In our previous study, we showed that Helicobacter pylori gamma-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclin-dependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.

Helicobacter pylori causes gastroduodenal disease, which is mediated in part by its outer membrane proteins (OMPs). To identify OMPs of H. pylori strain 26695, we performed a proteomic analysis. A sarcosine-insoluble outer membrane fraction was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 10 to 100 kDa, were visible on the gel in the alkaline pI regions (6.0 to 10.0). The proteome of the OMPs was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Of the 80 protein spots processed, 62 spots were identified; they represented 35 genes, including 16 kinds of OMP. Moreover, we identified 9 immunoreactive proteins by immunoblot analysis. This study contributes to the characterization of the H. pylori strain 26695 proteome and may help to further elucidate the biological function of H. pylori OMPs and the pathogenesis of H. pylori infection.Helicobacter pylori is a spiral-shaped, microaerophilic gramnegative bacterium that causes acute and chronic gastritis, gastroduodenal ulcers, and gastric cancer (3,7,21,40). More than half of the world's population has suffered from H. pylori infection (4, 5, 46). Surface proteins, including flagella, urease, and adhesin, are known to be involved in the pathogen-host relationship between H. pylori and the human gastric mucosa. A correlation between the motility state of some H. pylori isolates, and their ability to colonize the gastric epithelium has been established in experiments with gnotobiotic piglets (18). Urease enables H. pylori to survive in the acidic environment of the stomach (13) and plays a key role in colonizing the gastric mucosa (17). Adhesins, including BabA (25), AlpA/AlpB (42), HopZ (43), and SabA (26), are known to adhere to gastric epithelial cells.The genomes of two H. pylori strains have been sequenced (2, 49) and extensively compared (1). Of 64 theoretically predicted outer membrane proteins (OMPs), at least 8, including adhesins and porins, have been confirmed experimentally. However, it is unclear whether all of the predicted OMPs are expressed.Several methodological approaches have been applied to the identification of H. pylori surface proteins, including OMPs. Sarbarth et al. (48) selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. Exner et al. (19) purified OMP fractions by sucrose gradient centrifugation and identified heat-mobile OMPs, which may be porins, by using two-dimensional (2-DE) gel electrophoresis. Doig et al. (15) identified six OMPs in a sarcosine-insoluble OMP fraction and by using monoclonal antibodies, demonstrated that these proteins are located within or are associated with the outer membrane. In addition, by comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane fractions isolated by different isol...

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