| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2006: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In the last few years, the interest in exploring the proteome of biological fluids has expanded through the search for biomarkers of lifestyle related diseases. In this study, we investigated the proteome of pancreas in an animal model of metabolic syndrome, SHR/NDmcr-cp rat (SHR/NDmcr-cp). The pancreas tissues extirpated from twenty-week-old male SHR/NDmcr-cp and normative rats, Wistar-Kyoto/Izm (WKY/Izm). These two samples (1 g wet weight) were homogenized with 10 mL of a lysis buffer, containing of 7 M urea, 4% 3-[(cholamidopropyl) dimethyl ammonio]-1-propanesulfonate, 2 M thiourea, and 5 mM magnesium acetate / 30 mM Tris-HC1 buffer (pH 8.0). After centrifugation at 400,000 x g for 60 min at 4 0C, the supernatant was used for a two-dimensional electrophoresis (2-DE) sample. One hundred μg of a protein was diluted to 350 μL with a rehydration solution and applied onto Immobiline dry strips (linear gradient between pH 3 and 10, 13 cm (GE Healthcare Bio-sciences, NJ, USA)). The protein
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s were focused on a IPG-phor system (GE Healthcare Bio-sciences). After the first dimension electrophoresis, the strips were equilibrated for 15 min in a solution containing dithiothreitol, which was replaced by iodoacetamide. Separation in the second dimensional electrophoresis was carried out on a SE 600 Ruby (GE Healthcare Bio-sciences) using a 12.5% SDS-polyacrylamide gel. Pancreas proteins separated on a 2-DE gel were stained with coomassie brilliant blue. The 32 spots of the SHR/NDmcr-cp sample on the 2-DE gel were more stained than those of the WKY/Izm sample. Then those 32 spots were cut, and soaked with digestion buffer containing trypsin. After the overnight protein digestion, peptide fragments were desalted by Zip tips C18, extracted and analyzed with AXIMA-CFR plus MALDI TOF-MS. Thereafter, the protein was identified by a MASCOT program using Swiss-Prot and NCBInr databases limited to Rodents species. Ten proteins provided a significant match with databases' proteins. Those proteins included a-enolase, phosphoglycerate kinase, branched-chain-amino acid transferase, carboxy-peptidase Al precursor, α-protein, inositol monophosphatase chymotrypsinogen B precursor, hypoxanthine-guanine phosphoribosyltransferase, nucleoside diphosphate kinase B, elastase-2 precursor and annexin A5. Further study for the proteome of plasma in an animal model of metabolic syndrome is ongoing in order to search for biomarkers of lifestyle related diseases. Less
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