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r). The herpes simplex virus thymidine kinase cassette (tk) was cloned 3′ to the cGKII sequence. R1 embryonic stem (ES) cells [A. Nagy, J. Rossant, R. Nagy, W. Abramow-Newerly, J. C. Roder, Proc. Natl. Acad. Sci. U.S.A. 90, 8424 (1993)] were electroporated with the linearized vector and plated on irradiated G418-resistant embryonic feeder cells [R. Fässler and M. Meyer, Genes Dev. 9, 1896 (1995)]. Recombinant clones were selected with G418 (0.3 mg/ml) and 1-[2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-]-5-iodouracil (FIAU) (0.2 μM). Four of 100 double-resistant colonies showed homologous recombination at the cGKII locus. Germline chimeras were obtained by injection of mutant ES cell clones into C57BI/6 blastocysts. RT-PCR was performed with primers that amplify cGKII (nucleotides 508 to 1740) (5) and guanylin (nucleotides 331 to 521) [D. Sciaky, J. L. Kosiba, M. B. Cohen, Genomics 24, 583 (1994)], respectively, Immunoblot analysis was performed with antibody (Ab) B32-A3 to the COOH-terminal region of mouse cGKII, Ab A16-14 to cGKI, and Ab to the α, β, and γ catalytic subunits of the cAK (Santa Cruz Biotechnology, Santa Cruz, CA).
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r). The herpes simplex virus thymidine kinase cassette (tk) was cloned 3′ to the cGKII sequence. R1 embryonic stem (ES) cells [A. Nagy, J. Rossant, R. Nagy, W. Abramow-Newerly, J. C. Roder, Proc. Natl. Acad. Sci. U.S.A. 90, 8424 (1993)] were electroporated with the linearized vector and plated on irradiated G418-resistant embryonic feeder cells [R. Fässler and M. Meyer, Genes Dev. 9, 1896 (1995)]. Recombinant clones were selected with G418 (0.3 mg/ml) and 1-[2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-]-5-iodouracil (FIAU) (0.2 μM). Four of 100 double-resistant colonies showed homologous recombination at the cGKII locus. Germline chimeras were obtained by injection of mutant ES cell clones into C57BI/6 blastocysts. RT-PCR was performed with primers that amplify cGKII (nucleotides 508 to 1740) (5) and guanylin (nucleotides 331 to 521) [D. Sciaky, J. L. Kosiba, M. B. Cohen, Genomics 24, 583 (1994)], respectively, Immunoblot analysis was performed with antibody (Ab) B32-A3 to the COOH-terminal region of mouse cGKII, Ab A16-14 to cGKI, and Ab to the α, β, and γ catalytic subunits of the cAK (Santa Cruz Biotechnology, Santa Cruz, CA).
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r). The herpes simplex virus thymidine kinase cassette (tk) was cloned 3′ to the cGKII sequence. R1 embryonic stem (ES) cells [A. Nagy, J. Rossant, R. Nagy, W. Abramow-Newerly, J. C. Roder, Proc. Natl. Acad. Sci. U.S.A. 90, 8424 (1993)] were electroporated with the linearized vector and plated on irradiated G418-resistant embryonic feeder cells [R. Fässler and M. Meyer, Genes Dev. 9, 1896 (1995)]. Recombinant clones were selected with G418 (0.3 mg/ml) and 1-[2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-]-5-iodouracil (FIAU) (0.2 μM). Four of 100 double-resistant colonies showed homologous recombination at the cGKII locus. Germline chimeras were obtained by injection of mutant ES cell clones into C57BI/6 blastocysts. RT-PCR was performed with primers that amplify cGKII (nucleotides 508 to 1740) (5) and guanylin (nucleotides 331 to 521) [D. Sciaky, J. L. Kosiba, M. B. Cohen, Genomics 24, 583 (1994)], respectively, Immunoblot analysis was performed with antibody (Ab) B32-A3 to the COOH-terminal region of mouse cGKII, Ab A16-14 to cGKI, and Ab to the α, β, and γ catalytic subunits of the cAK (Santa Cruz Biotechnology, Santa Cruz, CA).
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4, pH 7.0, 2 mM EDTA, and 2 mM benzamidine (for kinase determination) or in 10 mM tris-HCI (pH 7.4), 300 mM sorbitol, 2 mM benzamidine, and leupeptin (20 μg/ ml) (for guanylate cyclase activity). The kinase activity was determined as described [P. Ruth et al., Eur. J. Biochem. 202, 1339 (1991)], with 10 μg of protein and in the presence or absence of 30 μM cGMP. GC activity in brush border membranes was determined as described [A. B. Vaandrager, S. Schulz, H. R. De Jonge, D. L. Garbers, J. Biol. Chem. 268, 2174 (1993)], with 35 μg of protein and 400 ng of STa. The samples were acetylated and cGMP concentrations were determined in an enzyme immunoassay (Cayman, Ann Arbor, MI). Values are expressed as the mean ± SEM with n = 8 for kinase activity and n = 4 for guanyly cyclase activity.
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4, pH 7.0, 2 mM EDTA, and 2 mM benzamidine (for kinase determination) or in 10 mM tris-HCI (pH 7.4), 300 mM sorbitol, 2 mM benzamidine, and leupeptin (20 μg/ ml) (for guanylate cyclase activity). The kinase activity was determined as described [P. Ruth et al., Eur. J. Biochem. 202, 1339 (1991)], with 10 μg of protein and in the presence or absence of 30 μM cGMP. GC activity in brush border membranes was determined as described [A. B. Vaandrager, S. Schulz, H. R. De Jonge, D. L. Garbers, J. Biol. Chem. 268, 2174 (1993)], with 35 μg of protein and 400 ng of STa. The samples were acetylated and cGMP concentrations were determined in an enzyme immunoassay (Cayman, Ann Arbor, MI). Values are expressed as the mean ± SEM with n = 8 for kinase activity and n = 4 for guanyly cyclase activity.
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note
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2, and were kept at 37°C.
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0021368822
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The suckling mouse model (10) was used to quantify the STa-induced diarrhea in vivo. STa (50 ng) was dissolved in 0.5 ml of isotonic PBS and injected intragastrically in 3- to 4-day-old mice. After a 2-hour incubation at 25°C, the whole intestine without stomach was carefully removed and weighed. The g/c ratio was calculated as the ratio of gut weight to remaining carcass weight. To evaluate the response to CT, we used the sealed mouse model [S. H. Richardson, J. C. Giles, K. S. Kruger, Infect. Immun. 43, 482 (1984)]. Data are expressed as mean ± SEM of five mice for each treatment. P < 0.05 versus control.
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26
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13344278032
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3H]Thymidine labeling of growth plates was performed as described [A. M. Reimold et al., Nature 379, 262 (1996)], and bone transplantation was performed as described by W. J. L. Felts [Transplant. Bull. 4, 5 (1957)]. Femurs of 1-week-old donors were implanted subcutaneously into 4- to 6-week-old recipients. After 14 days, the mice were killed and the length of the femurs was determined.
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A. Pfeifer et al., data not shown
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A. Pfeifer et al., data not shown.
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12644261392
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note
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We thank K. Kühn for his advice; M. Walter, M. Cuba, and I. Blumenstein for their help in the Ussing chamber experiments; K. Doerr, S. Kamm, and S. Benkert for technical assistance; and P. Klatt for help in quantifying the total body fat of the mice. Supported by grants from the Deutsche Forschungs Gemeinschaft, Bundesministerium für Forschung und Technologie, and Fonds der Chemie. R.F. was supported by the Hermann and Lilly Schilling Stiftung.
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