128 Substitutions in Penicillin-Binding Protein 1 in Amoxicillin-Resistant Helicobacter pylori Strains Isolated from Korean Patients

respectively (p<0.05). The expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1 were increased 6 hr after C. The expression of STAT3-induced feedback inhibitor, suppressor of cytokine signaling 3 - SOCS3, was elevated at 2 hr after C. We also demonstrated that egr-1 knockout mice which are more susceptible to C-induced duodenal ulcers had lower level of STAT3 expression, its phosphorylation, expression of importin α or β and STAT3/ DNA binding vs. wild type of mice in response to C. Conclusions: 1) C activated STAT3 signaling in rat duodenal mucosa through a) increased expression and its tyrosine phosphorylation; b) translocation to the nuclei probably with participation of importin α or β; c) enhanced STAT3 binding to its DNA with activation of its target anti-apoptotic gene expressions. 2) Inhibition of STAT3 expression in duodenal mucosa aggravated experimental duodenal ulcers. 3) Thus, STAT3 is a mediator of cell survival signaling during C-induced duodenal ulcer in rats. 127 Difluoromethylornithine, A Novel Treatment for Helicobacter pylori? David A. Leiman, Daniel P. Barry, Kathleen Fletcher, M. Blanca Piazuelo, Rupesh Chaturvedi, Keith T. Wilson Background: H. pylori induces a dysregulated host immune response that fails to eradicate the organism, and antibiotics are associated with treatment failures and recurrent infection. We have implicated polyamine synthesis by ornithine decarboxylase (ODC) in host cells in the immunopathogenesis of H. pylori infection, and shown that addition of difluoromethylornithine (DFMO), an inhibitor of ODC, reduces H. pylori colonization and gastritis in mice when added to drinking water as a 1% (w/v) solution. DFMO has antimicrobial effects on parasites by inhibiting their ODC. H. pylori does not possess ODC, but it expresses spermidine synthase that is required for proliferation. Our aim was to determine if DFMO affects H. pylori growth. Methods: H. pylori strain SS1 was grown in brucella broth or F12 medium, with 10% serum, and growth characteristics assessed by optical density every 2 to 4 h. 1% (w/v) DFMO was added to growth media at either the start of liquid culture or in the midlog phase of growth. In some experiments spermidine was added to the medium. Gramstaining and high-power brightfield microscopy was performed to assess morphology. Results: H. pylori exhibited exponential growth in brucella broth that was in the logarithmic phase from 4 h to 12 h with a doubling time of 3.8 h. In the presence of DFMO this growth was markedly attenuated with a linear growth pattern and a doubling time of 8.3 h. Microscopic assessment at 6 and 24 h revealed that at both time points bacteria exhibited predominantly coccoid morphology in the presence of DFMO, versus bacillary appearance in the absence of DFMO. DFMO caused a modest reduction of pH, but correction with NaOH did not enhance H. pylori growth. Addition of spermidine (25 to 625 μM) did not rescue H. pylori growth when added to bacteria exposed to DFMO. When DFMO was added at 6 h to growing cultures it immediately prevented exponential growth, and this resulted in coccoid morphology at 24 h. When bacteria exposed to DFMO from 0 h were transferred to medium lacking DFMO 6 h later, they were able to resume exponential growth, and recovery of the bacillary form. Growth of H. pylori was also attenuated by DFMO in F12 medium lacking polyamines. Conclusions: DFMO inhibits growth of H. pylori In Vitro, which is associated with conversion of the bacterium from the bacillary to the less virulent coccoid form. This occurs by a previously unrecognized effect that appears to be independent of an alteration in polyamine synthesis or transport. These findings indicate that DFMO or other agents that attenuate H. pylori growth and virulence could have an adjunctive therapeutic benefit in H. pylori eradication strategies.

125 M-Numb Expression Induced Gastric Mucosal Regeneration After Injury By Stimulating the Expression of Morphogenic Genes Tetsufumi Takahashi, Hidekazu Suzuki, Yoshimasa Saito, Hiroshi Nagata, Hideyuki Okano, Toshifumi Hibi m-Numb expression in the epithelium has been shown in early gland formation of the chicken stomach. However, the postnatal expression and role of this factor are unclear. In this context, m-Numb-mutant mice have been shown to exhibit embryonic lethality, while Musashi1-mutant mice (Msi1-KO) grow into adulthood (PNAS 99:15194-9,2002). Msi1 is known to bind to the m-Numb mRNA 3'-UTR region (Mol. Cell. Biol.; 21: 3888-900,2001). In the present study, the process of tissue repair after ethanol-induced acute gastric mucosal injury was investigated in Msi1-KO mice, in order to clarify the role of the Msi1-regulated m-Numb expression in gastric mucosal regeneration. Methods. Msi1-KO and wild-type ICR mice (male, 10 weeks old) were orally administered absolute ethanol (0.25 ml) after 18 hours of food deprivation, and the stomachs were resected 1, 3, 5 hours after the ethanol intoxication. m-Numb mRNA expression was analyzed by real-time PCR using specific primers for individual splicing variants. m-Numb protein expression was detected by Western blotting and immunohistochemical staining using anti-m-Numb antibody. The whole- mRNA expression in the stomach at 1 hour after the ethanol administration was compared by the microarray method. Results. Constitutively, the expression of m-Numb protein in the stomachs of the Msi1-KO mice was reduced as compared with that in the wild-type mice. After the ethanol administration, the expression of m-Numb mRNA and protein in the wildtype mice was upregulated for up to 5 hours. In particular, the phosphotyrosine binding site-conserved type of m-Numb mRNA was increased after the injury. In contrast, in the Msi1-KO mice, while m-Numb mRNA was expressed at the same level as that in the wildtype mice, m-Numb protein expression was comparatively very weak. These Msi1-KO mice showed delayed regeneration of the gastric epithelium after the injury. Immunohistochemical analysis revealed that m-Numb protein was normally expressed in the gastric epithelium and slightly in the zymogenic region, and did not coincide with that of H+, K+-ATPase. On the other hand, the induced m-Numb protein after the injury was expressed just under the pre-apoptotic epithelium, which may be the origin of regeneration. Microarray analysis indicated relatively higher expression levels of morphogenic genes such as TFF1, TFF2, Shh and Numb adaptor protein, Pon1, after gastric mucosal injury in wild-type mice. Conclusion. m-Numb expression appears to be a key factor in gastric epithelial regeneration after acute gastric mucosal injury, possibly via regulating the expression of morphogenic genes.

128 Substitutions in Penicillin-Binding Protein 1 in Amoxicillin-Resistant Helicobacter pylori Strains Isolated from Korean Patients Jae G. Kim, Ki S. Kim, Won S. Cheon, Jeong Wook Kim Multiple factors which confer amoxicillin resistance of Helicobacter pylori including pbp1 mutation has been tested but the exact mechanism has not been fully elucidated. In the present study, we assessed amoxicillin resistant rate in H. pylori strains isolated from Korean patients and investigated the correlation between amoxicillin resistance of H. pylori and substitutions in penicillin-binding protein (PBP) 1. Methods: One hundred fifty three H. pylori strains were isolated between 2005 and 2006 from Korean patients with chronic gastritis, peptic ulcer or stomach cancer. The minimum inhibitory concentrations (MICs) of the strains were determined with serial 2-fold agar dilution method. The resistance breakpoint for amoxicillin was defined as >0.5 μg/mL. The sequence of PBP1 was analyzed in three amoxicillin-susceptible strains including ATCC 700392 and compared with those of amoxicillin-resistant isolates. Result: Nine of one hundred fifty three H. pylori strains showed amoxicillin resistance (6.0%). The MICs values of resistant strains were ranged from 1 μg/mL to 4 μg/mL. PBP1 sequence analysis revealed six identical amino acid substitutions in all amoxicillin-resistant strains: Val16→Ile, Val45→Ile, Ser414→Arg, Asn562→Tyr, Thr593→Ala, and Gly595→Ser. Conclusion : We detected six identical PBP1 amino acid substitutions in all amoxicillin-resistant strains. Additional transformation study of mutated pbp1 gene would provide more information about pbp1 gene mutation that contributes to amoxicillin resistance.

126 STAT3 Tyrosine Phosphorylation and Its Nuclear Import Are Novel Molecular Mechanisms in Experiment Duodenal Ulceration in Rats and Egr-1 Knockout Mice Tetyana Khomenko, Longchuan Chen, Xiaoming Deng, Amrita Ahluwalia, Andrzej S. Tarnawski, Sandor Szabo Signal transducer and activator of transcription (STAT) 3 is a transcription factor which directly upregulates anti-apoptotic genes such as Bcl-xL, c-Myc, VEGF and Ref-1, and its function is critical for cellular transformation by tyrosine kinases. The role of STAT3 as a DNA-binding transcription factor depends on its ability to gain entrance to the nucleus. In this study, we hypothesize that duodenal ulcerogen cysteamine (C) activates STAT3 signaling and its passage through the nuclear pore to provide a critical protective role that is required for enterocytes survival during the early phases of C-induced duodenal ulcers. Methods: Sprague-Dawley rats were gavaged with saline or C-HCl (25 mg/100g x 3; 4 hr interval) and euthanized 0.5, 2, 6, 12, 24 or 48 hr later. Group of rats were pretreated with STAT3 antisense or scrambled control (0.5 mg/rats) or an inhibitor of STAT3/DNA binding, caffeic acid before C. Because of possible STAT3 and Egr-1 interactions we also used egr-1 knockout mice. Gene expression was determined by Affymetrix Assay, STAT3/DNA binding by Chemiluminescent TransFactor Kits (Clontech Inc.), protein levels by Western blotting and immunohistochemistry. Results: STAT3 antisense but not scrambled control aggravated duodenal ulcers by almost 2-fold, caffeic acid increased the size of duodenal ulcers by 1.8fold (p<0.05). C induced STAT3 protein translocation to nuclei and its tyrosine phosphorylation from 6 to 24 hr. At the same time we detected an increased expression and nuclear translocation of importin α and β in rat duodenal mucosa. C increased the binding of STAT3 to its DNA consensus sequences at 6, 12 and 24 hr after C by 1.5, 1.8 and 3.5- fold,

129 Ferric Uptake Regulator (FUR) Mutation in Helicobacter pylori Associated with the Development of Resistance to Metronidazole: Implication of the StructuralFunctional Relationships of Mutant-Type Fur Hitoshi Tsugawa, Hidekazu Suzuki, Izumi Nakagawa, Kenro Hirata, Toshihiro Nishizawa, Yoshimasa Saito, Juntaro Matsuzaki, Eisuke Iwasaki, Toshifumi Hibi Metronidazole (MNZ) is an important component of the currently employed therapeutic regimens for many bacterial infections, including Helicobacter pylori (H. pylori) infection. MNZ is a prodrug and is converted into its active form when its nitro group is reduced and superoxide radicals are generated. NADPH nitroreductase (RdxA) of H. pylori reduces MNZ

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AGA Abstracts

AGA Abstracts

acetic acid. Expression of mRNA for COX-2 and 15-PGDH in normal gastric tissue and ulcer tissue was assessed by real-time RT-PCR. Expression and localization of 15-PGDH protein was determined by western blotting and immunohistochemstry. To clarify the significance of the epidermal growth factor receptor (EGFR) signaling pathway in expression of 15-PGDH during gastric ulcer healing, three days after induction of ulcers (Day 0), some mice with ulcers were treated with EGFR kinase inhibitor (tyrphostin AG1478; 10 mg/kg BW) intraperitoneally on Days 3 and 4 and expression of mRNA for 15-PGDH was assessed. To determine the association of COX-2 with expression of 15-PGDH during gastric ulcer healing, selective COX-2 inhibitor (celecoxib; 10 mg/ kg BW) with or without 16, 16dimethyl PGE2 (5 μg/kg BW) was administered to mice with ulcers from Day 0 to Day 7 and expression of mRNA for 15-PGDH in ulcer tissue was evaluated. Results: 15-PGDH protein was expressed mainly in gastric epithelial cells in normal gastric mucosa, while it was markedly down-regulated at ulcer margins. Expression of mRNA for COX-2 was increased in ulcer tissue by 12.2-fold, whereas expression of mRNA for PGDH was reduced by 57% compared with normal gastric tissue. Consistent with this, protein level of 15-PGDH in ulcer tissue was decreased compared with that in normal gastric tissue. AG1478 reversed the down-regulation of mRNA expression of 15-PGDH in ulcer tissue. By Day 7, celecoxib had further decreased expression of mRNA for 15-PGDH in ulcer tissue compared with that in vehicle-treated mice with ulcers, and had impaired gastric ulcer healing, while restoration of 16,16-dimethyl PGE2 reversed the reduction of level of mRNA of 15-PGDH by celecoxib. Conclusion: These findings suggest that 15-PGDH is down-regulated during gastric ulcer healing at ulcer margins and is regulated by activation of the EGFR signaling pathway and PGE2 derived from COX-2.