Sephadex G-25 5,5 -dithiobis-2-nitrobenzoic acid. UV SNO-AGPa. (2) SNO-AGPa

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2 WHO (MRSA) First-in-Class ( ) (NO)NO NO NO NO 1-(AGP)183 5 N- 40 kda AGP 10~20 M 2~5 NO AGP S-AGPa (SNO-AGPa) in vitro in vivo (1) SNO-AGPa AGP A (Cys) -SH 3 1,4-dithiothreitol (DTT) 375min DTT SH DTT Sephadex G-25 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) SH A Cys SH DTT AGPa S- 10 S-Nitrosoglutathione (GS-NO) min S- S- Sephadex G-25 GS-NO -80 SNO-AGPa (2) SNO-AGPa Escherichia coli Salmonella typhimurium LT2 Bacillus subtilis Streptococcus pyogenes Pseudomonas aeruginosa PAO1 Klebsiella pneumoniae MGH78578 Staphylococcus aureus OM481 S. aureus OM584 S. aureus OM505 S.aureus OM623 M9 (Na2HPO4 12 g, KH2PO4 6 g, NaCl 1 g, NH4Cl 2 g/l) + 2 mm MgSO4, 0.2 % glucose,0.2 mm CaCl2, 0.02 % Vitamin B1 0.1% M9 OD630 = sample 25 M 379 h (OD630)PBS (%) SNO-AGPa 60 nm SNO-AGPa UV 2 h S-NO E. coli ATCC 9 h UV SNO-AGPa (3) NO M9 OD630 = RS-NOs 37 C 7 h DAF-FM DA (10 M) 371 h M9 OD630 SPECTRA FLUOR XFluor 4 (TECAN)485 nm 535 nm (4) SNO-AGP (in vivo) (CLP) Hubbard

3 (Hubbard WJ. Shock. 2005) ICR (6, 32 g) ml/ (5) SNO-AGPa 96 (Cellstar 96 well cell culture plate, greiner bio-one)96 well M9 (100 L) OD630 = 0.05 SNO-AGPa 9~48 37 C well 200 L 2%(w/v) L L 95% L nm (1) SNO-AGPa AGP RAW264.7 NO S- ph8 AGP 10 GS-NO 30 min A Cys-149 NO SNO-AGPa NO A 40% SNO-AGPa In vitro AGPa S- SNO-AGPa S- SNO-AGPa SNO-AGPa 3 SNO-AGPa SNO-AGPa in vivo AGPa AGPa S- (SNO-asialo-AGPa) SNO-AGPa 80 SNO-AGPs NO NO SNO-AGPa S- NO ROS SNO-AGPa ATP NO Carboxy-PTIO SNO-AGPa NO ROS ATP SNO-AGPa (2) AGP (K. pneumoniae MGH78578)(P. aeruginosa PAO1) (MRSA) (S. aureus OM623) SNO-AGPa ( -;, ; ; ; ; ) SNO-AGPa IC M 3 M 1 M 3 SNO-AGPa isobologram SNO-AGPa K. pneumoniae MGH78578; P. aeruginosa PAO1; S. aureus OM623; SNO-AGPa IC

4 SNO-AGPa SNO-AGPa 6G EtBr SNO-AGPa SNO-AGPa 6G SNO-AGPa SNO-AGPa AcrAB-TolC (K. pneumoniae ATCC10031) AcrAB-TolC AcrAB (K. pneumonia SKY2/pSTV28)AcrAB (K. pneumonia SKY2/pKAC28) SNO-AGPa AcrAB K. pneumonia SKY2/pKAC28 6G AcrAB K. pneumonia SKY2/pSTV28 SNO-AGPa AcrAB-TolC SNO-AGPa SNO-AGPa AGPa AGP NO SNO-AGPa SNO-AGPa NO ROS ATP SNO-AGPa SNO-AGPa AcrAB-TolC SNO-AGPa Ishima Y, Kragh-Hansen U, Maruyama T, Otagiri M. Poly-s-nitrosated albumin as a safe and effective multifunctional antitumor agent: characterization,biochemistry and possible future therapeutic applications. Biomed Res Int. 2013;2013: doi: /2013/ Watanabe K, Ishima Y, Akaike T, Sawa T, Kuroda T, Ogawa W, Watanabe H, Suenaga A, Kai T, Otagiri M, Maruyama T. S-nitrosated α-1-acid glycoprotein kills drug-resistant bacteria and aids survival in sepsis. FASEB J Jan;27(1): doi: /fj Ishima Y, Hoshino H, Shinagawa T, Watanabe K, Akaike T, Sawa T, Kragh-Hansen U, Kai T, Watanabe H, Maruyama T, Otagiri M. S-guanylation of human serum albumin is a unique posttranslational modification and results in a novel class of antibacterial agents. J Pharm Sci Sep;101(9): doi: /jps Komori H, Watanabe H, Shuto T, Kodama A, Maeda H, Watanabe K, Kai H, Otagiri M, Maruyama T. α(1)-acid glycoprotein up-regulates CD163 via TLR4/CD14 protein pathway: possible protection against hemolysis-induced oxidative stress. J Biol Chem Aug 31;287(36): doi: /jbc.M Komori H, Nishi K, Uehara N, Watanabe H, Shuto T, Suenaga A, Maruyama T, Otagiri M. Characterization of hepatic cellular uptake of α1-acid glycoprotein (AGP), part 2: involvement of hemoglobin β-chain on plasma membranes in the uptake of human AGP by liver parenchymal cells. J Pharm Sci Apr;101(4): doi: /jps Nishi K, Komori H, Kikuchi M, Uehara N, Fukunaga N, Matsumoto K, Watanabe H, Nakajou K, Misumi S, Suenaga A, Maruyama T, Otagiri M.

5 Characterization of the hepatic cellular uptake of α(1) -acid glycoprotein (AGP), part 1: a peptide moiety of human AGP is recognized by the hemoglobin β-chain on mouse liver parenchymal cells. J Pharm Sci Apr;101(4): doi: /jps S /3/ Alpha /12/ S-nitrosated a-1-acid glycoprotein kills drug-resistant bacteria and aids survival in sepsis Kaori Watanabe, Yu Ishima, Teruo Kuroda, Wakano Ogawa, Hiroshi Watanabe, Ayaka Suenaga, Toshiya Kai, Masaki Otagiri, and Toru Maruyama Asian Federation for Pharmaceutical Sciences 2013 (Jeju, Korea)2013/11/21-22 (Ramada Plaza Jeju Hotel) 4. A human serum albumin thioredoxin fusion protein prevents experimental contrast-induced nephropathy Hiroshi Watanabe, Azusa Kodama, Ryota Tanaka, Hisae Tanaka, Victor Tuan Giam Chuang, Yu Ishima, Masafumi Fukagawa, Masaki Otagiri, Toru Maruyama Asian Federation for Pharmaceutical Sciences 2013 (Jeju, Korea)2013/11/21-22 (Ramada Plaza Jeju Hotel) 5. S-NITROSATED α-1-acid GLYCOPROTEIN KILLS DRUG-RESISTANT BACTERIA AND AIDS SURVIVAL IN SEPSIS Kaori Watanabe, Yu Ishima, Teruo Kuroda, Wakano Ogawa, Hiroshi Watanabe, Ayaka Suenaga, Toshiya Kai, Masaki Otagiri, Toru Maruyama /10/ Poly-S-nitrosated Human Serum Albumin inhibits the Expression of P-glycoprotein Transporter in Human Multidrug-resistant Tumor Yu Ishima, Marie Hara, Hiroshi Watanabe, Masaki Otagiri, Toru Maruyama The 5th Asian Arden conference 2013/8/6-7 Aichi Gakuin University 7. Long-acting human serum albumin-thioredoxin fusion protein suppresses bleomycin-induced pulmonary fibrosis progression Ryota Tanaka, Hiroshi Watanabe, Yu Ishima, Masaki Otagiri, Toru Maruyama The 5th Asian Arden conference 2013/8/6-7 Aichi Gakuin University 8. S- EPR 13 NO 2013/6/ NO 2013/6/ /5/23-5/ /5/23-5/ S- EPR /5/23-5/ S-1-

6 S-1- (AGP) (2) Ishima Yu Otagiri Masaki (3) 15. S-1- (AGP) 12 NO S- 12 NO S- EPR Yakuzai/ (1) Maruyama Toru

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