Concentrated broth culture supernatants from 50 to 60% of Helicobacterpylori strains induce eukaryotic cell vacuolation in vitro. A quantitative assay for cell vacuolation was developed on the basis of the rapid uptake of neutral red dye into intracellular vacuoles. The neutral red dye uptake (NRU) of visibly vacuolated HeLa cells was significantly greater than that of nonvacuolated cells. By using the rapid NRU assay, we sought to determine the roles of H. pylori cytotoxin, urease, and ammonia in the vacuolation of HeLa cells. The NRU of HeLa cells incubated in medium containing ammonium chloride or ammonium sulfate was significantly greater than that of cells incubated in medium alone. In addition, ammonium salts augmented the NRU induced by H. pylori supernatants. The NRU induced by jack bean urease was augmented by the addition of urea to cell culture medium; this suggests that urease-mediated NRU occurs via the generation of ammonia. Acetohydroxamic acid blocked the NRU induced by jack bean urease and urea but failed to block the uptake induced by H. pyloni supernatants. Supernatant from a non-urease-producing H. pylori mutant strain induced NRU identical to that of the urease-positive parental strain. These observations indicate that the vacuolating activity in H. pyloni supernatants is not mediated solely by urease activity but that it may be potentiated by urease-mediated ammonia production. Helicobacter pylori infection has been strongly associated with type B gastritis and peptic ulcer disease, but the mechanisms whereby infection may lead to tissue inflammation and damage are poorly understood (3). Concentrated broth culture supernatants from 50 to 60% of H. pylori strains induce vacuolation of cells in vitro (5, 12), a phenomenon that has been attributed to cytotoxin activity. The vacuolating activity in supernatants is abolished by heating to 70°C or by incubation with proteases (12), and thus it is probable that vacuolation is mediated at least in part by a protein. Recently, the vacuolating activity in H. pylori culture supernatants has been attributed to urease activity rather than to a specific cytotoxin (25). The degradation of urea by urease liberates ammonia, which is known to induce cell injury (2, 22, 25); in addition, ammonia has long been known to induce cell vacuolation (6, 18, 20). To study further the vacuolation of cells induced by H. pylori supernatants, we developed a quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by cell vacuoles. By using this assay, we sought to clarify the roles of H. pylori cytotoxin, urease, and ammonia in the phenomenon of cell vacuolation. MATERIALS AND METHODS Bacterial strains. H. pylori 60190 and Tx3Oa, Tox+ and Tox-, respectively (5, 12), were used for most studies. These strains were cultured in brucella broth (BBL Microbiology Systems, Cockeysville, Md.) containing 5% fetal bovine serum for 48 h on a rotary shaker at 37°C in ambient air supplemented with 5% CO2. H. pylori 88-3887 (urease positive) and 26U1 (a...
Twenty adolescents with insulin-dependent diabetes mellitus were interviewed to obtain samples of problem situations that create obstacles to dietary adherence. The resulting 57 situations were analyzed using a reliable coding system to determine the presence or absence of 28 stimulus features. A hierarchical cluster analysis was used to identify 10 relatively homogeneous categories of obstacles to dietary adherence: being tempted to stop trying; negative emotional eating; facing forbidden foods; peer interpersonal conflict; competing priorities; eating at school; social events and holidays; food cravings; snacking when home, alone, or bored; and social pressure to eat. Diabetes educators should consider an individual's ability to cope with this array of obstacles to adherence when individualizing treatment. Dietary intervention then can be personalized to address specific situational obstacles.
During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein 1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1, mutations in each S1 gene resulted in changes in the deduced amino acid sequence of 1 protein. Using electrophoresis conditions that favor migration of 1 oligomers, we found that 1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas 1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in 1 protein affecting stability of 1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 1 protein, we used T1L ؋ PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein 3. This finding suggests that in some cases, mutations in 3 protein in the absence of 1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L ؋ PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the 1-encoding S1 gene. Given that the 1 and 3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.
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