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Expression of the C-terminal tail of DRA (down regulated in adenoma) as a recombinant antigen and to study protein-protein-interactions
A1l28 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
5199
5201
EFFECT OF ANTIOXIDATIVE AGENTS ON APOPTOSIS INDUCED BY ISCHEMIA-REPERFUSION IN RAT INTESTINAL MUCOSA. Masataka Kojima, Ryuichi Iwakiri, Yudai Goto, Bin Woo, Keiji Matsunaga, Hiroyoshi Utsumi, Sadatoshi Ishibashi, Kazuma Fuj imoto, Saga Med Sch, Saga, Japan. We reported that mucosal injury in the small intestine caused by occlusion of superior mesentric artery foilwed by reperfusion was partly associated with induction of apoptosis. This study was to examine if antioxidative agent could diminish mucosal apoptosis induced by ischemia-reperfu sion in rat intestine for evaluation of a role of oxidative stress on mucosal apoptosis. Methods: Under halothane anesthesia, male SD rats (200 3OOg) were inserted with a duoden al tube. Antioxidative agent, DMSO, rebarnipide, glutathione were infused into each rat through the duodenal tube for Ih before an ischemic insult. DMSO was administered at dose of 20mM 2oomM. Rebamipide was administered at dose of 3Ornglkg, loomglkg, dissolved in 0.5% carboxylmethyl cellulose. Glutathione was administered at a dose of 32mM. Control ischem ic animals were received with vehicles. Superior mesentric artery was occluded with a bulldog clamp for 30 min, followed by reperfusion for 60 min. Rats were sacrificed and small intestine was removed to examine apoptosis. Apoptosis was evaluated by % fragme nt DNA and immunohisto chemical stain. Results: Apoptosis in the intestinal mucosa increased in rats after ischemia-reperfusion(% fragmented DNA = 22.5 ±2.2%), compa red to the value in sham-treated rats(% fragmented DNA =2.2±0.8%). Administration of DMSO had no significant effect on the increased mucosal apoptosis at any dose tested. Infusion of glutathione did not reduce intestinal apoptosis followed by ischemiareperfusion. In contrast, rebamipide reduced % fragmented DNA induced by ischemia-reperfusion in a dose dependent manner(30mg/kg: l4 .9±2.2%, IOOmg/kg:7.4 ± 2.2%;p< 0.05). This result was confirmed by immunohistochemical study. Conclusion: DMSO, free radical scavenger, did not dim inished mucosal apoptosis caused by ischemia-reperfu sion, whreas rebamipide attenuated increasing % fragmented DNA in the intestinal mucosa after ischernia-reperfusion. suggesting that the scavenging effect of rebarnipide on htdroxyl radicals might reduce mucosal apoptosis caused by ischemia-reperfusion. Exogeno us administration of glutathione, a major cellular reductant, did not have any scavenging effect in this experiment. Organic source of glutathione might overbeat the scavenging effect of exogenous glutathione, that warrants an experiment under condition that organic glutathione is depleted. This experiment suggests possibility that increased mucosal apoptosis might be caused by hydroxyl radicals.
EXPRESSION OF THE C-TERMINAL TAIL OF DRA (DOWN REGULATED IN ADENOMA) AS A RECOMBINANT ANTIGEN AND TO STUDY PROTEIN-PROTEIN-INTERACTIONS. Georg Lamprecht, Elena Lin Wu, Michael Gregor, Ursula Seidler, Eberhard-Karls-University, Tuebingen, Tuebingen, Germany. Introduction: dra (dow n regulated in adenoma) is an intestinal anion exchanger that is involved in NaCI absorption. Mutations in the dra gene cause congenital chloride diarrhea. Other ion transporters (e.g. NHE3, the intestinal Na/Il- exchanger) interact with regulatory protein s through their cytoplasmic tail. Such interactions are conceivable for d ra as well and they may be studied in transfected cells or in vitro. Aims: I) To express the entire C-terminal tail of dra as a recombinant antigen to raise a polyclonal antiserum against dra . 2) To express the recombinant C-terminal tail as a Biotin tagged construct for future studies of protein-protein-interactions. Methods: The entire open reading frame of dra DNA was cloned from Caco-2 RNA by RT-PCR . The C-terminus beginning at aa 566 (C-dra) was expressed as a His- and S-tagged fusion protein (HS -C-dra) , purified on Nickel agarose, and used to immunize rabbits. Full-length dra and HS-Cdra were transfected into HEK293 cells to test the antiserum for sensitivity and specificity. C-dra was also expressed as an in vivo biotinylated construct (Biotin-C-dra ) using the PinPoint expression vector (Promega). Results: HS-C-dra was expressed and purified at high levels ( > 1 mg per 100 ml) and high purity (> 99% on Coomassie stain). The resulting antiserum recognizes HS-C -dra with a high titer (I :3000) in HEK293 cells. The sensitivity of the antiserum is about 10 x better than a commercially available probe against the S-tag. The antiserum can be further purified on the immobilized antigen. Despite demonstration of dra mRNA in transfected cells only the HS-C-dra construct but not the full-length dra construct was detected by western blot. The in vivo biotinylated C-d ra construct could be detected by HRP-conju gated streptavidin with comparable sensitivity to western blot. Summary and conclusions: We have raised a polyclonal antiserum against the C-terminus of dra that recognizes the antigen both in bacteria and in transfected HEK293 cells with high sensitivity. The antiserum needs further purification to increase specificity. Transfected full-length dra was not detected by the antiserum; this may be due to insufficient sensitivity of the antiserum or insufficient expression or stability of the transfected full-length protein. The HS -C-dra and the Bio tin-C-dra constructs will be tools to study interactions of dra with other proteins.
5200 DETECTION OF GASTRIC KI-M9 POSITIVE CELLS AS PUTA· TIVE ACCESSORY CELLS OF A LOCAL B-CELL STIMULA· TION. Matthias Kraft, Christian Maaser, Hans-Hubert Wacker, Susanne Riedel, Torsten Kucharzik, Reza Parwaresch, Wolfram Domschke, Markus Tiemann, Norbert Luegering , Dept of Medicine B, Muenster, Germany ; Dept of Hernatopathology , Kiel, Germany. Introduction: In low-grade MALT lymphomas occurrence of clonal expansion and somatic hypermutations of B-cells frequently can be found. Affinity maturation of B-cells by somatic hypermutation usually takes place within germinal centres. Follicular dendritic cells (FDC) are believed to function as accessory cells and to act on B-cells in shaping their immunoglobuline repertoire . Only recently, Ki-M9 positive B-cell accessory sinus lining cells have been detected in lymph nodes and were described as putative precursors of FDC, As somatic hypermutati on of B-cells in MALT lymphomas can regularly be found in mucosa devoid of germinal centers we addressed the question whether these B-cell accessory cells might also be found within gastric MALT lymphoma, We investigated 10 biopsies of chronic gastritis and MALT lymphoma by immunohistochem istry regarding the expression of Ki-M9, Methods: Ki-M9 is a monoclonal antibody (mAb) against a 70 kD protein which is selectively expressed on precursors of B-cells. The mAb was generated by immunisation of Balblc mice with fragmented human lymph nodes and subsequent transfection of myeloma cell lines. Immunohistochemistry was performed by APAAP method. Unaffected gastric mucosa was used as control tissue. Monoclonal antibodies investigated were as follows: B-cells (CD22 and CD19), T-cells (CD3, Leu 4), CD4 (OKT3) and CD8 (OKT8) as well as Ki-M 1 (cells of monocytoid lineage) and Ki-M4 (follicular dendritic cells). Results: Immunohistochemistry of low-grade MALTomas revealed Ki-M9 positive cells in the subepithelial compartment of all cases investigated. Examination of biopsy specimens of chronic gastritis showed Ki-M9 positive accessory cells in 5 of 10 cases, three of them in combination with IgM (s + ) B-cells. No co-reaction of plasma cells and monocytes was observed. Furthermore , intraepithelial Ki-M9 positive cells could not be detected. Conclusion: Ki-M9 positive cells frequently can be found within the subepithelial compartment of low-grade Malt lymphomas and in some cases of chronic gastritis with lymphatic hyperplasia. These precursor s of B-cell accessory cells, such as follicular dendritic cells, are surrounded by B-cells and morphologically point out a close interaction of these two cell populations within the subepithelial compartment. Ki-M9 positive cells of gastric mucosa may hold immunomodul ating functions by means of a Bcell accessory function by analogy with lymph nodes. The functional role of these cells remains to be investigated yet.
5202 OSTEOPENIA IN CELIAC DISEASE: DOES LACTOSE INTORANCE PLAYA MAJOR ROLE'! Francesco Lanzarotto, Francesco Crimi' , Vincenzo Villanacci, Mauro Panteghini, Franca Pagani, Alberto Lanzini, Dept Medicine, Brescia, Italy; Pathology, Brescia, lIaly; Clin Chemistry, Brescia, Italy. Background: osteopenia is a common condition in adult untreated celiac patients, and it may theoretically be aggravated by reduced dietary calcium intake as for patients with concomitant lactose intolerance. Aims of this study were to test the hypothesis that lactose intolerance may worsen osteopenia in adult celiac patients, and to assess the effect of gluten free diet on bone mineralization and on biochemical parameters of bone remodelling. Patients and Methods: we studied a total 97 celiac patients, 36 pre-gluten-free diet (MIF ratio 13/23 , mean age 36.9 ± 2.1 years, mean ± ES) and 61 on-diet for at least 1 year (MIF ratio 13/48, mean age 36. 1 ± 1.4 years). We measured bone mineral density (expressed as «Z" score) using dual photon X-ray absorptiometry on lumbar spine in addition to serum parameters of bone remodelling (deoxypyridinoline, osteocalcin and bone-ALP). Lactose tolerance was assessed using the H-breath test in 65 patients. Results: 23/36 pre-diet patients were osteopenic «
5203 ROLE OF NEUTROPHILS IN THE INCREASED SENSITIVITY TO OXIDATIVE STRESS IN DIABETES. Steve Lawson, David T. Ward, Scott T. Rehrig, Sherry D. Fleming, Daniel Mulloy, Rober t Hutton, Terez Shea-Donohue , Usuhs, Bethesda , MD; Usuhs, Bethesda, DC. We showed previously that diabetic rats exhibit a increased local and systemic injury to normally non-injurious periods of mesenteric ischemia Aim: To determine the mechanism of diabetes-induced sensitivity to in-
GASTROENTEROLOGY Vol. 118, No.4
5199
5201
EFFECT OF ANTIOXIDATIVE AGENTS ON APOPTOSIS INDUCED BY ISCHEMIA-REPERFUSION IN RAT INTESTINAL MUCOSA. Masataka Kojima, Ryuichi Iwakiri, Yudai Goto, Bin Woo, Keiji Matsunaga, Hiroyoshi Utsumi, Sadatoshi Ishibashi, Kazuma Fuj imoto, Saga Med Sch, Saga, Japan. We reported that mucosal injury in the small intestine caused by occlusion of superior mesentric artery foilwed by reperfusion was partly associated with induction of apoptosis. This study was to examine if antioxidative agent could diminish mucosal apoptosis induced by ischemia-reperfu sion in rat intestine for evaluation of a role of oxidative stress on mucosal apoptosis. Methods: Under halothane anesthesia, male SD rats (200 3OOg) were inserted with a duoden al tube. Antioxidative agent, DMSO, rebarnipide, glutathione were infused into each rat through the duodenal tube for Ih before an ischemic insult. DMSO was administered at dose of 20mM 2oomM. Rebamipide was administered at dose of 3Ornglkg, loomglkg, dissolved in 0.5% carboxylmethyl cellulose. Glutathione was administered at a dose of 32mM. Control ischem ic animals were received with vehicles. Superior mesentric artery was occluded with a bulldog clamp for 30 min, followed by reperfusion for 60 min. Rats were sacrificed and small intestine was removed to examine apoptosis. Apoptosis was evaluated by % fragme nt DNA and immunohisto chemical stain. Results: Apoptosis in the intestinal mucosa increased in rats after ischemia-reperfusion(% fragmented DNA = 22.5 ±2.2%), compa red to the value in sham-treated rats(% fragmented DNA =2.2±0.8%). Administration of DMSO had no significant effect on the increased mucosal apoptosis at any dose tested. Infusion of glutathione did not reduce intestinal apoptosis followed by ischemiareperfusion. In contrast, rebamipide reduced % fragmented DNA induced by ischemia-reperfusion in a dose dependent manner(30mg/kg: l4 .9±2.2%, IOOmg/kg:7.4 ± 2.2%;p< 0.05). This result was confirmed by immunohistochemical study. Conclusion: DMSO, free radical scavenger, did not dim inished mucosal apoptosis caused by ischemia-reperfu sion, whreas rebamipide attenuated increasing % fragmented DNA in the intestinal mucosa after ischernia-reperfusion. suggesting that the scavenging effect of rebarnipide on htdroxyl radicals might reduce mucosal apoptosis caused by ischemia-reperfusion. Exogeno us administration of glutathione, a major cellular reductant, did not have any scavenging effect in this experiment. Organic source of glutathione might overbeat the scavenging effect of exogenous glutathione, that warrants an experiment under condition that organic glutathione is depleted. This experiment suggests possibility that increased mucosal apoptosis might be caused by hydroxyl radicals.
EXPRESSION OF THE C-TERMINAL TAIL OF DRA (DOWN REGULATED IN ADENOMA) AS A RECOMBINANT ANTIGEN AND TO STUDY PROTEIN-PROTEIN-INTERACTIONS. Georg Lamprecht, Elena Lin Wu, Michael Gregor, Ursula Seidler, Eberhard-Karls-University, Tuebingen, Tuebingen, Germany. Introduction: dra (dow n regulated in adenoma) is an intestinal anion exchanger that is involved in NaCI absorption. Mutations in the dra gene cause congenital chloride diarrhea. Other ion transporters (e.g. NHE3, the intestinal Na/Il- exchanger) interact with regulatory protein s through their cytoplasmic tail. Such interactions are conceivable for d ra as well and they may be studied in transfected cells or in vitro. Aims: I) To express the entire C-terminal tail of dra as a recombinant antigen to raise a polyclonal antiserum against dra . 2) To express the recombinant C-terminal tail as a Biotin tagged construct for future studies of protein-protein-interactions. Methods: The entire open reading frame of dra DNA was cloned from Caco-2 RNA by RT-PCR . The C-terminus beginning at aa 566 (C-dra) was expressed as a His- and S-tagged fusion protein (HS -C-dra) , purified on Nickel agarose, and used to immunize rabbits. Full-length dra and HS-Cdra were transfected into HEK293 cells to test the antiserum for sensitivity and specificity. C-dra was also expressed as an in vivo biotinylated construct (Biotin-C-dra ) using the PinPoint expression vector (Promega). Results: HS-C-dra was expressed and purified at high levels ( > 1 mg per 100 ml) and high purity (> 99% on Coomassie stain). The resulting antiserum recognizes HS-C -dra with a high titer (I :3000) in HEK293 cells. The sensitivity of the antiserum is about 10 x better than a commercially available probe against the S-tag. The antiserum can be further purified on the immobilized antigen. Despite demonstration of dra mRNA in transfected cells only the HS-C-dra construct but not the full-length dra construct was detected by western blot. The in vivo biotinylated C-d ra construct could be detected by HRP-conju gated streptavidin with comparable sensitivity to western blot. Summary and conclusions: We have raised a polyclonal antiserum against the C-terminus of dra that recognizes the antigen both in bacteria and in transfected HEK293 cells with high sensitivity. The antiserum needs further purification to increase specificity. Transfected full-length dra was not detected by the antiserum; this may be due to insufficient sensitivity of the antiserum or insufficient expression or stability of the transfected full-length protein. The HS -C-dra and the Bio tin-C-dra constructs will be tools to study interactions of dra with other proteins.
5200 DETECTION OF GASTRIC KI-M9 POSITIVE CELLS AS PUTA· TIVE ACCESSORY CELLS OF A LOCAL B-CELL STIMULA· TION. Matthias Kraft, Christian Maaser, Hans-Hubert Wacker, Susanne Riedel, Torsten Kucharzik, Reza Parwaresch, Wolfram Domschke, Markus Tiemann, Norbert Luegering , Dept of Medicine B, Muenster, Germany ; Dept of Hernatopathology , Kiel, Germany. Introduction: In low-grade MALT lymphomas occurrence of clonal expansion and somatic hypermutations of B-cells frequently can be found. Affinity maturation of B-cells by somatic hypermutation usually takes place within germinal centres. Follicular dendritic cells (FDC) are believed to function as accessory cells and to act on B-cells in shaping their immunoglobuline repertoire . Only recently, Ki-M9 positive B-cell accessory sinus lining cells have been detected in lymph nodes and were described as putative precursors of FDC, As somatic hypermutati on of B-cells in MALT lymphomas can regularly be found in mucosa devoid of germinal centers we addressed the question whether these B-cell accessory cells might also be found within gastric MALT lymphoma, We investigated 10 biopsies of chronic gastritis and MALT lymphoma by immunohistochem istry regarding the expression of Ki-M9, Methods: Ki-M9 is a monoclonal antibody (mAb) against a 70 kD protein which is selectively expressed on precursors of B-cells. The mAb was generated by immunisation of Balblc mice with fragmented human lymph nodes and subsequent transfection of myeloma cell lines. Immunohistochemistry was performed by APAAP method. Unaffected gastric mucosa was used as control tissue. Monoclonal antibodies investigated were as follows: B-cells (CD22 and CD19), T-cells (CD3, Leu 4), CD4 (OKT3) and CD8 (OKT8) as well as Ki-M 1 (cells of monocytoid lineage) and Ki-M4 (follicular dendritic cells). Results: Immunohistochemistry of low-grade MALTomas revealed Ki-M9 positive cells in the subepithelial compartment of all cases investigated. Examination of biopsy specimens of chronic gastritis showed Ki-M9 positive accessory cells in 5 of 10 cases, three of them in combination with IgM (s + ) B-cells. No co-reaction of plasma cells and monocytes was observed. Furthermore , intraepithelial Ki-M9 positive cells could not be detected. Conclusion: Ki-M9 positive cells frequently can be found within the subepithelial compartment of low-grade Malt lymphomas and in some cases of chronic gastritis with lymphatic hyperplasia. These precursor s of B-cell accessory cells, such as follicular dendritic cells, are surrounded by B-cells and morphologically point out a close interaction of these two cell populations within the subepithelial compartment. Ki-M9 positive cells of gastric mucosa may hold immunomodul ating functions by means of a Bcell accessory function by analogy with lymph nodes. The functional role of these cells remains to be investigated yet.
5202 OSTEOPENIA IN CELIAC DISEASE: DOES LACTOSE INTORANCE PLAYA MAJOR ROLE'! Francesco Lanzarotto, Francesco Crimi' , Vincenzo Villanacci, Mauro Panteghini, Franca Pagani, Alberto Lanzini, Dept Medicine, Brescia, Italy; Pathology, Brescia, lIaly; Clin Chemistry, Brescia, Italy. Background: osteopenia is a common condition in adult untreated celiac patients, and it may theoretically be aggravated by reduced dietary calcium intake as for patients with concomitant lactose intolerance. Aims of this study were to test the hypothesis that lactose intolerance may worsen osteopenia in adult celiac patients, and to assess the effect of gluten free diet on bone mineralization and on biochemical parameters of bone remodelling. Patients and Methods: we studied a total 97 celiac patients, 36 pre-gluten-free diet (MIF ratio 13/23 , mean age 36.9 ± 2.1 years, mean ± ES) and 61 on-diet for at least 1 year (MIF ratio 13/48, mean age 36. 1 ± 1.4 years). We measured bone mineral density (expressed as «Z" score) using dual photon X-ray absorptiometry on lumbar spine in addition to serum parameters of bone remodelling (deoxypyridinoline, osteocalcin and bone-ALP). Lactose tolerance was assessed using the H-breath test in 65 patients. Results: 23/36 pre-diet patients were osteopenic «
5203 ROLE OF NEUTROPHILS IN THE INCREASED SENSITIVITY TO OXIDATIVE STRESS IN DIABETES. Steve Lawson, David T. Ward, Scott T. Rehrig, Sherry D. Fleming, Daniel Mulloy, Rober t Hutton, Terez Shea-Donohue , Usuhs, Bethesda , MD; Usuhs, Bethesda, DC. We showed previously that diabetic rats exhibit a increased local and systemic injury to normally non-injurious periods of mesenteric ischemia Aim: To determine the mechanism of diabetes-induced sensitivity to in-