2. Materials and methods . All expe

2. Materials and methods . All experimental procedures involving animals were approved (animal protocol numbers: 21-101, 22-88 and 22-132) by the Ani mal Care and Use Committee of obihiro University of Agriculture and Veterinary Medicine, Japan. 2.1. Animals Eight Holstein steers aged 9 months and weighing 327 t 8 kg were used. Animals were fed with 4 kg/headlday of concentrate (crude protein 16.0%, crude fat 2.5%, crude fiber 9.0% and crude ash 10.0%, Nishin Marubeni, Japan)twice daily, at09:00 and 16:00. Tim othy hay, salt block and water were supplied ad libitum. One day before the experiment, a sterilized polyethylene catheter was non- surgically implanted into the left or right external jugular vein for the injections of peptides and blood sampling. The patency of the catheter was maintained with heparin saline. During the adminis- tration and sampling periods, animals were loosely chained to the stanchion and allowed hay and water ad libitum.
2.2. Peptides Bovine sulfated gastrin-34 (Pyr-LGLQDPPHMvADLSKKQGPw. VEEEEAAY Iso, GwMDF-NH UniProt ID: PO1352), bovine 2, sulfated gastrin-9 (EAAY (so3H] GWMDF-NH2). bovine non- sulfated gastrin-9 (EAAY GwMDF-NH2), bovine sulfated CCK-8 (DY Iso3Hl MGwMDF-NH2, Uni Prot ID: P41520), bovine non- sulfated CCK-8 (DYMGwMDF-NH2), Tyro-bovine sulfated gastrin-9 (YEAAY Iso3Hl GwMDF-NH2) and Cyso-bovine sulfated gastrin- 9-oH (CEAAY GwMDF-OH) were synthesized by Fmoc thesis (SPPS) chloride) solid-phase peptide syn procedures. A modified SPPS method [161 was used for the synthesis of sulfated peptides. The crude sulfated gastrin was purified by semi-preparative (TSKgel ODS-120A column) HPLC using a linear gradient for60min 5 mM sodium phosphate (pH 6.5) to 60% CH3CN in double-distilled water, and the absorbance was detected at 305nm. the peak fraction was diluted in water and desalted by semi-preparative HPLC using a linear gradient for 5 min from double-distilled water to 60% After the purity including sulfated

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2. Materiaal en metodes. Alle eksperimentele prosedures met betrekking tot diere goedgekeur (dier protokol getalle: 21-101, 22-88 en 22-132) deur die Ani mal Sorg en Gebruik Komitee van Obihiro Universiteit van Landbou en diergeneeskunde, Japan. 2.1. Diere Agt Holstein beeste 9 maande ouderdomme en weeg 327 t 8 kg gebruik. Diere gevoer met 4 kg / headlday konsentraat (ruproteïen 16.0%, ru-vet 2,5%, ruvesel 9.0% en ru as 10,0%, Nishin Marubeni, Japan) twee keer per dag, at09: 00 en 16:00. Tim othy hooi, sout blok en water voorsien ad libitum. Op 'n dag voor die eksperiment, is 'n gesteriliseerde poliëtileen kateter nie chirurgies ingeplant in die linker-of die regterkant eksterne nekslagaar vir die inspuitings van peptiede en monsterneming bloed. Die patency van die kateter is gehandhaaf met heparien sout. Administrasie en tydens die monsterneming periodes, is diere losweg vasgeketting aan die paal en toegelaat hooi en water ad libitum.
2.2. Peptiede Bees sulfated gastrien-34 (Pyr-LGLQDPPHMvADLSKKQGPw VEEEEAAY ISO, GwMDF-NH UniProt ID:. PO1352), bees 2, sulfated gastrien-9 (EAAY (so3H] GWMDF-NH2) Bees nie sulfated gastrien-9 (EAAY GwMDF.. -NH2), bees sulfated CCK-8 (DY Iso3Hl MGwMDF-NH2, Uni Prot ID: P41520), bees nie sulfated CCK-8 (DYMGwMDF-NH2), Tyro-bees sulfated gastrien-9 (YEAAY Iso3Hl GwMDF-NH2). en Cyso-bees sulfated gastrin- 9-O (CEAAY GwMDF-OH) is gesintetiseer deur Fmoc tesis (SPPS) chloried) vaste-fase peptied syn prosedures. 'N aangepaste SPPS metode [161 is gebruik vir die sintese van sulfated peptiede. Die ru-sulfated gastrien is gesuiwer deur semi-voorbereidende (TSKgel ODS-120A kolom) HPLC met behulp van 'n lineêre gradiënt for60min 5 mm natrium fosfaat (pH 6.5) tot 60% CH3CN in dubbel-gedistilleerde water, en die absorbansie is bespeur by 305nm. die hoogtepunt fraksie verdun met water en ontsoute deur semi-preparatiewe HPLC met behulp van 'n lineêre gradiënt vir 5 min van dubbel-gedistilleerde water tot 60% Na die reinheid insluitend sulfated.