- Email: [email protected]
The mechanism of gastric mucosal apoptosis induced by ammonia
A734 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
3980
3982
IDENTIFICATION OF COMH: A NOVEL, HEUCOBACTER pyLORI SPECIFIC TRANSFORMATION GENE, AND COMPLE· MENTATION OF COMH MUTANTS WITH THE NEW RDXA· VECTOR SYSTEM. Leonard C. Smeets, Jetta J. Bijlsma, Sacha Y. Boomkens, Christina Mje Vandenbroucke-Grauls, Johannes G. Kusters, Vrije Univ, Amsterdam, Netherlands.
THE MECHANISM OF GASTRIC MUCOSAL APOPTOSIS IN· DUCED BY AMMONIA. Hideo Suzuki, Akinori Yanaka, Akira Nakahara, Naomi Tanaka, Hiroshi Muto, Yasuo Uchiyama, Clin medicine, Univ of Tsukuba, Tsukuba, Ibaraki, Japan; Cell Biology and Anatomy, Osaka Univ, Suita, Osaka, Japan. Helicobacter pylori (HP) has been shown to exert as the pathogen of gastritis, peptic ulcer, and gastric cancer. HP produces high concentrations of ammonia within the gastric lumen. We have previously shown that ammonia is toxic especially at high ambient pHs. It has also been reported that prolonged acid inhibition accelerates progress of gastric atrophy in HP-infected subjects. Gastric atrophy is defined as the loss of gastric glands and associates gastric cell apoptosis, In this study, we examined the mechanism of ammonia-induced apoptosis of gastric epithelial cells at various ambient pHs. Methods : GSM06 cells, established from gastric surface mucous cells of transgenic mice harboring SV 40 large T -antigen gene (Digestion 57:141,'96), were cultured in the presence or absence of 10-30 mM NH4CI at various ambient pH (5.0, 6.0, and 7.0). Apoptosis of the GSM06 cells were evaluated by elctron microscopy, by detecting DNA ladder formation, by counting the number of cells stained with antibody of caspase-3, and -9, and by fluorometrical measurement of caspase-3 activity (Nature letter 380:723, '96). Leakage of mitochondrial cytochrome c into cytosol was examined by Western blot analysis. Results: I. In NH4CItreated cells, morphology of EM showed shrinkage of cellular nucleus, accompanied by condensation of nuclear chromation. In addition, a number of vacuoles were observed within the cytoplasm. DNA ladder formation was detected most prominently at pH 7.0. The number of the cells stained with caspase-3, or caspase-9 increased in a pH dependent manner. Activity of casapase-3 was highest at pH 7.0. Leakage of cytochrome c into cytosol was also detected at pH 7.0. 2. In the absence of NH4CI, none of these changes were demonstrated at all the ambient pHs. Conclusions: Ammonia, at concentrations detected in Hp-infected subjects, induces apoptosis of gastric epithelial cells by mitochondrial membrane destruction and by enhancing activities of caspase-3 and caspase-9 especially at high ambient pHs.
H pylori is naturally competent for transformation, which means that it can take up extracellular DNA and incorporate it in its genome. To identify genes that are essential for transformation, we screened a random mutant library of H. pylori for transformation-deficient mutants. A H. pylori strain 1061 library consisting of random mutants, created by insertion of a suicide vector, was screened for competence deficient mutants by natural transformation with chromosomal DNA of a clarithromycin resistant strain (Cla R DNA). This screen resulted in the identification of one mutant that did not give rise to Cla" transformants. In this mutant the vector had disrupted ORF HPI527 (strain 26695 genomic sequence). Site-directed mutants in HPI527 were constructed in strain 1061 and 26695 by insertion of the aphA-3 kanamycin resistance cassette. These mutants were not competent for uptake of Cla" DNA (like the original vector-insertion mutant) nor for uptake of plasmid pHEL2. To assess whether disruption of HPI527 or a polar effect of the HPI527 mutation is responsible for this phenotype, complementation of HPI527 was performed with a newly developed strategy: the rdxA-vector. This vector takes advantage from the Metronidazole (Mtz) resistance that is caused by disruption of the rdxA gene. The rdxA-vector contains a multiple cloning site for insertion of DNA fragments, flanked by two fragments of the rdxA gene. After transformation to H pylori, the cloned DNA is inserted into the rdxA gene and these mutants can easily be selected by their Mtz-resistance. A copy of HPI527 was cloned into the rdxA-vector and transformed to strain 26695. A Mtz R transformant with a second co~ of HP1527 was transformed with the 1527-knockout construct. A Mtz KmR colony with the aphA-3 insertion in the original HP1527 gene (as determined by PCR) showed a natural transformation efficiency equal to the parental strain 26695. The HP1527 complementation in trans shows that this gene is essential for transformation. Therefore, we propose the name comH (competence of Helicobacter) for gene HP1527. No homologs of this gene are present in any other sequenced bacterial species, including many transformable species, which indicates that the competence system of H. pylori has unique properties. In addition, we showed that the new rdxA-vector is a small and therefore easy transformable tool for single-copy insertion into the H pylori genome, without the use of additional resistance markers.
3981 HETEROGENEITY OF H.PYLORI CAG GENOTYPES IN EXPER· IMENTALLY INFECTED MICE. Michele Sozzi, Sung-Kook Kim, Marialuisa Crosatti, Judith Romero, Martin J. Blaser, Div of Gastroenterology and Digest Endoscopy, C R 0, Aviano, Italy; Div of Infectious Diseases, Vanderbilt Univ, Nashville, TN; Vanderbilt Univ, Nashville, TN. Background. Our hypothesis is that the H.pylori (Hp) cag island is unstable in vivo, and that passage from a human to an animal host might select for different genotypes. Aim. To assess whether the Hp population recovered from experimentally infected mice show heterogeneity in cag genotypes. Methods. FVBIN (wt) mice were orally infected by strain HP-1 (infecting strain = IS) from a Peruvian ulcer patient and 5 were sacrified 8 weeks later. Direct PCR on mouse gastric tissue was performed using primers for glmM (a marker of Hp) and cagA. From mice with discrepant results for glmM (+) and cagA (-), chromosomal DNA was extracted from 10 single colonies (SC) isolated from the mouse stomach, and from a sweep (SW) of colonies. PCR for cagA, picB (both located in the right half of cag), virBll (located in the left half of cag), and glmM was performed using DNA from the IS and the emerging strains (ES). Serial dilution PCRs of glmM and cagA were done on selected DNA templates. Results. The gastric tissues of 2 (wt 6 and wt 9) of 5 mice were PCR+ for glmM but not cagA. For the IS, the PCRs for all 4 genes studied were strongly positive, but the ES sweeps gave weaker signals for cagA and picB (Table). Examination of 10 SC from mouse 9 showed full signal for glmM and virBll, but reduced or absent signals for cagA and picB in each case. Serial dilution PCR of SW isolates showed a 10- to 100-fold decrease in cagA signal compared to the IS. RAPD-PCR confirmed the identity of all strains. Conclusions. The decrease of cagA and picB, but not virBll, amplification indicates a selective loss of the right half of the cagA island during murine infection. Although the mechanism for this phenomenon is presently unknown, it is consistent with host-induced adaptive changes of cag genotype.
3983 ASSOCIATION BETWEEN HEUCOBACTER PYLORI INFECTION AND FETAL INTRAUTERINE GROWTH RETARDATION (IUGR). P. Yan, H. Murray, B. Spurrett, H. H-X Xia, G. Eslick, N. 1. Talley, Univ of Sydney, Penrith, Australia. Aim: H. pylori infection has been associated with growth retardation in young children. We aimed to determine if there is an association between H. pylori infection and IUGR. Methods: 451 consecutive pregnant women (aged 15-44 years) attending for routine examinations in the third trimester were enrolled. They consisted of 402 Caucasians, 25 Asians, 17 Aborigines and 7 Pacific Islanders. Together with other prenatal screening blood tests, anti-H pylori IgG was tested using a validated kit, Pylori Dtect ELISA (sensitivity 96% and specificity 94%). Clinical, demographic and previous obstetric data as well as smoking history were collected. At delivery, weight, height, gender and status of the neonate were recorded; IUGR was defined if the birth weight was below the 10th percentile according to gestational age for infants born in Australia. Results: 89 (20%) women were seropositive for H. pylori. The rate was significantly lower in Caucasians (17%) than Asians (40%,p=0.OO4), Aboriginal (35%, p=0.05) and Pacific Islanders (57%, p=0.02). Seropositive women were significantly older than seronegative women (28.6 vs 26.7 years, p=O.OO4). Women with H pylori infection were significantly shorter (162 vs 165 ern, p=0.OO3), and less likely to be smokers, compared with seronegative women (13% vs 23%, p=0.02). There were 34 (7.5%) IUGR cases; the rate was not significantly different between the ethnic groups (7% Caucasians, 16% Asians, 12% Aborigines and 0% Pacific Islanders). IUGR was more common in seropositive women than seronegative women (13.5% vs 6%, OR=2.41, 95%CI 1.14-5.08, p=0.018), and in smokers than non-smokers (13.3% vs 5.3%, OR=2.8, 95%CI 1.37-5.62, p=0.004). Women with IUGR were significantly shorter than those without (160 em vs 165 cm, p=O.OOI). IUGR was not associated with neonatal gender, numbers of pregnancies or births, pre-eclampsia, hypertension or previous gastrointestinal disease. A multiple logistic regression model including H. pylori status, smoking, maternal age ~d heig~t, and ethnicity revealed .~at smoking (p=O.OOI), maternal height (p=O.005) and H pylori seroposttrvity (p=O.03) were all independent risk factors for IUGR. Conclusions: H. pylori infection in pregnant women may affect fetal intrauterine growth.
3984
Gene glmM
cagA pieS virS11
Emerging strains (ES) wt6 wt9 wt9 IS SW SW SC1 SC2 SC3 SC4 SC5 SC6 SCl +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ ++ + ++ + ++ + + + + + +++ +++ +++ +++ +++ +++
+++ ++ +++
+++ ++ + +++
sca
SC9 SC10
+++ +++ +++ ++ + ++ + + + +++ +++ +++
+++ ++ + +++
SIGNIFICANT ENHANCEMENT OF HEUCOBACTER PYLORIINDUCED GASTRITIS BY FISH MEAL IN MONGOLIAN GER· BILS. Tetsuya Tanigawa, Toshihiko Kawamori, Masaki Iimuro, Kazuhide Higuchi, Tetsuo Arakawa, Takashi Sugimura, Keiji Wakabayashi, Osaka City Univ Med Sch, Osaka, Japan; National Cancer Ctr Research Institute, Tokyo, Japan. Gastric cancer continues to be one of the most common malignancies in the world. Recent reports demonstrated the relationship between Hipylori infection and gastric carcinogenesis in Mongolian gerbils. This animal
GASTROENTEROLOGY Vol. 118, No.4
3980
3982
IDENTIFICATION OF COMH: A NOVEL, HEUCOBACTER pyLORI SPECIFIC TRANSFORMATION GENE, AND COMPLE· MENTATION OF COMH MUTANTS WITH THE NEW RDXA· VECTOR SYSTEM. Leonard C. Smeets, Jetta J. Bijlsma, Sacha Y. Boomkens, Christina Mje Vandenbroucke-Grauls, Johannes G. Kusters, Vrije Univ, Amsterdam, Netherlands.
THE MECHANISM OF GASTRIC MUCOSAL APOPTOSIS IN· DUCED BY AMMONIA. Hideo Suzuki, Akinori Yanaka, Akira Nakahara, Naomi Tanaka, Hiroshi Muto, Yasuo Uchiyama, Clin medicine, Univ of Tsukuba, Tsukuba, Ibaraki, Japan; Cell Biology and Anatomy, Osaka Univ, Suita, Osaka, Japan. Helicobacter pylori (HP) has been shown to exert as the pathogen of gastritis, peptic ulcer, and gastric cancer. HP produces high concentrations of ammonia within the gastric lumen. We have previously shown that ammonia is toxic especially at high ambient pHs. It has also been reported that prolonged acid inhibition accelerates progress of gastric atrophy in HP-infected subjects. Gastric atrophy is defined as the loss of gastric glands and associates gastric cell apoptosis, In this study, we examined the mechanism of ammonia-induced apoptosis of gastric epithelial cells at various ambient pHs. Methods : GSM06 cells, established from gastric surface mucous cells of transgenic mice harboring SV 40 large T -antigen gene (Digestion 57:141,'96), were cultured in the presence or absence of 10-30 mM NH4CI at various ambient pH (5.0, 6.0, and 7.0). Apoptosis of the GSM06 cells were evaluated by elctron microscopy, by detecting DNA ladder formation, by counting the number of cells stained with antibody of caspase-3, and -9, and by fluorometrical measurement of caspase-3 activity (Nature letter 380:723, '96). Leakage of mitochondrial cytochrome c into cytosol was examined by Western blot analysis. Results: I. In NH4CItreated cells, morphology of EM showed shrinkage of cellular nucleus, accompanied by condensation of nuclear chromation. In addition, a number of vacuoles were observed within the cytoplasm. DNA ladder formation was detected most prominently at pH 7.0. The number of the cells stained with caspase-3, or caspase-9 increased in a pH dependent manner. Activity of casapase-3 was highest at pH 7.0. Leakage of cytochrome c into cytosol was also detected at pH 7.0. 2. In the absence of NH4CI, none of these changes were demonstrated at all the ambient pHs. Conclusions: Ammonia, at concentrations detected in Hp-infected subjects, induces apoptosis of gastric epithelial cells by mitochondrial membrane destruction and by enhancing activities of caspase-3 and caspase-9 especially at high ambient pHs.
H pylori is naturally competent for transformation, which means that it can take up extracellular DNA and incorporate it in its genome. To identify genes that are essential for transformation, we screened a random mutant library of H. pylori for transformation-deficient mutants. A H. pylori strain 1061 library consisting of random mutants, created by insertion of a suicide vector, was screened for competence deficient mutants by natural transformation with chromosomal DNA of a clarithromycin resistant strain (Cla R DNA). This screen resulted in the identification of one mutant that did not give rise to Cla" transformants. In this mutant the vector had disrupted ORF HPI527 (strain 26695 genomic sequence). Site-directed mutants in HPI527 were constructed in strain 1061 and 26695 by insertion of the aphA-3 kanamycin resistance cassette. These mutants were not competent for uptake of Cla" DNA (like the original vector-insertion mutant) nor for uptake of plasmid pHEL2. To assess whether disruption of HPI527 or a polar effect of the HPI527 mutation is responsible for this phenotype, complementation of HPI527 was performed with a newly developed strategy: the rdxA-vector. This vector takes advantage from the Metronidazole (Mtz) resistance that is caused by disruption of the rdxA gene. The rdxA-vector contains a multiple cloning site for insertion of DNA fragments, flanked by two fragments of the rdxA gene. After transformation to H pylori, the cloned DNA is inserted into the rdxA gene and these mutants can easily be selected by their Mtz-resistance. A copy of HPI527 was cloned into the rdxA-vector and transformed to strain 26695. A Mtz R transformant with a second co~ of HP1527 was transformed with the 1527-knockout construct. A Mtz KmR colony with the aphA-3 insertion in the original HP1527 gene (as determined by PCR) showed a natural transformation efficiency equal to the parental strain 26695. The HP1527 complementation in trans shows that this gene is essential for transformation. Therefore, we propose the name comH (competence of Helicobacter) for gene HP1527. No homologs of this gene are present in any other sequenced bacterial species, including many transformable species, which indicates that the competence system of H. pylori has unique properties. In addition, we showed that the new rdxA-vector is a small and therefore easy transformable tool for single-copy insertion into the H pylori genome, without the use of additional resistance markers.
3981 HETEROGENEITY OF H.PYLORI CAG GENOTYPES IN EXPER· IMENTALLY INFECTED MICE. Michele Sozzi, Sung-Kook Kim, Marialuisa Crosatti, Judith Romero, Martin J. Blaser, Div of Gastroenterology and Digest Endoscopy, C R 0, Aviano, Italy; Div of Infectious Diseases, Vanderbilt Univ, Nashville, TN; Vanderbilt Univ, Nashville, TN. Background. Our hypothesis is that the H.pylori (Hp) cag island is unstable in vivo, and that passage from a human to an animal host might select for different genotypes. Aim. To assess whether the Hp population recovered from experimentally infected mice show heterogeneity in cag genotypes. Methods. FVBIN (wt) mice were orally infected by strain HP-1 (infecting strain = IS) from a Peruvian ulcer patient and 5 were sacrified 8 weeks later. Direct PCR on mouse gastric tissue was performed using primers for glmM (a marker of Hp) and cagA. From mice with discrepant results for glmM (+) and cagA (-), chromosomal DNA was extracted from 10 single colonies (SC) isolated from the mouse stomach, and from a sweep (SW) of colonies. PCR for cagA, picB (both located in the right half of cag), virBll (located in the left half of cag), and glmM was performed using DNA from the IS and the emerging strains (ES). Serial dilution PCRs of glmM and cagA were done on selected DNA templates. Results. The gastric tissues of 2 (wt 6 and wt 9) of 5 mice were PCR+ for glmM but not cagA. For the IS, the PCRs for all 4 genes studied were strongly positive, but the ES sweeps gave weaker signals for cagA and picB (Table). Examination of 10 SC from mouse 9 showed full signal for glmM and virBll, but reduced or absent signals for cagA and picB in each case. Serial dilution PCR of SW isolates showed a 10- to 100-fold decrease in cagA signal compared to the IS. RAPD-PCR confirmed the identity of all strains. Conclusions. The decrease of cagA and picB, but not virBll, amplification indicates a selective loss of the right half of the cagA island during murine infection. Although the mechanism for this phenomenon is presently unknown, it is consistent with host-induced adaptive changes of cag genotype.
3983 ASSOCIATION BETWEEN HEUCOBACTER PYLORI INFECTION AND FETAL INTRAUTERINE GROWTH RETARDATION (IUGR). P. Yan, H. Murray, B. Spurrett, H. H-X Xia, G. Eslick, N. 1. Talley, Univ of Sydney, Penrith, Australia. Aim: H. pylori infection has been associated with growth retardation in young children. We aimed to determine if there is an association between H. pylori infection and IUGR. Methods: 451 consecutive pregnant women (aged 15-44 years) attending for routine examinations in the third trimester were enrolled. They consisted of 402 Caucasians, 25 Asians, 17 Aborigines and 7 Pacific Islanders. Together with other prenatal screening blood tests, anti-H pylori IgG was tested using a validated kit, Pylori Dtect ELISA (sensitivity 96% and specificity 94%). Clinical, demographic and previous obstetric data as well as smoking history were collected. At delivery, weight, height, gender and status of the neonate were recorded; IUGR was defined if the birth weight was below the 10th percentile according to gestational age for infants born in Australia. Results: 89 (20%) women were seropositive for H. pylori. The rate was significantly lower in Caucasians (17%) than Asians (40%,p=0.OO4), Aboriginal (35%, p=0.05) and Pacific Islanders (57%, p=0.02). Seropositive women were significantly older than seronegative women (28.6 vs 26.7 years, p=O.OO4). Women with H pylori infection were significantly shorter (162 vs 165 ern, p=0.OO3), and less likely to be smokers, compared with seronegative women (13% vs 23%, p=0.02). There were 34 (7.5%) IUGR cases; the rate was not significantly different between the ethnic groups (7% Caucasians, 16% Asians, 12% Aborigines and 0% Pacific Islanders). IUGR was more common in seropositive women than seronegative women (13.5% vs 6%, OR=2.41, 95%CI 1.14-5.08, p=0.018), and in smokers than non-smokers (13.3% vs 5.3%, OR=2.8, 95%CI 1.37-5.62, p=0.004). Women with IUGR were significantly shorter than those without (160 em vs 165 cm, p=O.OOI). IUGR was not associated with neonatal gender, numbers of pregnancies or births, pre-eclampsia, hypertension or previous gastrointestinal disease. A multiple logistic regression model including H. pylori status, smoking, maternal age ~d heig~t, and ethnicity revealed .~at smoking (p=O.OOI), maternal height (p=O.005) and H pylori seroposttrvity (p=O.03) were all independent risk factors for IUGR. Conclusions: H. pylori infection in pregnant women may affect fetal intrauterine growth.
3984
Gene glmM
cagA pieS virS11
Emerging strains (ES) wt6 wt9 wt9 IS SW SW SC1 SC2 SC3 SC4 SC5 SC6 SCl +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ ++ + ++ + ++ + + + + + +++ +++ +++ +++ +++ +++
+++ ++ +++
+++ ++ + +++
sca
SC9 SC10
+++ +++ +++ ++ + ++ + + + +++ +++ +++
+++ ++ + +++
SIGNIFICANT ENHANCEMENT OF HEUCOBACTER PYLORIINDUCED GASTRITIS BY FISH MEAL IN MONGOLIAN GER· BILS. Tetsuya Tanigawa, Toshihiko Kawamori, Masaki Iimuro, Kazuhide Higuchi, Tetsuo Arakawa, Takashi Sugimura, Keiji Wakabayashi, Osaka City Univ Med Sch, Osaka, Japan; National Cancer Ctr Research Institute, Tokyo, Japan. Gastric cancer continues to be one of the most common malignancies in the world. Recent reports demonstrated the relationship between Hipylori infection and gastric carcinogenesis in Mongolian gerbils. This animal