RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach.

The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [(35)S]Met-labeled RACK1 protein fragments.

VacA genotyping directly from gastric biopsy specimens and estimation of mixed Helicobacter pylori infections in patients with duodenal ulcer and gastritis.

BACKGROUND: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. METHODS: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. RESULTS: The ml subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. CONCLUSIONS: These results suggest that vacA s1 genotype and cagA+ status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region.

Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells.

The molecular basis for putative aberrant splicing of hypoxanthine (guanine) phosphoribosyltransferase (hprt) pre-mRNA in Chinese hamster V-79 cells was determined for 75 independent (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+)-BPDE]-induced and 6 spontaneous 8-azaguanine-resistant mutant clones that had exon deletions in their hprt cDNA. Genomic DNA fragments corresponding to the missing exons and their flanking intron regions were amplified by PCR and sequenced. The results indicated that each of these mutants generated a normal-sized PCR product and resulted from aberrant splicing. For (+)-BPDE-induced aberrant splicing mutants, 81% (61 of 75 clones) had base substitution mutations, 5% (4 of 75 clones) had a single base deletion, and 13% (10 of 75 clones) lacked a detectable mutation in the skipped exon, its flanking intron sequences, or in the upstream donor site of the preceding intron. All mutations at a splice donor site resulted in skipping of the entire upstream neighboring exon, whereas alterations at a splice acceptor site caused skipping of the downstream neighboring exon or activation of a cryptic acceptor site in the downstream exon. Fifty-nine % of the splicing mutants had a mutation occurring at the splice site consensus sequence in the intron, and 28% of the splicing mutants had mutations within exon sequences. Among 21 aberrant splicing mutant clones with a mutation inside an exon sequence, seven were in exon 2, two were in exon 3, and twelve were in exon 4. Evidence is presented that a stemloop structure sequesters the splice donor site of exon 2 in pre-mRNA and plays a role in exon 2 skipping. Mutant clones with mutations stabilizing the proposed stemloop structure inhibited the use of the normal exon 2 splice site which resulted in exon 2 skipping in the hprt mRNA. These mutant clones expressed a mixed population of mRNAs, and both normal-sized and truncated mRNA were formed. Similar to our earlier finding that treatment of V-79 cells with (+)-BPDE resulted in a dose-dependent mutation profile within the coding region of the hprt gene, we also observed the presence of dose-dependence in the profile of (+)-BPDE-induced base substitutions in aberrant splicing mutants. As the dose of (+)-BPDE was decreased, the proportion of base substitution mutations at AT base pairs that affected RNA splicing was increased.

Modifications of microsomal and nuclear fractions by butylated hydroxyanisole and its effect on benzo(a)pyrene metabolites binding to macromolecules.

The effect of butylated hydroxyanisole (BHA) administration on the hepatic components of the monooxygenase system and lipid peroxidation in microsomal and nuclear fractions was investigated in male Swiss mice. Addition of BHA to the diet for 8 days increased significantly the content of cytochrome P-450 (by 50%) and two times the specific activities of NADH- and NADPH-cytochrome c reductases in liver microsomes and lowered the concentration of cytochrome P-450 in liver nuclei. Lipid peroxidation of liver microsomes obtained from BHA fed mice was higher (by 70%) as compared with the control. The inhibition of peroxidation was shown when BHA was added to the incubation mixture containing control microsomal fraction or liver homogenate. When benzo(a)pyrene (BP) was incubated with liver microsomes from BHA fed mice the binding of BP metabolites to microsomal macromolecules increased 5.5-fold compared with control. However, there was no such effect in case of liver nuclei. In view of these results it has been postulated that BHA can play not only preventive role in chemical carcinogenesis.

Effect of dietary butylated hydroxyanisole on the mouse hepatic monooxygenase system of nuclear and microsomal fractions.

The effect of butylated hydroxyanisole (BHA) administration on the hepatic monooxygenase system of nuclear and microsomal fraction was investigated in male mice. Addition of BHA to the diet significantly lowered the content of cytochrome P-450 in liver nuclei and increased the specific activity of NADPH-cytochrome c reductase and the content of cytochrome b5 in liver microsomes. Incubation of benzo[a]pyrene (BP) with liver nuclei from BHA-fed mice resulted in inhibition of binding of BP metabolites to nuclear macromolecules by 50% compared with control. However, there was no effect of BHA on the binding of BP metabolites to macromolecules when BP was incubated with added DNA and liver microsomes from BHA-fed mice. It has been postulated that modification of nuclear monooxygenases by BHA may play a role in the inhibitory effect of BHA on BP carcinogenesis.