牛乳に含まれるアディポサイトカインの定量と機能探索
Contents
1. Casabie11 Pineiro et al 19979 Houseknecht McGuire et al 19979 Aoki Kawamura et al 1999 9 Bronsky Karpisek et al 2006 Martin Woo et al 2006 Ilcol Hizli et al 2008 Lage Baldelli et al 20029 0 Connor Funanage et al 2003 62. 10 11 12 Aoki N S Jin no et al 2007 Identification and characterization of microvesicles secreted by 313 L1 adipocytes redox and hormone dependent induction of milk fat globule epidermal growth factor 8 associated microvesicles Endocrinology 148 8 3850 62 Aoki N M Kawamura et al 1999 Lactation dependent down regulation of leptin production in mouse mammary gland Biochim Biophys Acta 1427 2 298 306 Aoki N M Kishi et al 1995 Molecular cloning of glycoprotein antigens MGP57 53 recognized by monoclonal antibodies raised against bovine milk fat globule membrane Biochim Biophys Acta 1245 3 385 91 Bronsky J M Karpisek et al 2006 Adiponectin adipocyte fatty acid binding protein and epidermal fatty acid binding protein proteins newly identified in human breast milk Clin Chem 52 9 1763 70 Casabiell X V Pineiro et al 1997 Presence of leptin in colostrum and or breast milk from lactating mothers a potential role in the regulation of neonatal food intake J Clin Endocrinol Metab 82 12 4270 3 Houseknecht K L M K McGuire et al 1997 Leptin is present in human milk and is r
3. ELISA ELISA 7 not shown 4 C 15 DSTFTGFLLYHNIVE keyhole limp
4. 8 0 Yoneda Tomimoto et al 2008 1 cDNA cDNA Aoki
5. 1 pGEM T Easy TAC 4 6 RT TCR cDNA 2 pEF1 Myc His A C Myc 2 5 EcoRI 3 Xbal 1 cDNA PCR EcoRI
6. cDNA PCR cDNA 1 cDNA Aoki Kishi et al 1995 cDNA RNAA RT PCR RNA RT PCR cDNA RNA 28S 185 RNA 2 RNA o1igo dT cDNA 1 PCR 3
7. CN 5 30 TY OCR EO 2 OFT v CHIR LL 32 vector EcoRI Sall sense primer 5 3 antisense primer 5 3 BL21 0 1mM AIT OT 12 000 rpm 3min 50 mM buffer pH7 02 0 05 gs 500 gL 15 10 put 1 Duty40 1 min 3 8 5 6 mL g RT 1h 4C 12 000 rpm 30 73 1 AMR RA 11 4 L 2 M 1 L 4C 2h 50 mM buffer pH7 0 500 mL 4 1h buffer 1 5L 4 4
8. Cane 2 1ane 1 cDNA CBB 15 C Ni SDS PAGE CBB 1 4 1 cDNA sas a sponses AMEE b Adipo S ATGCTGCTGCAGGGAGCT 69 86 NM 174742 b Adipo AS TCATTCAACAATGTTATGGTA 771 791 b Lep S ATGCGCTGTGGACCCCTG 48 65 NM 173928 b Lep AS TCAGCACCCGGGACTGAGGTC 531 551 330 BC148022 b Res AS CTACTGGATATGCAGGCGGCA 358 378 2
9. 3 6 NIH 3T3 ELISA BLISA LISAN ELISA ELISA BUUO0P UI0 Resistin mouse ELISA kit
10. 2 80C 4C 5 000 g 30 3T3 L1 10 FCS DMEM LE 6 well plate 50 2 mL wel1 2 10ng mL 0 25 uM dexamethasone 0 5 mM 3 isobuty1 1 methylxanthine Day 0 2 5 ng mL 2 3 Day 8
11. 7 90 VY DEEDES LIB AVY b RRR L ZED LMELISAY y 1 ELISA 8 ng mL 10ng mL Tes
12. cDNA Query L Blast serch cDNA Sbjct 35 Query 1 ATGCGCTGTGGACCCCTGTATCGATTCCTGTGGCTTTGGCCCTATCTGTCTTACGTGGAG 60 Sbjct 48 ATGCGCTGTGGACCCCTGTATCGATTCCTGTGGCTTTGGCCCTATCTGTCTTACGTGGAG 107 Query 61 GCTGTGCCCATCTGCAAGGTCCAGGATGACACCAAAACCCTCATCAAGACAATTGTCACC 120 Sbjct 108 GCTGTGCCCATCTGCAAGGTCCAGGATGACACCAAAACCCTCATCAAGACAATTGTCACC 167 Query 121 AGGATCAATGACATCTCACACACGCAGTCCGTCTCCTCCAAACAGAGGGTCACTGGTTTG 180 Sbjct 168 AGGATCAATGACATCTCACACACGCAGTCCGTCTCCTCCAAACAGAGGGTCACTGGTTTG 227 Query 181 GACTTCATCCCTGGGCTCCACCCTCTCCTGAGTTTGTCCAAGATGGACCAGACATTGGCG 240 Sbjct 228 GACTTCATCCCTGGGCTCCACCCTCTCCTGAGTTTGTCCAAGATGGACCAGACATTGGCG 287 Query 241 ATCTACCAACAGATCCTCACCAGTCTGCCTTCCAGAAATGTGGTCCAAATATCCAATGAC 300 Sbjct 288 347 Query 301 CTGGAGAACCTCCGGGACCTTCTCCACCTGCTGGCCGCCTCCAAGAGCTGCCCCTTGCCG 360 Sbjct 348 CTGGAGAACCTCCGGGACCTTCTCCACCTGCTGGCCGCCTCCAAGAGCTGCCCCTTGCCG 407 Query 361 CAGGTCAGGGCCCTGGAGAGCTTGGAGAGCTTGGGCGTTGTCCTGGAAGCTTCCCTCTAC 420 Sbjct 408 CAGGTCAGGGCCCTGGAGAGCTTGGAGAGCTTGGGCGTTGTCCTGGAAGCTTCCCTCTAC 467 Query 421 TCCACCGAGGTGGTGGCCCTGAGCCGGCTGCAGGGGTCACTACAGGACAT
13. e 22 b Adipo S EcoRI ATGAATTC CACC ATGCTGCTGCAGGGAGCT 69 86 NM 174742 521 NM_173928 b Lep AS Xbal ATTCTAGA GCACCCGGGACTGAGGTC 531 551 b Res S EcoRI ATGAATIC CACC ATGAAGGCICTICTCCIICG 49 66 347 BC148022 b Res AS Xbal ATTCTAGA CTGGATATGCAGGCGGCA 358 378
14. 2 Trizol RNA Vf RICE Bet WRN PF Cine 0 75 ee 1000 bp gt 500 bp gt BS 2 RNA cDNA 1 2 3 pGEM T Easy sie Query 1 ATGCTGCTGCAGGGAGCTCTTCTACTGCTACTAGCCTTACCCAGTCATGGCGAGGACAAC 60 Sbjct 69 ATGCTGCTGCAGGGAGCTCTTCTACTGCTACTAGCCTTACCCAGTCATGGCGAGGACAAC 128 Query 61 ATGGAAGATCCCCCGCTGCCCAAGGGGGCCTGCGCAGGTTGGATGGCAGGCATCCCAGGA 120 Sbjct 129 ATGGAAGATCCCCCGCTGCCCAAGGGGGCCTGCGCAGGTTGGATGGCAGGCATCCCAGGA 188 Query 121 CATCCTGGCCACAATGGCACACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAG 180 Sbjct 189 CATCCTGGCCACAATGGCACACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAG 248 Query 181 GGAGAGAAAGGAGATGCAGGTCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATG 240 Sbjct 249 GGAGAGAAAGGAGATGCAGGTCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATG 308 Query 241 ACAGGAGCTGAAGGGCCACG
15. 1 5 ELISA 93 7 cDNA
16. A B 3T3 L1 NIH 3T3 n 3 41 g mL 13 ELISA RED 3T3 L1 NIH 3T3 n 3 210 210 210 140 140 140 95 95 95 70 55 43 36 5 28 19 3 14 His Myc NIH 3T3 SDS PAGE
17. NH TKFIMLNTPGLYYFYHITVYMKIVKVSLFK ive human seq HITVYMEDVEVSLFE 277 mouse seg 24 HAPT KI YHOO its TK HALI L a bovine sed 232 huran seg moise seg 248 bovine seq 233 24 human seq 238 JIHT 244 mouse seg 24 BEF OTA 247 BS GENETYX MAC Multiple Alignment 3 2 FET 38 bovine seqg ou human seg 5 5 5 GL Mouse seq R LET W IKVODOTKTLIKTIVTRINDISHT V NKO 6a bawine 128 human seq 128 Muse 128 bovine seq 21 QVRABESLEARBGV 1 human seg boy WSTEVVALSRLOGSLOO LOOLORSP 167 mouse seg LOGS Pit 16 9 GENETYX MAC Multiple Alignment 3 2 bovine
18. Xba I pEF1 Myc His A Genopure Plasmid Midi Kit NIH 3T3 10 DMEM FuGENE HD FuGENE a ES 02 2 10 35mm dish 72 PBS 2 Aoki Jin no et al 2007 28 NIH 3T3 7 SDS PAGE
19. 13 000 rpm 30 minke DHE ITV Ni 1 mL 4C 50mM buffer pH7 0 500 mM 50 mM buffer pH7 0 CAH 37 Hydropathy plot C BD Ni 100
20. Kishi et al 1995 RNA AGPC Trizol ExTaq DNA polymerase Pyrobest DNA polymerase GenBank Accession NMM 174742 GenBank Accession No NM_173928 GenBank Accession No BC148022 cDNA 1 QIAEX II Gel Extraction Kit pGEM T Easy vector TA BigDyee Terminators vl 1 Cycle Sequencing Kit BigDye Terminators 1 Cycle Sequencing Kit ABI PRISM 3100 Genetic Analyzer 5 cDNA PCR cDNA Aoki Kawamura et al 1999 2 2 cDNA
21. Day 10 48 ELISA ic Eqn cDNA NIH 3T3 ELISA ELISA 8 Es FAO AOT T APPA FAIA EE UL CHE FOV Y 3 3 1 ELISA Casabiell Pineiro et al 19979 Houseknecht McGuire et al 19979 Smith Kirwin 0 Connor et al 19981 Aoki Kawamura et al 1999 2 dI EFLI DF vy 11 1 3 2 ELISA
22. GAGACCACGT GCCACT GCCAGT GCGCAGGCAT GGACTGGACTGGA TGGGACGTACGTGCTGAGACCACGT GCCACT GCCAGT GCGCAGGCAT GGACTGGACTGGA GCTCGCTGCTGCCGCCTGCATATCCAGTAG 330 GCTCGCTGCTGCCGCCTGCATATCCAGTAG 378 60 108 120 168 180 228 240 288 300 348 serch cDNA Sb jct 3 7 210 210 140 210 140 95 140 95 95 70 70 70 55 55 43 43 36 5 36 5 28 28 193 19 3 7 His Myc NIH 3T3 72 bR zaJ T ane 1 2 1ane 3 SDS PAGE His lane 1 cDNA Vic bovine seq ara aA e 52 human seq QETT TOGPGYVIEL PL 57 mouse seg EDOVTTTEELAPAIVPP ttc bayine 5 5 114 seq NVYRSMFSV iiy mouse seg MAYVYRS 28 bovine
23. GGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGA 300 Sbjct 309 ACAGGAGCTGAAGGGCCACGGGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGA 368 Query 301 GAAGCCGCTTATGTGTATCGCTCAGCGTTCAGTGTGGGGCTGGAGACCCGCGTCACTGTT 360 Sbjct 369 GAAGCCGCTTATGTGTATCGCTCAGCGTTCAGTGTGGGGCTGGAGACCCGCGTCACTGTT 428 Query 361 CCCAATGTACCCATTCGCTTTACTAAGATCTTCTACAACCAACAGAATCATTATGACGGC 420 Sbjct 429 CCCAATGTACCCATTCGCTTTACTAAGATCTTCTACAACCAACAGAATCATTATGACGGC 488 Query 421 AGCACTGGCAAGTTCTACTGCAACATTCCGGGACTCTACTACTTCTCTTACCACATCACG 480 Sbjct 489 AGCACTGGCAAGTTCTACTGCAACATTCCGGGACTCTACTACTTCTCTTACCACATCACG 548 Query 481 GTGTACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACC 540 Sbjct 549 GTGTACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACC 608 Query 541 TACGACCAGTATCAGGAAAAGAATGTGGACCAGGCCTCTGGCTCTGTGCTCCTCCATCTG 600 Sbjct 609 TACGACCAGTATCAGGAAAAGAATGTGGACCAGGCCTCTGGCTCTGTGCTCCTCCATCTG 668 Query 601 GAGGTGGGAGACCAAGTCTGGCTCCAGGTGTACGAGGGTGAAAATCACAATGGGGTCTAT 660 Sbjct 669 GAGGTGGGAGACCAAGTCTGGCTCCAGGTGTACGAGGGTGAAAATCACAATGGGGTCTAT 728 Query 661 GCAGATAATGTCAATGACTCCACCTTCACAGGCTTCCTTCTCTACCATAACATTGTTGAA 720 Sbjct 729 GCAGATAATGTCAATGACTCCACCTTCACAGGCTTCCTTCTCTACCATAACATTGTTGAA 788 Query 721 TGA 723 Sbjct 789 TGA 791 4 cDNA RT PCR
24. GTTGCGGCAG 480 Sbjct 468 TCCACCGAGGTGGTGGCCCTGAGCCGGCTGCAGGGGTCACTACAGGACATGTTGCGGCAG 527 Query 481 CTGGACCTCAGTCCCGGGTGCTGA 504 Sbjct 528 CTGGACCTCAGTCCCGGGTGCTGA 551 5 cDNA RT PCR cDNA Query Blast serch cDNA Sb jct 36 Query Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct 6 cDNA RT PCR cDNA Query Blast 1 49 61 109 121 169 181 209 241 289 301 349 ATGAAGGCTCTCTCCTTCCTCTTCATCCCAGT CCT GGGGCTGCTGGTGTGTGGCCAGTCG ATGAAGGCTCTCTCCTTCCTCTTCATCCCAGT CCT GGGGCTGCTGGTGTGTGGCCAGTCG CTGTGCCCCATAGATAAAGCCATCAGTGAGAAGATCCAGGAGGTCACCACCTCCCTAGTT CTGTGCCCCATAGATAAAGCCATCAGTGAGAAGATCCAGGAGGTCACCACCTCCCTAGTT CCTGGGGCAGTAAGGATCATTGGCCTGGACTGCCGGAGTGTCACCTCTAGGGGGTCCCTG CCT GGGGCAGTAAGGATCATTGGCCTGGACTGCCGGAGTGTCACCTCTAGGGGGTCCCTG GTCACCTGCCCTTCAGGCTTCGCCGTCACT GGCTGCACGT GT GGCTCCGCCTGTGGCTCG GTCACCTGCCCTTCAGGCTTCGCCGTCACT GGCT GCACGT GT GGCTCCGCCTGTGGCTCG TGGGACGTACGT GCT
25. Seq 56 human seq 5 Mouse seg ba bovine s g 5 TTG TT TA Th ML GE tea LT 189 human seq 57 eC PNG AVIA ie GSACGSWDUR VE AS DWT AA ROP 195 modse seg 1 i 114 10 GENETYX MAC Multiple Alignment 3 2 _ 39 CO B11 ELISA A 4 B 3T3 L1 NIH 3T3 n 3 40 ng mL ng mL 1 2 ELISA
26. elated to maternal plasma leptin concentration and adiposity Biochem Biophys Res Commun 240 3 742 7 Ilcol Y 0 Z B Hizli et al 2008 Resistin is present in human breast milk and it correlates with maternal hormonal status and serum level of C reactive protein Clin Chem Lab Med 46 1 118 24 Lage M R Baldelli et al 2002 Presence of bovine leptin in edible commercial milk and infant formula J Endocrinol Invest 25 8 670 4 Martin L J J G Woo et al 2006 Adiponectin is present in human milk and is associated with maternal factors Am J Clin Nutr 83 5 1106 11 0 Connor D Funanage et al 2003 Leptin is not present in infant formulas J Endocrinol Invest 26 5 490 Smith Kirwin 5 M D 0 Connor et al 1998 Leptin expression in human mammary epithelial cells and breast milk J Clin Endocrinol Metab 83 5 1810 3 Yoneda A Tomimoto et al 2008 Expression of adiponectin receptors AdipoR1 and AdipoR2 in normal colon epithelium and colon cancer tissue Oncol Rep 20 3 479 83 ee 1000 bp gt 500 bp gt 1 cDNA CT 200 NTI ee 2 3 285 gt 185 gt 2 RNA
27. et hemocyanin KHL C 15 SLQDMLRQLDLSPGC KHL C 15 GMDWTGARCCRLHTQ KHL 7 14 2
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