Histoautoradiography: After perfusion of the hearts with 48 |J.Ci 14C-AAP10, they were perfused with 5% formalin (pH 7, phosphate-buffered) for fixation; the position of the electrodes was marked by injection of ink. After a seven-day postfixation period in 5% formalin, the marked ventricular areas under the electrode grids were excised, embedded in hydroxyethylmethacrylate (Technovit 7100; Heraeus-Kulzer, Wehrheim, Germany), cut into 2 |j,m thick sections, fixed to slides and dipped in NTB2 emulsion (emulsion:water in 2:1 proportion at 42°C, Kodak, Rochester, New York, USA). After 201 days of exposure at -20°C the slides were developed, and photographs were taken using a Zeiss Axiolab (Koln, Germany) with Achroplan objectives and a Nikon F3 camera (Tokyo, Japan) (magnification 400x).
Chemical synthesis: The natural antiarrhythmic peptide, which was isolated from bovine atria and first described by Aonuma and colleagues , had the following amino acid sequence:
This and two related peptides with the following sequences were synthesized:
H2N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH2 (C26N7O8H37) (AAP10)
H2N-(14C-Gly)-Ala-Gly-4Hyp-Pro-Tyr-CONH2 (C26N7O8H37) (14C-AAP10)
H2N-Gly-Ala-Gly-Pro-Pro-Tyr-CONH2 (C26N7O7H36) (AAP8TT)
For chemical synthesis, a modified Merrifield technique was employed using the Fmoc strategy as described by Atherton and Sheppard . The peptides were purified using semipreparative reversed-phase high performance liquid chromatography . Purity was 99% or greater. There is a wonderful pharmacy offering Advair Diskus for Asthma buy here, and you can shop with it.