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Amplification of urease gene cluster of Helicobacter pylori by long-PCR
April 1995
• ACTIVATION OF PARIETAL CELL CALPAIN DISRUPTS ACTIN-BASED CYTOSKELETON AND PLASMA MEMBRANES. S. Ogihara, X. Yao, J. G. Forte, Dept. of Molecular & Cell Biology, Univ. of California, Berkeley, CA Stimulation of parietal cells by carbachol involves dynamic remodeling of actin-based cytoskeleton and elevation of intracellular Ca 2+ concentration ([Ca2+]i). Our previous work established an interaction between parietal cell calpain (Ca2+-dependent protease) and ezrin (a membrane-cytoskeleton linker), i.e., rapid hydrolysis of ezrin when [Ca2+]i was elevated (Am. J. Physiol. 265:C36, 1993), and cytochemical data suggested that the state of actin polymerization was also changed. The present experiments were performed to specifically evaluate and quantitate changes in monomeric(G)-actin and filamentous (F)-actin when [Ca~+]i was elevated with the ionophore ionomycin. Rabbit gastric glands were incubated in miniman essential medium and treated with 2 ~tM ionomycio for 6 rain, sufficient to raise [Ca2+]i to ~1 ~M and hydrolyze 20-30% of parietal cell ezrin. Incubation was with or without pretteatment with calpaio inhibitor I (CPI, 50 0.g/ml), followed by centrifugation to separate glands from medium. Integrity of plasma membranes after ionomycin-t~atment was evaluated by measuring protein, LDH, and actin released into the medium. The packed glands were treated with 0.1% NP-40 to extract G-actin and loosely associated F-actin. Amount of G- and F-actin in the NP-40 extract and NP-40 pellet were measured by the DNase I-inhibition assay. Results as follows: Extracellular medium NP-40 Extract NP-40 Pellet Protein LDH G+Factin G-actin F-acfin G-actin F-actin Control 0.4-+0 7.7_+0.6 2.3_+0.2' 16+1.5 14+_2.6 <0.1 70-+1.3 Ionomycin 12-1-0.1 34-+2.0 2.9i0.3 25-+3.2 32+--5.7 <0.I 44_+4.3 Ionomycin+CPI 0.4_+0 19+_2.5 2.4_+0.4 17+2.0 14+_2.1 <0.1 69_+1.8 Values represent mean -+sefor % of total amount of protein, LDH, or actin (n=6) Elevation of [Ca2+]i led to substantial decrease in cytostmcmral actin: decreased F-actin in the NP-40 pellet; a corresponding increase in actin content of the NP40 extract, both in G-acfin and loosely associated P-acfin; and a loss of protein from glandular cells. We also found increased loss of a 120 kDa cytoplasmic protein specific for parietal cells, but relatively little actin was lost from the cells. All observed changes, including ezrin hydrolysis, change in the state of actiu and release of protein, were prevented or attenuated when CPI was included along with ionomvcin, whereas CPI did not inhibit Ca2+ antry to the cytosol. These data demonstrate both fragmentation and depolymerization of cytostructural Factin, as well as loss of cytosolic protein, in response to sustained [Ca2+]i increase. Furthermore, the data suggest that the effects are primarily due to activation of calpaln. Currently we suspect that G-actin may be sequestered by an actin binding protein which might play some role in regulating actin cytoskeleton remodeling.
GASTRIC MUCOSAL DAMAGE WITH CHANGES IN MUCIN INDUCED BY HISTAMINE IN RATS. ~gsumu OHARA. Tomoe WATANABE*, Takafumi ICHIKAWA, and Kyoko HOTTA. Depts. of Biochem. and *Int. Med., Kitasato Univ., Sch. of Med., Sagamihara 228, JAPAN Intrinsic factors must induce acute and chronic gastric damage. However, they have not been identified but it has Deen reported that histamine inauced gastric mucosal damage in animals. The aim OTour stuay is to ctadTy whether gastric mucin cha.ng.es quantitative!y a.nd qua!itatively with. gastric mucosal injury oy nismmme. M e t n o c l s : M a l e wistar ram Tasted for 24 hours intrap..eritoneally received histamine (80.mg/kg). The rats were sacndcea at 0.5, 1 3 4 5 and 7 hours rarer injection. The control group was Qiven. venic!e aria sacrificed. 3 hours after injection. The mucosai surmce pH was measuraa with pH testpaper, and mucosal damage was macroscopically a s s e s s e d . . The .corpus region was exciseo aria mucin was extractea ano puririea as previously described1)... Qualitative changes ot mucin were detected using enzyme-linKed lectin assay (ELLA). Results: Mucosal surface pH drol0ped to 2.6 at 0.5 hour and remained at this value for one hour aft(~r histamine injection, within tour hours it regained the value of the control state (pH - 4.3). Hemorrhagic erosmns and linear ulcers became apparent rater 0.5 hour and the most striking changes were noted one hour after histamine injection. A gradual return to control conditions was Rater observed. Fluctuation in mucin content and binding activity to some lectins was correlated to gastric damage. A ~:lecrease in mucin content was ~qreatest one hour after histamine injection but recovery to the original level.occurred,within 3 hours~ The relative amounts of fucose and galactose aiminisneu and the average oligosaccharide chain length had decreased to 1 t sugars at 0.5 hour and one hour after histamine injection, out resumption of the control level (13-14 sugars) was noted by 3 hours. Based on ELLA relative amounts of T-antigen and lactosamine structure in the mucin increased and the WGA binding stru .cture of the mucin decreased aner histamine injection and these cnanges continued for 4-5 hours after injection. On th.e other hand, the Tn antigen and the UEA-I binding structure of the mucin revealed no changes at all due to histamine injection. Conclusions: One injection of 80 mg/K~qhistamine caused the secret.ion .of HCI to retain a low pH ror one hour. Histamine also inouceu a oecrease in gastric mucin content and modification of oligosaccharide chains. The increase in HCI and the change in mucm induced by histamine may be the cause of astric damage. Ref. 1) O~ara S., et al. Comp. Biochem. Physiol. 1993, 106B. 147
Esophageal, Gastric, and Duodenal Disorders A181
AMPLIFICATION HELICOBACTER
OF UREASE GENE CLUSTER O F PYLORI BY LONG-PCR. K.Ogura,
T.Shimada, S.Ota, M.Takahashi, E.Hamada, M.Matsumura, and M.Omata. Second Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan. Helicobacter pylori (H. pylori) is c l o s e l y related to the pathogenesis of gastroduodenal diseases. H. pylori produces a potent urease which is considered to be one of the most important factors for H. pylori pathogenicity. Urease gene cluster of H. pylori consists of ureC, ureD, ureA, ureB, ureI, ureE, ureF, ureG and ureH genes. It is reported that not only the structural genes but also other genes are necessary for the u r e a s e expression. For the genetic analysis among different strains, we tried to amplify almost whole range of urease genes (8.6 kbp) by Iong-PCR method. M E T H O D S : DNA was isolated from six strains of H. pylori (ATCC43526 and 5 clinical isolates). Several pairs of PCR-primers were designed and tested for the long-PeR, and we finally used t h e following pair: 5'-AAAACGCCCCAAAACTAAGCGAGACG-3' (located on ureC) and 5'-GATTTCACTCACGGCTCCATCCACTC-3' (located on ureH) (product length 8.6 kbp). The PCR-reaction was carried out for 40 cycles (93°C, 1 min; 60°C, 1 min; 72°C, 10 min), and Taq Extender PCR Additive (Stratagene) was used to enhance amplification. PCR products were digested with restriction enzymes (EcoR 1, EcoR V and Xho 1) to see RFLPS. R E S U L T S : PCR products of expected size were obtained from all strains e x a m i n e d . EcoR 1 and Xho 1 failed to digest PCR products, while EcoR V digestion showed different RFLPs among 6 strains. DISCUSSION: Long-PeR method will be useful to analyze the structure and diversity of urease gene cluster in relation to H. pylori pathogenicity, and this method may be applicable to other important genes of H. pylori.
• CHANGES OF SERUM PEPSINOGEN AND GASTRIN IN HELICOBACTER PYLORI POSITIVE GASTRIC ULCER AFTER CLEARANCE OF THE INFECTION T.Ohkusa, K.Fujiki, I.Takashimizu, S.Tokoi, S.Endo, A.Araki, K.Honda, K.Shimoi. First Department of Internal Medicine,Tokyo Medical and Dental University School of Medicine, Tokyo 113, Japan AIM: Although changes of serum pepsinogen and gastrin in Helicobacter pylori (HP) positive gastritis and duodenal ulcer by eradication have been reported by a few studies, there are no reports on the changes in HP positive gastric ulcer. In this study, we have investigated the effect of therapy for HP positive gastric ulcer on serum pepsinogen (PG) I and II, and gastrin. METHODS: Thirty patients (age 59,14, M/F:23/7) with HP positive gastric ulcer were treated by lansoprazole and antibiotics (AMPC,clarithromycin, or roxithromycin) for 2 weeks. Serum PG I and II concentrations (DINABOT), serum gastrin (DINABOT), and IgG titres to HP (RADIUM) were measured before the treatment, one month, and 3 months after stopping the treatment. The presence and clearance of HP were determined by rapid urease test and histological HP staining. Statistical analysis were evaluated by paired t test and ANOVA. RESULTS: In 15 patients (age 58,13, M/F:12/3) of in whom HP was successfully cleared, the PG I/II ratio was significantly increased (p<0.001) and the anti-HP IgG was significantly decreased (p<0.01) when compared between before, one month and 3 months after the treatment. The PGII was significantly decreased when compared between before, one month and 3 months after the treatment (p<0.05). There were no significant changes in the gastrin. In 15 patients (age 58e15, M/F:11/4) in whom the therapy failed to clear the infection, there were no significant changes in the PG I/II ratio, PGI, PGII, anti-HP IgG and gastrin. CONCLUSION: An increase in the PGI~PGII ratio could be used as a predictor for the clearance of HP infection in gastric ulcer as well as the serum IgG antibody to HP.
• ACTIVATION OF PARIETAL CELL CALPAIN DISRUPTS ACTIN-BASED CYTOSKELETON AND PLASMA MEMBRANES. S. Ogihara, X. Yao, J. G. Forte, Dept. of Molecular & Cell Biology, Univ. of California, Berkeley, CA Stimulation of parietal cells by carbachol involves dynamic remodeling of actin-based cytoskeleton and elevation of intracellular Ca 2+ concentration ([Ca2+]i). Our previous work established an interaction between parietal cell calpain (Ca2+-dependent protease) and ezrin (a membrane-cytoskeleton linker), i.e., rapid hydrolysis of ezrin when [Ca2+]i was elevated (Am. J. Physiol. 265:C36, 1993), and cytochemical data suggested that the state of actin polymerization was also changed. The present experiments were performed to specifically evaluate and quantitate changes in monomeric(G)-actin and filamentous (F)-actin when [Ca~+]i was elevated with the ionophore ionomycin. Rabbit gastric glands were incubated in miniman essential medium and treated with 2 ~tM ionomycio for 6 rain, sufficient to raise [Ca2+]i to ~1 ~M and hydrolyze 20-30% of parietal cell ezrin. Incubation was with or without pretteatment with calpaio inhibitor I (CPI, 50 0.g/ml), followed by centrifugation to separate glands from medium. Integrity of plasma membranes after ionomycin-t~atment was evaluated by measuring protein, LDH, and actin released into the medium. The packed glands were treated with 0.1% NP-40 to extract G-actin and loosely associated F-actin. Amount of G- and F-actin in the NP-40 extract and NP-40 pellet were measured by the DNase I-inhibition assay. Results as follows: Extracellular medium NP-40 Extract NP-40 Pellet Protein LDH G+Factin G-actin F-acfin G-actin F-actin Control 0.4-+0 7.7_+0.6 2.3_+0.2' 16+1.5 14+_2.6 <0.1 70-+1.3 Ionomycin 12-1-0.1 34-+2.0 2.9i0.3 25-+3.2 32+--5.7 <0.I 44_+4.3 Ionomycin+CPI 0.4_+0 19+_2.5 2.4_+0.4 17+2.0 14+_2.1 <0.1 69_+1.8 Values represent mean -+sefor % of total amount of protein, LDH, or actin (n=6) Elevation of [Ca2+]i led to substantial decrease in cytostmcmral actin: decreased F-actin in the NP-40 pellet; a corresponding increase in actin content of the NP40 extract, both in G-acfin and loosely associated P-acfin; and a loss of protein from glandular cells. We also found increased loss of a 120 kDa cytoplasmic protein specific for parietal cells, but relatively little actin was lost from the cells. All observed changes, including ezrin hydrolysis, change in the state of actiu and release of protein, were prevented or attenuated when CPI was included along with ionomvcin, whereas CPI did not inhibit Ca2+ antry to the cytosol. These data demonstrate both fragmentation and depolymerization of cytostructural Factin, as well as loss of cytosolic protein, in response to sustained [Ca2+]i increase. Furthermore, the data suggest that the effects are primarily due to activation of calpaln. Currently we suspect that G-actin may be sequestered by an actin binding protein which might play some role in regulating actin cytoskeleton remodeling.
GASTRIC MUCOSAL DAMAGE WITH CHANGES IN MUCIN INDUCED BY HISTAMINE IN RATS. ~gsumu OHARA. Tomoe WATANABE*, Takafumi ICHIKAWA, and Kyoko HOTTA. Depts. of Biochem. and *Int. Med., Kitasato Univ., Sch. of Med., Sagamihara 228, JAPAN Intrinsic factors must induce acute and chronic gastric damage. However, they have not been identified but it has Deen reported that histamine inauced gastric mucosal damage in animals. The aim OTour stuay is to ctadTy whether gastric mucin cha.ng.es quantitative!y a.nd qua!itatively with. gastric mucosal injury oy nismmme. M e t n o c l s : M a l e wistar ram Tasted for 24 hours intrap..eritoneally received histamine (80.mg/kg). The rats were sacndcea at 0.5, 1 3 4 5 and 7 hours rarer injection. The control group was Qiven. venic!e aria sacrificed. 3 hours after injection. The mucosai surmce pH was measuraa with pH testpaper, and mucosal damage was macroscopically a s s e s s e d . . The .corpus region was exciseo aria mucin was extractea ano puririea as previously described1)... Qualitative changes ot mucin were detected using enzyme-linKed lectin assay (ELLA). Results: Mucosal surface pH drol0ped to 2.6 at 0.5 hour and remained at this value for one hour aft(~r histamine injection, within tour hours it regained the value of the control state (pH - 4.3). Hemorrhagic erosmns and linear ulcers became apparent rater 0.5 hour and the most striking changes were noted one hour after histamine injection. A gradual return to control conditions was Rater observed. Fluctuation in mucin content and binding activity to some lectins was correlated to gastric damage. A ~:lecrease in mucin content was ~qreatest one hour after histamine injection but recovery to the original level.occurred,within 3 hours~ The relative amounts of fucose and galactose aiminisneu and the average oligosaccharide chain length had decreased to 1 t sugars at 0.5 hour and one hour after histamine injection, out resumption of the control level (13-14 sugars) was noted by 3 hours. Based on ELLA relative amounts of T-antigen and lactosamine structure in the mucin increased and the WGA binding stru .cture of the mucin decreased aner histamine injection and these cnanges continued for 4-5 hours after injection. On th.e other hand, the Tn antigen and the UEA-I binding structure of the mucin revealed no changes at all due to histamine injection. Conclusions: One injection of 80 mg/K~qhistamine caused the secret.ion .of HCI to retain a low pH ror one hour. Histamine also inouceu a oecrease in gastric mucin content and modification of oligosaccharide chains. The increase in HCI and the change in mucm induced by histamine may be the cause of astric damage. Ref. 1) O~ara S., et al. Comp. Biochem. Physiol. 1993, 106B. 147
Esophageal, Gastric, and Duodenal Disorders A181
AMPLIFICATION HELICOBACTER
OF UREASE GENE CLUSTER O F PYLORI BY LONG-PCR. K.Ogura,
T.Shimada, S.Ota, M.Takahashi, E.Hamada, M.Matsumura, and M.Omata. Second Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan. Helicobacter pylori (H. pylori) is c l o s e l y related to the pathogenesis of gastroduodenal diseases. H. pylori produces a potent urease which is considered to be one of the most important factors for H. pylori pathogenicity. Urease gene cluster of H. pylori consists of ureC, ureD, ureA, ureB, ureI, ureE, ureF, ureG and ureH genes. It is reported that not only the structural genes but also other genes are necessary for the u r e a s e expression. For the genetic analysis among different strains, we tried to amplify almost whole range of urease genes (8.6 kbp) by Iong-PCR method. M E T H O D S : DNA was isolated from six strains of H. pylori (ATCC43526 and 5 clinical isolates). Several pairs of PCR-primers were designed and tested for the long-PeR, and we finally used t h e following pair: 5'-AAAACGCCCCAAAACTAAGCGAGACG-3' (located on ureC) and 5'-GATTTCACTCACGGCTCCATCCACTC-3' (located on ureH) (product length 8.6 kbp). The PCR-reaction was carried out for 40 cycles (93°C, 1 min; 60°C, 1 min; 72°C, 10 min), and Taq Extender PCR Additive (Stratagene) was used to enhance amplification. PCR products were digested with restriction enzymes (EcoR 1, EcoR V and Xho 1) to see RFLPS. R E S U L T S : PCR products of expected size were obtained from all strains e x a m i n e d . EcoR 1 and Xho 1 failed to digest PCR products, while EcoR V digestion showed different RFLPs among 6 strains. DISCUSSION: Long-PeR method will be useful to analyze the structure and diversity of urease gene cluster in relation to H. pylori pathogenicity, and this method may be applicable to other important genes of H. pylori.
• CHANGES OF SERUM PEPSINOGEN AND GASTRIN IN HELICOBACTER PYLORI POSITIVE GASTRIC ULCER AFTER CLEARANCE OF THE INFECTION T.Ohkusa, K.Fujiki, I.Takashimizu, S.Tokoi, S.Endo, A.Araki, K.Honda, K.Shimoi. First Department of Internal Medicine,Tokyo Medical and Dental University School of Medicine, Tokyo 113, Japan AIM: Although changes of serum pepsinogen and gastrin in Helicobacter pylori (HP) positive gastritis and duodenal ulcer by eradication have been reported by a few studies, there are no reports on the changes in HP positive gastric ulcer. In this study, we have investigated the effect of therapy for HP positive gastric ulcer on serum pepsinogen (PG) I and II, and gastrin. METHODS: Thirty patients (age 59,14, M/F:23/7) with HP positive gastric ulcer were treated by lansoprazole and antibiotics (AMPC,clarithromycin, or roxithromycin) for 2 weeks. Serum PG I and II concentrations (DINABOT), serum gastrin (DINABOT), and IgG titres to HP (RADIUM) were measured before the treatment, one month, and 3 months after stopping the treatment. The presence and clearance of HP were determined by rapid urease test and histological HP staining. Statistical analysis were evaluated by paired t test and ANOVA. RESULTS: In 15 patients (age 58,13, M/F:12/3) of in whom HP was successfully cleared, the PG I/II ratio was significantly increased (p<0.001) and the anti-HP IgG was significantly decreased (p<0.01) when compared between before, one month and 3 months after the treatment. The PGII was significantly decreased when compared between before, one month and 3 months after the treatment (p<0.05). There were no significant changes in the gastrin. In 15 patients (age 58e15, M/F:11/4) in whom the therapy failed to clear the infection, there were no significant changes in the PG I/II ratio, PGI, PGII, anti-HP IgG and gastrin. CONCLUSION: An increase in the PGI~PGII ratio could be used as a predictor for the clearance of HP infection in gastric ulcer as well as the serum IgG antibody to HP.