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villus axis and modulation of expression in response to polyamines
GASTROENTEROLOGYVol. 114, No. 4
A880 AGAABSTRACTS G3606
NEW INSIGHT ON PEG TUBE PLACEMENT IN NEUROLOGICALLY IMPAIRED PATIENTS. CA Garcia-Bueno, TM Rossi, A Tjota, CA Camacho, M Yuwono. Department of Pediatrics, Division of Gastroenterology and Nutrition, State University of New York at Buffalo, School of Medicine & Biomedical Sciences, Buffalo, New York. Nutritional rehabilitation in neurologically disabled children has been improved with the use of gastrostomy feeds, but unfortunately some of these patients develop complications or worsening of previous symptoms. The aim of the present study is to review the complications related to GER in PEG placement in children with discrete/focal neurologically impairment vs those with severe neurologic impairment. Methods: 27 neurologically impaired patients underwent PEG placement for nutritional support. Studies (Scintiscan, pH probe or UGI) which were done prior to the PEG placement were reviewed. Results: A total of 27 patients who had PEG placement. 54% developed problems related to GER. The patients with focal involvement (3/27) had marked clinical improvement after PEG placement and had no complications related to GER. In patients with severe impairment, 54% had subsequent problems with GER and/or aspiration while 42% had documented clinical improvement. One patient (4%) was lost to follow-up. Of the 54% with further problems 29% needed Nissen Fundoplication, while the other 25% did not. In addition, of those neurologically handicapped patients with problems after PEG placement, only 53% had a preceding abnormal study. Conclusion: In our experience 54% of profound neurologically disabled patients presented with postoperative complications of GER after PEG placement, and 29% of these underwent Nissen Fundoplication. This data does not support "protective anti-refiux procedure" in all patients. However, certain groups of neurologic impaired patients, particularly those with global impairment should be considered for a more invasive procedure rather than only a feeding gastrostomy. • G3607 TRANSACTIVATION BY THE GUT-ENRICHED KR~IPPEL-LIKE FACTOR (GKLF) DEPENDS ON ACIDIC AMINO ACID RESIDUES AND ON PROTEIN-PROTEIN INTERACTION. DE Geiman, H Ton-That, JM Shields, and VW Yang. Dept. of Med., The Johns Hopkins Univ. Sch. of Med., Baltimore, MD. Backeround: GKLF is a recently identified transcription factor containing 3 C2H2 zinc fingers with homology to the Drosophila Krtippel protein [Shields, Christy and Yang (1996) J. Biol. Chem. 271, 20009; Shields and Yang (1997) J. Biol. Chem. 272, 18504; Shields and Yang (1998) Nucl. Acids Res., in press]. Expression of GKLF is enriched in the gut epithelium and is associated with growth arrest. Recent studies suggest that the N terminal 109 aa residues of GKLF contain a transactivation domain capable of activating gene expression [Ton-That et al. (1997) Gastroenterology 112, A911]. Aim: The aim of the present study is to define the aa residues required for GKLF to transactivate gene expression and to determine whether the ability of GKLF to transactivate depends on protein-protein interaction. Methods: The N-terminal 109 aa residues of GKLF was fused to the DNA-binding domain of the yeast transcription factor Gal4. The chimeric Gal4-GKLF(1-109) protein was examined for its ability to transactivate a luciferase reporter gene driven by 5 tandem copies of the Gal4-binding sequence linked to either a minimal promoter (G5Elb-LUc) or the herpes virus thymidine kinase promoter (G5TK-Luc) in transiently transfected Chinese hamster ovary (CHO) cells. A mutated Ga14-GKLF(1-109[E93/94/96V]) construct was generated by sitedirected mutageneais in which valine residues were substituted for three clustered glutamate residues between aa 93-96 of GKLF and analyzed as above. Protein-protein interaction was examined by cotransfection experiments involving Ga14-GKLF(1-109), a luciferase reporter, and increasing amounts of an expression construct containing GKLF, the adenovirus EIA protein, or the transcription co-activator p300/CBP. Results: In transfected CHO cells Ga14-GKLF(1-109) increased the reporter activity of G5Elb-Luc and G5TK-Luc by 10- and 30-fold, respectively. In contrast, the Ga14-GKLF(1-109[E93/94/96V]) mutant construct failed to activate either reporter gene. The activation of the two reporters by Gal4-GKLF (1-109) was inhibited by the presence of an expression construct containing full-length GKLF in a dose-dependent fashion. This abrogation of transactivation was also observed with a construct containing truncated GKLF that retained the Nterminal but not the C-terminal portion of GKLF. An expression construct containing the wild-type adenovirus E1A also inhibited transactivation by Ga14-GKLF(1-109), while a mutated EIA that no longer interacted with p300/CBP did not. Conversely, an expression construct containing full-length p300/CBP increased transaetivation by GaI4-GKLF(1-109). Conclusions: (1) Three clustered glutamate residues located between aa 93-96 are critical for GKLF to transactivate gene expression. (2) The ability of GKLF to activate gene expression depends on protein-protein interaction which very likely involves the recently described transcription co-activator p300/CBP. This work was supported by grants from the NIH.
• G3608
ANTIZYME mRNA DISTRIBUTION ALONG THE CRYPT/VILLUS AXIS AND MODULATION OF EXPRESSION IN RESPONSE TO POLYAMINES. John Gill, Lyndon Kirby and ER Seidel. Department of Physiology, School of Medicine, East Carolina University, Greenville, NC. Polyamines are essential for the maintenance of all cellular growth, in particular the rapidly proliferating cells of the intestinal epithelia which are exposed to high concentrations of polyamines in the gut lumen. The protein antizyme (AZ) has a dual function in polyamine metabolism. It is responsible for the regulating 1) degradation of ornithine decarboxylase (ODC), a ratelimiting enzyme in the polyamine biosynthetic pathway and 2) transport of polyamines across the plasma membrane. Rat small intestinal epithelial cells were isolated by incubation of everted sacs of intestine in Ca+2-free/EDTA buffer. Confirmation of a crypt/villus orientation was confirmed by measurment of alkaline phosphatase activity. The mRNA for AZ was detected by Northern blot in all cells isolated from the crypt/villus axis which included eight fractions representing cells from the tip of the villus to the base of the crypt. In the longitudinal axis, AZ transcripts were identified in cells from duodenum, jejunum and ileum. In a second experiment, intracellular polyamines were depleted in cultured intestinal epithelial cells (IEC-6) by 72 hr incubation with the specific, irreversible inhibitor of ODC, difluoromethylomithine (DFMO, 5 raM). DFMO treatment results in polyamine depletion and cell growth inhibition; the subsequent addition of the polyamine, putrescine (10 ~M) restores normal cellular growth. Total RNA was collected over a period of 24 hours (seven time points) after putrescine treatment, and AZ mRNA levels were analyzed by the Ribonuclease Protection Assay (RPA). Four hours after putrescine treatment, AZ mRNA levels began to increase with a maximum change of four-fold observed at the 12-hour time point with respect to the control (no putrescine). These results show that AZ transcript is present in epithelial cells along the entire crypt/villus and longitudinal axes of the intestine. The inhibitory action of AZ on both polyamine synthesis and absorption may be dependent on polyamine-induced regulation of AZ gene transcription or mRNA stability. • G3609 IN VIVO REGULATION OF GUANYLIN GENE EXPRESSION. P Goday, J Hochman, M Pontoglio, D Witte, MB Cohen, Children's Hosp Med Ctr & Univ of Cincinnati, Cincinnati, OH, and Pasteur Institute, Paris, France. Guanylin is a mammalian peptide, which binds to intestinal guanylate cyclase-C and mediates C1- effiux via the cystic fibrosis transmembrane regulator. We have recently shown that HNFlot is required for transcriptional activation of guanylin gene-luciferase reporter constructs in intestinal cell lines (Am. J. Physiol. 273:G833-G841, 1997). To further test the hypothesis that HNFlc~ is required for guanylin expression we subsequently evaluated guanylin mRNA expression in mice lacking HNFI~. These mice showed a decrease in intestinal guanylin mRNA expression by Northern analysis but in situ hybridization showed that guanylin message was present in the small intestine and colon. Thus, HNFlcz may influence but is not required for guanylin gene expression in the intestine. To identify additional regulatory elements that are required for in vivo guanylin gene expression, we created five transgenic mouse lines with a pGL3 luciferase reporter gene linked to a -1886 to + 39 bp guanylin promoter; this promoter element had yielded maximal reporter gene expression in transient transfection assays. In four out of five mouse lines, expression of the reporter gene was primarily localized to the distal jejunum and ileum indicating that the -1886 bp promoter was capable of preferentially driving reporter expression in the intestine of transgenic mice. However, unlike the endogenous pattern of guanylin expression, there was aberrant expression in the stomach and a relative lack of expression in the colon. Although reporter gene expression was not copynumber dependent, it is unlikely that this pattern results from a site of integration-position effect since a similar pattern of expression was seen in multiple lines. We conclude:(1) that the -1886bp construct may contain binding sites for transcription factors expressed in the stomach but may exclude the suppressor elements that normally prevent significant guanylin expression in this site (2) that the -1886 bp promoter can direct transgene expression to the intestine however additional regulatory elements must be present, outside this immediate 5'-flanking sequence, which contribute to the tissue-specific pattern of guanylin gene expression. Supported by PHS grants DK47318 and DK07727. G3610
DEVELOPMENT OF PROPRANOLOL METABOLISM IN THE ISOLATED PERFUSED NEONATAL SHEEP LIVER. P.J. Gow 1, H. Ghabrial 1, M.S. Ching 1, A. Shulkes 2, S. Treepongkaruna 2, R.A. Smallwood 1, D.I. Morgan 3. Department of Medicine I and Surgery 2, Austin & Repatriation Medical Centre, Heidelberg, Victoria 3081 and Victorian College of Pharmacy 3, Parkville, Victoria, 3052, Australia. We have developed an isolated perfused neonatal sheep liver preparation to study the maturation of propranolol metabolism in vivo in the first 10 days of life and to compare it to previous work investigating propranolol metabolism in fetal liver in late gestation. Methods: The livers of 6 lambs (ages: 2x2days, 2x6 days, 2xl0 days) were perfused via the portal vein in an oxygenated
A880 AGAABSTRACTS G3606
NEW INSIGHT ON PEG TUBE PLACEMENT IN NEUROLOGICALLY IMPAIRED PATIENTS. CA Garcia-Bueno, TM Rossi, A Tjota, CA Camacho, M Yuwono. Department of Pediatrics, Division of Gastroenterology and Nutrition, State University of New York at Buffalo, School of Medicine & Biomedical Sciences, Buffalo, New York. Nutritional rehabilitation in neurologically disabled children has been improved with the use of gastrostomy feeds, but unfortunately some of these patients develop complications or worsening of previous symptoms. The aim of the present study is to review the complications related to GER in PEG placement in children with discrete/focal neurologically impairment vs those with severe neurologic impairment. Methods: 27 neurologically impaired patients underwent PEG placement for nutritional support. Studies (Scintiscan, pH probe or UGI) which were done prior to the PEG placement were reviewed. Results: A total of 27 patients who had PEG placement. 54% developed problems related to GER. The patients with focal involvement (3/27) had marked clinical improvement after PEG placement and had no complications related to GER. In patients with severe impairment, 54% had subsequent problems with GER and/or aspiration while 42% had documented clinical improvement. One patient (4%) was lost to follow-up. Of the 54% with further problems 29% needed Nissen Fundoplication, while the other 25% did not. In addition, of those neurologically handicapped patients with problems after PEG placement, only 53% had a preceding abnormal study. Conclusion: In our experience 54% of profound neurologically disabled patients presented with postoperative complications of GER after PEG placement, and 29% of these underwent Nissen Fundoplication. This data does not support "protective anti-refiux procedure" in all patients. However, certain groups of neurologic impaired patients, particularly those with global impairment should be considered for a more invasive procedure rather than only a feeding gastrostomy. • G3607 TRANSACTIVATION BY THE GUT-ENRICHED KR~IPPEL-LIKE FACTOR (GKLF) DEPENDS ON ACIDIC AMINO ACID RESIDUES AND ON PROTEIN-PROTEIN INTERACTION. DE Geiman, H Ton-That, JM Shields, and VW Yang. Dept. of Med., The Johns Hopkins Univ. Sch. of Med., Baltimore, MD. Backeround: GKLF is a recently identified transcription factor containing 3 C2H2 zinc fingers with homology to the Drosophila Krtippel protein [Shields, Christy and Yang (1996) J. Biol. Chem. 271, 20009; Shields and Yang (1997) J. Biol. Chem. 272, 18504; Shields and Yang (1998) Nucl. Acids Res., in press]. Expression of GKLF is enriched in the gut epithelium and is associated with growth arrest. Recent studies suggest that the N terminal 109 aa residues of GKLF contain a transactivation domain capable of activating gene expression [Ton-That et al. (1997) Gastroenterology 112, A911]. Aim: The aim of the present study is to define the aa residues required for GKLF to transactivate gene expression and to determine whether the ability of GKLF to transactivate depends on protein-protein interaction. Methods: The N-terminal 109 aa residues of GKLF was fused to the DNA-binding domain of the yeast transcription factor Gal4. The chimeric Gal4-GKLF(1-109) protein was examined for its ability to transactivate a luciferase reporter gene driven by 5 tandem copies of the Gal4-binding sequence linked to either a minimal promoter (G5Elb-LUc) or the herpes virus thymidine kinase promoter (G5TK-Luc) in transiently transfected Chinese hamster ovary (CHO) cells. A mutated Ga14-GKLF(1-109[E93/94/96V]) construct was generated by sitedirected mutageneais in which valine residues were substituted for three clustered glutamate residues between aa 93-96 of GKLF and analyzed as above. Protein-protein interaction was examined by cotransfection experiments involving Ga14-GKLF(1-109), a luciferase reporter, and increasing amounts of an expression construct containing GKLF, the adenovirus EIA protein, or the transcription co-activator p300/CBP. Results: In transfected CHO cells Ga14-GKLF(1-109) increased the reporter activity of G5Elb-Luc and G5TK-Luc by 10- and 30-fold, respectively. In contrast, the Ga14-GKLF(1-109[E93/94/96V]) mutant construct failed to activate either reporter gene. The activation of the two reporters by Gal4-GKLF (1-109) was inhibited by the presence of an expression construct containing full-length GKLF in a dose-dependent fashion. This abrogation of transactivation was also observed with a construct containing truncated GKLF that retained the Nterminal but not the C-terminal portion of GKLF. An expression construct containing the wild-type adenovirus E1A also inhibited transactivation by Ga14-GKLF(1-109), while a mutated EIA that no longer interacted with p300/CBP did not. Conversely, an expression construct containing full-length p300/CBP increased transaetivation by GaI4-GKLF(1-109). Conclusions: (1) Three clustered glutamate residues located between aa 93-96 are critical for GKLF to transactivate gene expression. (2) The ability of GKLF to activate gene expression depends on protein-protein interaction which very likely involves the recently described transcription co-activator p300/CBP. This work was supported by grants from the NIH.
• G3608
ANTIZYME mRNA DISTRIBUTION ALONG THE CRYPT/VILLUS AXIS AND MODULATION OF EXPRESSION IN RESPONSE TO POLYAMINES. John Gill, Lyndon Kirby and ER Seidel. Department of Physiology, School of Medicine, East Carolina University, Greenville, NC. Polyamines are essential for the maintenance of all cellular growth, in particular the rapidly proliferating cells of the intestinal epithelia which are exposed to high concentrations of polyamines in the gut lumen. The protein antizyme (AZ) has a dual function in polyamine metabolism. It is responsible for the regulating 1) degradation of ornithine decarboxylase (ODC), a ratelimiting enzyme in the polyamine biosynthetic pathway and 2) transport of polyamines across the plasma membrane. Rat small intestinal epithelial cells were isolated by incubation of everted sacs of intestine in Ca+2-free/EDTA buffer. Confirmation of a crypt/villus orientation was confirmed by measurment of alkaline phosphatase activity. The mRNA for AZ was detected by Northern blot in all cells isolated from the crypt/villus axis which included eight fractions representing cells from the tip of the villus to the base of the crypt. In the longitudinal axis, AZ transcripts were identified in cells from duodenum, jejunum and ileum. In a second experiment, intracellular polyamines were depleted in cultured intestinal epithelial cells (IEC-6) by 72 hr incubation with the specific, irreversible inhibitor of ODC, difluoromethylomithine (DFMO, 5 raM). DFMO treatment results in polyamine depletion and cell growth inhibition; the subsequent addition of the polyamine, putrescine (10 ~M) restores normal cellular growth. Total RNA was collected over a period of 24 hours (seven time points) after putrescine treatment, and AZ mRNA levels were analyzed by the Ribonuclease Protection Assay (RPA). Four hours after putrescine treatment, AZ mRNA levels began to increase with a maximum change of four-fold observed at the 12-hour time point with respect to the control (no putrescine). These results show that AZ transcript is present in epithelial cells along the entire crypt/villus and longitudinal axes of the intestine. The inhibitory action of AZ on both polyamine synthesis and absorption may be dependent on polyamine-induced regulation of AZ gene transcription or mRNA stability. • G3609 IN VIVO REGULATION OF GUANYLIN GENE EXPRESSION. P Goday, J Hochman, M Pontoglio, D Witte, MB Cohen, Children's Hosp Med Ctr & Univ of Cincinnati, Cincinnati, OH, and Pasteur Institute, Paris, France. Guanylin is a mammalian peptide, which binds to intestinal guanylate cyclase-C and mediates C1- effiux via the cystic fibrosis transmembrane regulator. We have recently shown that HNFlot is required for transcriptional activation of guanylin gene-luciferase reporter constructs in intestinal cell lines (Am. J. Physiol. 273:G833-G841, 1997). To further test the hypothesis that HNFlc~ is required for guanylin expression we subsequently evaluated guanylin mRNA expression in mice lacking HNFI~. These mice showed a decrease in intestinal guanylin mRNA expression by Northern analysis but in situ hybridization showed that guanylin message was present in the small intestine and colon. Thus, HNFlcz may influence but is not required for guanylin gene expression in the intestine. To identify additional regulatory elements that are required for in vivo guanylin gene expression, we created five transgenic mouse lines with a pGL3 luciferase reporter gene linked to a -1886 to + 39 bp guanylin promoter; this promoter element had yielded maximal reporter gene expression in transient transfection assays. In four out of five mouse lines, expression of the reporter gene was primarily localized to the distal jejunum and ileum indicating that the -1886 bp promoter was capable of preferentially driving reporter expression in the intestine of transgenic mice. However, unlike the endogenous pattern of guanylin expression, there was aberrant expression in the stomach and a relative lack of expression in the colon. Although reporter gene expression was not copynumber dependent, it is unlikely that this pattern results from a site of integration-position effect since a similar pattern of expression was seen in multiple lines. We conclude:(1) that the -1886bp construct may contain binding sites for transcription factors expressed in the stomach but may exclude the suppressor elements that normally prevent significant guanylin expression in this site (2) that the -1886 bp promoter can direct transgene expression to the intestine however additional regulatory elements must be present, outside this immediate 5'-flanking sequence, which contribute to the tissue-specific pattern of guanylin gene expression. Supported by PHS grants DK47318 and DK07727. G3610
DEVELOPMENT OF PROPRANOLOL METABOLISM IN THE ISOLATED PERFUSED NEONATAL SHEEP LIVER. P.J. Gow 1, H. Ghabrial 1, M.S. Ching 1, A. Shulkes 2, S. Treepongkaruna 2, R.A. Smallwood 1, D.I. Morgan 3. Department of Medicine I and Surgery 2, Austin & Repatriation Medical Centre, Heidelberg, Victoria 3081 and Victorian College of Pharmacy 3, Parkville, Victoria, 3052, Australia. We have developed an isolated perfused neonatal sheep liver preparation to study the maturation of propranolol metabolism in vivo in the first 10 days of life and to compare it to previous work investigating propranolol metabolism in fetal liver in late gestation. Methods: The livers of 6 lambs (ages: 2x2days, 2x6 days, 2xl0 days) were perfused via the portal vein in an oxygenated