中文摘要
鱟是古老的無脊椎海洋動物，缺乏後天性免疫系統，因此完全依賴由血漿及血球組成的先天性免疫系統抵抗病原菌入侵。由台灣品種鱟 (Taiwanese Tachypleus tridentatus) 的血漿中分離之血漿凝集素具有辨識革蘭氏陽性和陰性菌、以及其他病源菌相關分子型態(pathogen associated molecular patterns, PAMPs) 的能力。以往利用嗜甲醇酵母菌 (Pichia pastoris) 可於胞外表現鱟血漿凝集素，但其表現量及產率皆低；而目前尚無利用原核生物系統表現並純化可溶性鱟血漿凝集素的成功案例。本研究利用蛋白質工程技術設計及修飾重組鱟血漿凝集素胜肽序列，並利用大腸桿菌表現此新穎之重組蛋白。其生物功能性分析顯示重組鱟血漿凝集素可識別特定的細菌及病原相關分子型態、亦能結合多種臨床分離之病源菌，並發現此結合細菌的能力可能與病源菌細胞壁中特殊醣分子相關。此外，本研究亦首次發現重組鱟血漿凝集素具有抑制綠膿桿菌生長的能力。本論文探討重組鱟血漿凝集素與細菌、病原菌及糖類的特定分子交互作用關係，將有助於發展偵測臨床微生物致病菌或內毒素殘留的新穎方法。
 

Table of Contents
中文摘要…………………………………………………………………………..…..I
Abstract…......................................................... II
Acknowledgement III
Abbreviation XI
Chapter 1 Introduction 1
1.1 Innate immunity of invertebrates 1
1.2 Horseshoe crab 3
1.3 Hemolymph coagulation 4
1.4 Endotoxin detection assay–from Limulus amebocyte lysate (LAL) to recombinant Factor C (rFC) 6
1.5 Endotoxin removal kit-ToxinEraser and EndoTrap® 8
1.6 Tachylectin 9
1.7 Tachypleus plasma lectin 1 and Tachypleus plasma lectin 2 12
1.8 Starch binding domain (SBD) 16
1.9 Solubility-enhancing peptide (SEP) 16
1.10 ySBD-SEP-TPL2 17
1.11 Characteristics and detection methods of clinically important pathogens 18
1.12 Research motivation 23
Chapter 2 Materials and methods 25
2.1 Microbial strains, media and plasmid 25
2.2 Construction of plasmid 25
2.3 Competent cell preparation 26
2.4 Escherichia coli heat-shock transformation 27
2.5 Confirmation of colonies by in situ PCR 27
2.6 Mini-preparation of plasmid 28
2.7 Small scale expression of recombinant TPL2 in E. coli 28
2.8 Purification of recombinant SEP-TPL2 and removal endotoxin 29
2.9 Protein buffer exchange 30
2.10 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 30
2.11 Bicinchoninic acid (BCA) assay 30
2.12 Enzyme-linked imunosorbent assay (ELISA) for PAMPs 31
2.13 Enzyme-linked immunosorbent assay (ELISA) for bacteria 31
2.14 Magnetic reduction (MR) assay 33
2.15 Inhibition and competitive ELISA 33
2.16 Antibacterial activity assay 34
2.17 Analytical ultracentrifugation 34
2.18 Mass spectrometry determination 35
2.19 Statistical analysis 35
Chapter 3 Results 36
3.1 Small scale expression of recombinant TPL2 36
3.2 Design of different recombinant TPL2 36
3.3 Construction of pET23a-n7-tpl2 and pET23a-c7-tpl2 37
3.4 Small scale expression of recombinant TPL2 37
3.5 Purification of SEP-TPL2 and removal endotoxin 38
3.6 Binding activity of SEP-TPL2 to PAMPs 39
3.7 Bacteria-binding activity of SEP-TPL2 40
3.8 Clinically isolated bacteria recognition of SEP-TPL2 41
3.9 L-rhamnose binding activity of SEP-TPL2 42
3.10 Binding activity of SEP-TPL2 to L-rhamnose 43
3.11 Binding activity of SEP-TPL2 to Rhamnosoft® HP 43
3.12 Antibacterial activity of SEP-TPL2 45
3.13 Determination of SEP-TPL2 forms 45
Chapter 4 Discussion 47
4-1 Expression and purification of TPL2 47
4-2 PAMP-binding activity of TPL2 48
4-3 Bacteria-binding activity of TPL2 49
4-4 Functional roles of rhamnose in pathogens 50
4-5 Antibacterial activity of SEP-TPL2 54
4-6 Characterization of SEP-TPL2 forms 55
4-7 Application of SEP-TPL2 for endotoxin detection kit and endotoxin removal kit 56
4-8 Comparison between SEP-TPL2 and rhamnose binding lectins (RBLs) 57
4-9 Conclusion 58
References… 61
Figures…… 72
Tables…….. 98
Appendices… 105

 
List of Figures
Fig. 1-1 Structures of bacterial cell wall and pathogen associated molecular patterns (PAMPs) 3
Fig. 1-2 Innate immune system of horseshoe crab 4
Fig. 1-3 Schematic illustration of hemolymph coagulation cascade system in Lmulus 6
Fig. 1-4 Schematic illustration of endotoxin detection mechanisms in LAL system and rFC system 8
Fig. 1-5 Three dimensional structures of (A) TL-2 and (B) TL-5A 11
Fig. 1-6 Sequence alignment of tachylectins and Tachypleus plasma lectins 15
Fig. 1-7 Scanning electron micrographs of pathogens 20
Fig. 3-1 Plasmid map and cartoon maps of recombinant TPL2 73
Fig. 3-2 Small-scale expression of recombinant TPL2s 76
Fig. 3-3 Purification of SEP-TPL2 77
Fig. 3-4 Purification of endotoxin-free SEP-TPL2 78
Fig. 3-5 Molecular weight determination of SEP-TPL2 79
Fig. 3-6 Binding activity of SEP-TPL2 to LPS 80
Fig. 3-7 Binding activity of SEP-TPL2 to S. aureus LTA and S. faecalis LTA 81
Fig. 3-8 Comparison of PAMP-binding activity between SEP-TPL2 and endotoxin-free SEP-TPL2 82
Fig. 3-9 Binding activity of SEP-TPL2 to Gram-negative bacteria 83
Fig. 3-10 Binding activity of SEP-TPL2 to Gram-positive bacteria 84
Fig. 3-11 Dose-dependent binding of SEP-TPL2 to bacteria 85
Fig. 3-12 Binding activity of SEP-TPL2 to clinically isolated bacteria 86
Fig. 3-13 Binding activity of SEP-TPL2 to L-rhamnose 88
Fig. 3-14 P. aeruginosa binding activity of SEP-TPL2 in the presence of L-rhamnose 89
Fig. 3-15 P. aeruginosa binding activity of SEP-TPL2 in the presence of Rhamnosoft® HP 90
Fig. 3-16 L. monocytogenes binding activity of SEP-TPL2 in the presence of Rhamnosoft® HP 92
Fig. 3-17 Binding activity of SEP-TPL2 to Rhamnosoft® HP 94
Fig. 3-18 Antibacterial activity of SEP-TPL2 95
Fig. 3-19 Characterization of SEP-TPL2 species by SDS-PAGE and analytical ultracentrifugation 96

 
List of Tables
Table 1-1 Native tachylectins from Japanese T. tridentatus 12
Table 1-2 Fusion partners for solubility enhancement of recombinant proteins 17
Table 1-3 Summary of native and two recombinant TPL2s 18
Table 1-4 Summary of characteristics of pathogens 21
Table 1-5 Summary of pathogen detection methods 23
Table 3-1 Chemical structures of PAMPs of SEP-TPL2 binding bacteria 98
Table 4-1 Comparison among purification of TPL2 99
Table 4-2 Comparison PAMP binding activity between ySBD-SEP-TPL2 and SEP-TPL2 99
Table 4-3 Summary of antibiotic resistance of clinical pathogens 100
Table 4-4 Analytical methods of PAMP components of rhamnose-containing bacteria 101
Table 4-5 Summary of antibacterial lectins 102
Table 4-6 Prices of endotoxin detection kits 103
Table 4-7 Summary of tachylectins and Tachypleus plasma lectins 104


List of Appendices
Appendix Fig. 1 Small scale expression of recombinant TPL2 105
Appendix Fig. 2 Amplification of DNA fragment encoding n7-tpl2 and c7-tpl2 by PCR 106
Appendix Fig. 3 Verification of pET23a-n7-tpl2 and pET23a-c7-tpl2 by in situ PCR 108
Appendix Fig. 4 Comparison of Roche blocking reagent and BSA in LPS-binding ELISA for blocking 110
Appendix Fig. 5 Comparison binding activity of ySBD-SEP-TPL2 and SEP-TPL2 to PAMPs 111
Appendix Fig. 6 Characterization of ySBD-SEP-TPL2 species by SDS-PAGE and analytical ultracentrifugation 112
Appendix Fig. 7 Structure of Rhamnosoft® HP 114
Appendix Fig. 8 Multiple sequence alignment of SEP-TPL2 and RBLs 115

References
1 Hoebe, K., Janssen, E. and Beutler, B. (2004) The interface between innate and adaptive immunity. Nature immunology. 5, 971-974
2 Iwanaga, S. and Lee, B. L. (2005) Recent advances in the innate immunity of invertebrate animals. Journal of biochemistry and molecular biology. 38, 128-150
3 Bang, F. B. (1956) A bacterial disease of Limulus polyphemus. Bulletin of the Johns Hopkins Hospital. 98, 325-351
4 Medzhitov, R. and Janeway, C., Jr. (2000) Innate immune recognition: mechanisms and pathways. Immunological reviews. 173, 89-97
5 Vollmer, W., Blanot, D. and de Pedro, M. A. (2008) Peptidoglycan structure and architecture. FEMS microbiology reviews. 32, 149-167
6 Ulevitch, R. J. (1993) Recognition of bacterial endotoxins by receptor-dependent mechanisms. Advances in immunology. 53, 267-289
7 Morath, S., von Aulock, S. and Hartung, T. (2005) Structure/function relationships of lipoteichoic acids. Journal of endotoxin research. 11, 348-356
8 Sugumaran, M. (2002) Comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society. 15, 2-9
9 Iwanaga, S., Morita, T., Harada, T., Nakamura, S., Niwa, M., Takada, K., Kimura, T. and Sakakibara, S. (1978) Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis. 7, 183-188
10 Fujita, T. (2002) Evolution of the lectin-complement pathway and its role in innate immunity. Nature reviews. Immunology. 2, 346-353
11 Aderem, A. and Ulevitch, R. J. (2000) Toll-like receptors in the induction of the innate immune response. Nature. 406, 782-787
12 Bogdan, C., Rollinghoff, M. and Diefenbach, A. (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Current opinion in immunology. 12, 64-76
13 Rensen, P. C., de Vrueh, R. L., Kuiper, J., Bijsterbosch, M. K., Biessen, E. A. and van Berkel, T. J. (2001) Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting. Advanced drug delivery reviews. 47, 251-276
14 Morath, S., Geyer, A. and Hartung, T. (2001) Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J Exp Med. 193, 393-397
15 Xia, X. (2000) Phylogenetic relationship among horseshoe crab species: effect of substitution models on phylogenetic analyses. Systematic biology. 49, 87-100
16 Muta, T. and Iwanaga, S. (1996) The role of hemolymph coagulation in innate immunity. Current opinion in immunology. 8, 41-47
17 Iwanaga, S. and Kawabata, S. (1998) Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab. Frontiers in bioscience : a journal and virtual library. 3, D973-984
18 Iwanaga, S., Kawabata, S. and Muta, T. (1998) New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. Journal of biochemistry. 123, 1-15
19 Iwanaga, S. (1993) The limulus clotting reaction. Current opinion in immunology. 5, 74-82
20 Levin, J., Tomasulo, P. A. and Oser, R. S. (1970) Detection of endotoxin in human blood and demonstration of an inhibitor. The Journal of laboratory and clinical medicine. 75, 903-911
21 Ding, J. L. and Ho, B. (2001) A new era in pyrogen testing. Trends in biotechnology. 19, 277-281
22 Widener, J. W. and Barlow, R. B. (1999) Decline of a horseshoe crab population on Cape Cod. The Biological bulletin. 197, 300-302
23 Morrison, D. C. and Jacobs, D. M. (1976) Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry. 13, 813-818
24 Weigele, P. R., Scanlon, E. and King, J. (2003) Homotrimeric, beta-stranded viral adhesins and tail proteins. Journal of bacteriology. 185, 4022-4030
25 Ali, S. A., Iwabuchi, N., Matsui, T., Hirota, K., Kidokoro, S., Arai, M., Kuwajima, K., Schuck, P. and Arisaka, F. (2003) Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4. Biophysical journal. 85, 2606-2618
26 Kuo, T. H., Chuang, S. C., Chang, S. Y. and Liang, P. H. (2006) Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping. The Biochemical journal. 393, 757-766
27 Saito, T., Kawabata, S., Hirata, M. and Iwanaga, S. (1995) A novel type of limulus lectin-L6. Purification, primary structure, and antibacterial activity. The Journal of biological chemistry. 270, 14493-14499
28 Okino, N., Kawabata, S., Saito, T., Hirata, M., Takagi, T. and Iwanaga, S. (1995) Purification, characterization, and cDNA cloning of a 27-kDa lectin (L10) from horseshoe crab hemocytes. The Journal of biological chemistry. 270, 31008-31015
29 Beisel, H. G., Kawabata, S., Iwanaga, S., Huber, R. and Bode, W. (1999) Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus. The EMBO journal. 18, 2313-2322
30 Inamori, K., Saito, T., Iwaki, D., Nagira, T., Iwanaga, S., Arisaka, F. and Kawabata, S. (1999) A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides. The Journal of biological chemistry. 274, 3272-3278
31 Saito, T., Hatada, M., Iwanaga, S. and Kawabata, S. (1997) A newly identified horseshoe crab lectin with binding specificity to O-antigen of bacterial lipopolysaccharides. The Journal of biological chemistry. 272, 30703-30708
32 Honda, S., Kashiwagi, M., Miyamoto, K., Takei, Y. and Hirose, S. (2000) Multiplicity, structures, and endocrine and exocrine natures of eel fucose-binding lectins. The Journal of biological chemistry. 275, 33151-33157
33 Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S. N., Iwanaga, S. and Kawabata, S. (1999) Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen. Proceedings of the National Academy of Sciences of the United States of America. 96, 10086-10091
34 Norman Kairies*†, H.-G. B., Pablo Fuentes-Prior*, Ryoko Tsuda‡, Tatsushi Muta‡, Sadaaki Iwanaga‡, and Wolfram Bode*, R. H., and Shun-ichiro Kawabata‡§. (2001) The 2.0-Å crystal structure of tachylectin 5A provides evidence for the common origin of the innate immunity and the blood coagulation systems. Biochemistry. 98, 13519-13524
35 Chiou, S. T., Chen, Y. W., Chen, S. C., Chao, C. F. and Liu, T. Y. (2000) Isolation and characterization of proteins that bind to galactose, lipopolysaccharide of Escherichia coli, and protein A of Staphylococcus aureus from the hemolymph of Tachypleus tridentatus. J Biol Chem. 275, 1630-1634
36 Chen, S. C., Yen, C. H., Yeh, M. S., Huang, C. J. and Liu, T. Y. (2001) Biochemical properties and cDNa cloning of two new lectins from the plasma of Tachypleus tridentatus: Tachypleus plasma lectin 1 and 2+. J Biol Chem. 276, 9631-9639
37 Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J. and Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics (Oxford, England). 23, 2947-2948
38 Chou, W. I., Pai, T. W., Liu, S. H., Hsiung, B. K. and Chang, M. D. (2006) The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding. The Biochemical journal. 396, 469-477
39 Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstorff, P. and Svensson, B. (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochimica et biophysica acta. 1543, 275-293
40 Tung, J. Y., Chang, M. D., Chou, W. I., Liu, Y. Y., Yeh, Y. H., Chang, F. Y., Lin, S. C., Qiu, Z. L. and Sun, Y. J. (2008) Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path. The Biochemical journal. 416, 27-36
41 Lin, S. C., Lin, I. P., Chou, W. I., Hsieh, C. A., Liu, S. H., Huang, R. Y., Sheu, C. C. and Chang, M. D. (2009) CBM21 starch-binding domain: a new purification tag for recombinant protein engineering. Protein expression and purification. 65, 261-266
42 Low, E.-L. (2010) Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2. National Tsing Hua University Master Thesis
43 Hsieh, C.-A. (2010) Expression and purification of recombinant human interferon-γ and interleukin-12 employing starch binding domain. National Tsing Hua University Master Thesis
44 Ghosh, S., Rasheedi, S., Rahim, S. S., Banerjee, S., Choudhary, R. K., Chakhaiyar, P., Ehtesham, N. Z., Mukhopadhyay, S. and Hasnain, S. E. (2004) Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli. BioTechniques. 37, 418, 420, 422-413
45 Esposito, D. and Chatterjee, D. K. (2006) Enhancement of soluble protein expression through the use of fusion tags. Current opinion in biotechnology. 17, 353-358
46 Georgiou, G. and Valax, P. (1996) Expression of correctly folded proteins in Escherichia coli. Current opinion in biotechnology. 7, 190-197
47 Qing, G., Ma, L. C., Khorchid, A., Swapna, G. V., Mal, T. K., Takayama, M. M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G. T., Ikura, M. and Inouye, M. (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nature biotechnology. 22, 877-882
48 di Guana, C., Lib, P., Riggsa, P. D. and Inouyeb, H.
49 Nygren, P. A., Stahl, S. and Uhlen, M. (1994) Engineering proteins to facilitate bioprocessing. Trends in biotechnology. 12, 184-188
50 LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F. and McCoy, J. M. (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/technology (Nature Publishing Company). 11, 187-193
51 Davis, G. D., Elisee, C., Newham, D. M. and Harrison, R. G. (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnology and bioengineering. 65, 382-388
52 Marblestone, J. G., Edavettal, S. C., Lim, Y., Lim, P., Zuo, X. and Butt, T. R. (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein science : a publication of the Protein Society. 15, 182-189
53 Zhang, Y. B., Howitt, J., McCorkle, S., Lawrence, P., Springer, K. and Freimuth, P. (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein expression and purification. 36, 207-216
54 Lin, S. C., Liu, W. T., Liu, S. H., Chou, W. I., Hsiung, B. K., Lin, I. P., Sheu, C. C. and Dah-Tsyr Chang, M. (2007) Role of the linker region in the expression of Rhizopus oryzae glucoamylase. BMC Biochem. 8, 9
55 Santos, R. L., Zhang, S., Tsolis, R. M., Kingsley, R. A., Garry Adams, L. and Bäumler, A. J. (2001) Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes and Infection. 3, 335-1344
56 Hook, E. W. (1961) Salmonellosis: certain factors influencing the interaction of Salmonella and the human host. Bulletin of the New York Academy of Medicine. 37, 499-512
57 Brisse, S. and Verhoef, J. (2001) Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. International journal of systematic and evolutionary microbiology. 51, 915-924
58 Hawser, S. P., Bouchillon, S. K., Hoban, D. J., Badal, R. E., Hsueh, P. R. and Paterson, D. L. (2009) Emergence of high levels of extended-spectrum-beta-lactamase-producing gram-negative bacilli in the Asia-Pacific region: data from the Study for Monitoring Antimicrobial Resistance Trends (SMART) program, 2007. Antimicrobial agents and chemotherapy. 53, 3280-3284
59 Högenauer, C., Langner, C., Beubler, E., Lippe, I. T., Schicho, R., Gorkiewicz, G., Krause, R., Gerstgrasser, N., Krejs, G. J. and Hinterleitner, T. A. (2006) Klebsiella oxytoca as a Causative Organism of Antibiotic-Associated Hemorrhagic Colitis. New England Journal of Medicine. 355, 2418-2426
60 Supparatpinyo, K. S., T. . (2005) Pandrug-Resistant Acinetobacter baumannii at Maharaj Nakorn Chiang Mai Hospital. 22, 1-8
61 Levin, A. S., Barone, A. A., Penco, J., Santos, M. V., Marinho, I. S., Arruda, E. A., Manrique, E. I. and Costa, S. F. (1999) Intravenous colistin as therapy for nosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 28, 1008-1011
62 Perez, F., Hujer, A. M., Hujer, K. M., Decker, B. K., Rather, P. N. and Bonomo, R. A. (2007) Global Challenge of Multidrug-Resistant Acinetobacter baumannii. Antimicrobial agents and chemotherapy. 51, 3471-3484
63 Boulbitch, A., Quinn, B. and Pink, D. (2000) Elasticity of the rod-shaped gram-negative eubacteria. Physical review letters. 85, 5246-5249
64 Murphy, T. F., Brauer, A. L., Eschberger, K., Lobbins, P., Grove, L., Cai, X. and Sethi, S. (2008) Pseudomonas aeruginosa in chronic obstructive pulmonary disease. American journal of respiratory and critical care medicine. 177, 853-860
65 Roche, O., Beuhorry-Sassus, F., Boillot, A., Dupont, M. J., Plesiat, P., Talon, D., Cahn, J. Y. and Michel-Briand, Y. (1995) [Pseudomonas aeruginosa septicemia. Host-related risk factors in 82 episodes]. Presse medicale (Paris, France : 1983). 24, 1164-1166
66 Croce, M. A. (2000) Diagnosis of acute respiratory distress syndrome and differentiation from ventilator-associated pneumonia. American journal of surgery. 179, 26S-29S; discussion 30S
67 Wang, J., Liu, Y., Wan, D., Fang, X., Li, T., Guo, Y., Chang, D., Su, L., Wang, Y., Zhao, J. and Liu, C. (2012) Whole-genome sequence of Staphylococcus aureus strain LCT-SA112. Journal of bacteriology. 194, 4124
68 Sethi, S., Evans, N., Grant, B. J. and Murphy, T. F. (2002) New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. The New England journal of medicine. 347, 465-471
69 Chickering, H. T., Park, J. H. and Jr. (1919) STaphylococcus aureus pneumonia. Journal of the American Medical Association. 72, 617-626
70 Aguilar J Fau - Urday-Cornejo, V., Urday-Cornejo V Fau - Donabedian, S., Donabedian S Fau - Perri, M., Perri M Fau - Tibbetts, R., Tibbetts R Fau - Zervos, M. and Zervos, M. Staphylococcus aureus meningitis: case series and literature review.
71 Baudouin, S. V. The pulmonary physician in critical care . 3: critical care management of community acquired pneumonia.
72 Eller, J. r., Ede, A., Schaberg, T., Niederman, M. S., Mauch, H. and Lode, H. (1998) Infective exacerbations of chronic bronchitis : Relation between bacteriologic etiology and lung function. CHEST Journal. 113, 1542-1548
73 Sethi S Fau - Evans, N., Evans N Fau - Grant, B. J. B., Grant Bj Fau - Murphy, T. F. and Murphy, T. F. New strains of bacteria and exacerbations of chronic obstructive pulmonary disease.
74 Bogaert, D., De Groot, R. and Hermans, P. W. (2004) Streptococcus pneumoniae colonisation: the key to pneumococcal disease. The Lancet infectious diseases. 4, 144-154
75 Hu R Fau - Li, J., Li J Fau - Yao, K., Yao K Fau - Miao, M., Miao M Fau - Zhu, K., Zhu K Fau - Liu, Z. and Liu, Z. Listeria septicemia accompanied by central nervous system involvement in a patient with multiple myeloma and secondary diabetes.
76 Bannister, B. A. (1987) Listeria monocytogenes meningitis associated with eating soft cheese. The Journal of infection. 15, 165-168
77 Garcia-Montero M Fau - Rodriguez-Garcia, J. L., Rodriguez-Garcia Jl Fau - Calvo, P., Calvo P Fau - Gonzalez, J. M., Gonzalez Jm Fau - Fernandez-Garrido, M., Fernandez-Garrido M Fau - Loza, E., Loza E Fau - Serrano, M. and Serrano, M. Pneumonia caused by Listeria monocytogenes.
78 Volkmann, M., Skiebe, E., Kerrinnes, T., Faber, F., Lepka, D., Pfeifer, Y., Holland, G., Bannert, N. and Wilharm, G. (2010) Orbus hercynius gen. nov., sp. nov., isolated from faeces of wild boar, is most closely related to members of the orders 'Enterobacteriales' and Pasteurellales. International journal of systematic and evolutionary microbiology. 60, 2601-2605
79 Bharali, P., Saikia, J. P., Ray, A. and Konwar, B. K. (2013) Rhamnolipid (RL) from Pseudomonas aeruginosa OBP1: a novel chemotaxis and antibacterial agent. Colloids and surfaces. B, Biointerfaces. 103, 502-509
80 de Breij, A., Dijkshoorn, L., Lagendijk, E., van der Meer, J., Koster, A., Bloemberg, G., Wolterbeek, R., van den Broek, P. and Nibbering, P. (2010) Do biofilm formation and interactions with human cells explain the clinical success of Acinetobacter baumannii? PloS one. 5, e10732
81 Choi, U. K., Lee, O. H., Lim, S. I. and Kim, Y. C. (2010) Optimization of Antibacterial Activity of Perilla frutescens var. acuta Leaf against Pseudomonas aeruginosa Using the Evolutionary Operation-Factorial Design Technique. International journal of molecular sciences. 11, 3922-3932
82 McLeod, J. W. and Gordon, J. (1922) Production of Hydrogen Peroxide by Bacteria. The Biochemical journal. 16, 499-506
83 Dieuleveux, V., Lemarinier, S. and Gueguen, M. (1998) Antimicrobial spectrum and target site of D-3-phenyllactic acid. International journal of food microbiology. 40, 177-183
84 Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K. and Adley, C. (2010) An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnology advances. 28, 232-254
85 Ayçiçek, H., Aydoğan, H., Küçükkaraaslan, A., Baysallar, M. and Başustaoğlu, A. C. (2004) Assessment of the bacterial contamination on hands of hospital food handlers. Food Control. 15, 253-259
86 Al-Ghazali, M. R. and Al-Azawi, S. K. (1986) Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq. The Journal of applied bacteriology. 60, 251-254
87 Bennett, R. W. (2005) Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology. Journal of food protection. 68, 1264-1270
88 Abdel-Hamid, I., Ivnitski, D., Atanasov, P. and Wilkins, E. (1999) Highly sensitive flow-injection immunoassay system for rapid detection of bacteria. Analytica Chimica Acta. 399, 99-108
89 Magliulo, M., Simoni, P., Guardigli, M., Michelini, E., Luciani, M., Lelli, R. and Roda, A. (2007) A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria. Journal of agricultural and food chemistry. 55, 4933-4939
90 Taylor, A. D., Ladd, J., Yu, Q., Chen, S., Homola, J. and Jiang, S. (2006) Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor. Biosensors & bioelectronics. 22, 752-758
91 Iqbal, S. S., Mayo, M. W., Bruno, J. G., Bronk, B. V., Batt, C. A. and Chambers, J. P. (2000) A review of molecular recognition technologies for detection of biological threat agents. Biosensors & bioelectronics. 15, 549-578
92 Chen, C.-S. and Durst, R. A. (2006) Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles. Talanta. 69, 232-238
93 Abdel-Hamid, I., Ivnitski, D., Atanasov, P. and Wilkins, E. (1999) Highly sensitive flow-injection immunoassay system for rapid detection of bacteria. Analytica Chimica Acta. 399, 99-108
94 Toze, S. (1999) PCR and the detection of microbial pathogens in water and wastewater. Water Research. 33, 3545-3556
95 Rahn, K., De Grandis, S. A., Clarke, R. C., McEwen, S. A., Galan, J. E., Ginocchio, C., Curtiss, R., 3rd and Gyles, C. L. (1992) Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and cellular probes. 6, 271-279
96 McConnell, M. J., Perez-Ordonez, A., Perez-Romero, P., Valencia, R., Lepe, J. A., Vazquez-Barba, I. and Pachon, J. (2012) Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment. Journal of clinical microbiology. 50, 1412-1414
97 Zhang, Y., Isaacman, D. J., Wadowsky, R. M., Rydquist-White, J., Post, J. C. and Ehrlich, G. D. (1995) Detection of Streptococcus pneumoniae in whole blood by PCR. Journal of clinical microbiology. 33, 596-601
98 Chen, Y. and Knabel, S. J. (2007) Multiplex PCR for simultaneous detection of bacteria of the genus Listeria, Listeria monocytogenes, and major serotypes and epidemic clones of L. monocytogenes. Applied and environmental microbiology. 73, 6299-6304
99 Reichelt, P., Schwarz, C. and Donzeau, M. (2006) Single step protocol to purify recombinant proteins with low endotoxin contents. Protein expression and purification. 46, 483-488
100 Jain, K. K. (2005) The role of nanobiotechnology in drug discovery. Drug Discov Today. 10, 1435-1442
101 Hong, C.-Y., Wu, C. C., Chiu, Y. C., Yang, S. Y., Horng, H. E. and Yang, H. C. (2006) Magnetic susceptibility reduction method for magnetically labeled immunoassay. Applied Physics Letters. 88, 212512
102 Hong, C.-Y., Wu, C. C., Chiu, Y. C., Yang, S. Y., Horng, H. E. and Yang, H. C. (2006) Magnetic susceptibility reduction method for magnetically labeled immunoassay. Applied Physics Letters. 88, 212512
103 Giraud, M. F. and Naismith, J. H. (2000) The rhamnose pathway. Current opinion in structural biology. 10, 687-696
104 Rocchetta, H. L., Burrows, L. L. and Lam, J. S. (1999) Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol Mol Biol Rev. 63, 523-553
105 Eckstein, T. M., Silbaq, F. S., Chatterjee, D., Kelly, N. J., Brennan, P. J. and Belisle, J. T. (1998) Identification and recombinant expression of a Mycobacterium avium rhamnosyltransferase gene (rtfA) involved in glycopeptidolipid biosynthesis. J Bacteriol. 180, 5567-5573
106 Collins, B. E. and Paulson, J. C. (2004) Cell surface biology mediated by low affinity multivalent protein-glycan interactions. Curr Opin Chem Biol. 8, 617-625
107 Chieh, J. J., Yang, S. Y., Jian, Z. F., Wang, W. C., Horng, H. E. (2008) Hyper-high-sensitivity wash-free magnetoreduction assay on biomolecules using high-Tc superconducting quantum interference devices. Journal of Applied Physics. 103, 014703
108 Yang, S. Y., Chieh, J. J., Wang, W. C., Yu, C. Y., Lan, C. B., Chen, J. H., Horng, H. E., Hong, C. Y., Yang, H. C. and Huang, W. (2008) Ultra-highly sensitive and wash-free bio-detection of H5N1 virus by immunomagnetic reduction assays. J Virol Methods. 153, 250-252
109 Yang, S. Y., Wang, W. C., Lan, C. B., Chen, C. H., Chieh, J. J., Horng, H. E., Hong, C. Y., Yang, H. C., Tsai, C. P., Yang, C. Y., Cheng, I. C. and Chung, W. C. (2010) Magnetically enhanced high-specificity virus detection using bio-activated magnetic nanoparticles with antibodies as labeling markers. J Virol Methods. 164, 14-18
110 Andres, E., Molinari, J., Peterszegi, G., Mariko, B., Ruszova, E., Velebny, V., Faury, G. and Robert, L. (2006) Pharmacological properties of rhamnose-rich polysaccharides, potential interest in age-dependent alterations of connectives tissues. Pathol Biol (Paris). 54, 420-425
111 Riou-Gotta, M. O., Fournier, E., Danzon, A., Pelletier, F., Levang, J., Mermet, I., Blanc, D., Humbert, P. and Aubin, F. (2009) Rare skin cancer: a population-based cancer registry descriptive study of 151 consecutive cases diagnosed between 1980 and 2004. Acta Oncol. 48, 605-609
112 Faury, G., Ruszova, E., Molinari, J., Mariko, B., Raveaud, S., Velebny, V. and Robert, L. (2008) The alpha-L-Rhamnose recognizing lectin site of human dermal fibroblasts functions as a signal transducer: modulation of Ca2+ fluxes and gene expression. Biochim Biophys Acta. 1780, 1388-1394
113 Hardy, C. M. and Mobbs, K. J. (1999) Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine. Molecular reproduction and development. 52, 216-224
114 Yigit, H., Queenan, A. M., Rasheed, J. K., Biddle, J. W., Domenech-Sanchez, A., Alberti, S., Bush, K. and Tenover, F. C. (2003) Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing beta-lactamase KPC-2. Antimicrobial agents and chemotherapy. 47, 3881-3889
115 Huys, G., Cnockaert, M., Vaneechoutte, M., Woodford, N., Nemec, A., Dijkshoorn, L. and Swings, J. (2005) Distribution of tetracycline resistance genes in genotypically related and unrelated multiresistant Acinetobacter baumannii strains from different European hospitals. Research in microbiology. 156, 348-355
116 Gales, A. C., Reis, A. O. and Jones, R. N. (2001) Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. Journal of clinical microbiology. 39, 183-190
117 Vila, J., Ruiz, J., Goni, P. and Jimenez de Anta, T. (1997) Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. The Journal of antimicrobial chemotherapy. 39, 757-762
118 Poole, K., Krebes, K., McNally, C. and Neshat, S. (1993) Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. Journal of bacteriology. 175, 7363-7372
119 Lonks, J. R., Garau, J., Gomez, L., Xercavins, M., Ochoa de Echaguen, A., Gareen, I. F., Reiss, P. T. and Medeiros, A. A. (2002) Failure of macrolide antibiotic treatment in patients with bacteremia due to erythromycin-resistant Streptococcus pneumoniae. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 35, 556-564
120 Burrows, L. L., Charter, D. F. and Lam, J. S. (1996) Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster. Mol Microbiol. 22, 481-495
121 Li, Q. and Reeves, P. R. (2000) Genetic variation of dTDP-L-rhamnose pathway genes in Salmonella enterica. Microbiology. 146 ( Pt 9), 2291-2307
122 Chiang, S. L. and Mekalanos, J. J. (1999) rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization. Infection and immunity. 67, 976-980
123 Yamashita, Y., Tomihisa, K., Nakano, Y., Shimazaki, Y., Oho, T. and Koga, T. (1999) Recombination between gtfB and gtfC is required for survival of a dTDP-rhamnose synthesis-deficient mutant of Streptococcus mutans in the presence of sucrose. Infection and immunity. 67, 3693-3697
124 Ma, Y., Stern, R. J., Scherman, M. S., Vissa, V. D., Yan, W., Jones, V. C., Zhang, F., Franzblau, S. G., Lewis, W. H. and McNeil, M. R. (2001) Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose. Antimicrobial agents and chemotherapy. 45, 1407-1416
125 Lam, M. Y., McGroarty, E. J., Kropinski, A. M., MacDonald, L. A., Pedersen, S. S., Hoiby, N. and Lam, J. S. (1989) Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa. Journal of clinical microbiology. 27, 962-967
126 Bozue, J. A., Parthasarathy, N., Phillips, L. R., Cote, C. K., Fellows, P. F., Mendelson, I., Shafferman, A. and Friedlander, A. M. (2005) Construction of a rhamnose mutation in Bacillus anthracis affects adherence to macrophages but not virulence in guinea pigs. Microb Pathog. 38, 1-12
127 Hall, E. A. and Knox, K. W. (1965) Properties of the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei. The Biochemical journal. 96, 310-318
128 Coyette, J. and Ghuysen, J. M. (1970) Structure of the walls of Lactobacillus acidophilus strain 63 AM Gasser. Biochemistry. 9, 2935-2943
129 Wicken, A. J., Ayres, A., Campbell, L. K. and Knox, K. W. (1983) Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus. Journal of bacteriology. 153, 84-92
130 Sarkar, S., Lombardo, S. A., Herner, D. N., Talan, R. S., Wall, K. A. and Sucheck, S. J. (2010) Synthesis of a single-molecule L-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-L-rhamnose antibodies. Journal of the American Chemical Society. 132, 17236-17246
131 Chen, W., Gu, L., Zhang, W., Motari, E., Cai, L., Styslinger, T. J. and Wang, P. G. (2011) L-rhamnose antigen: a promising alternative to alpha-gal for cancer immunotherapies. ACS chemical biology. 6, 185-191
132 Lee, R. T. and Lee, Y. C. (2000) Affinity enhancement by multivalent lectin-carbohydrate interaction. Glycoconjugate journal. 17, 543-551
133 Janeway, C. A., Jr. (1989) Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor symposia on quantitative biology. 54 Pt 1, 1-13
134 Cheng, C. M., Martinez, A. W., Gong, J., Mace, C. R., Phillips, S. T., Carrilho, E., Mirica, K. A. and Whitesides, G. M. (2010) Paper-based ELISA. Angewandte Chemie (International ed. in English). 49, 4771-4774
135 Martinez, A. W., Phillips, S. T., Butte, M. J. and Whitesides, G. M. (2007) Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angewandte Chemie (International ed. in English). 46, 1318-1320
136 Carrilho, E., Phillips, S. T., Vella, S. J., Martinez, A. W. and Whitesides, G. M. (2009) Paper microzone plates. Analytical chemistry. 81, 5990-5998
137 Marques, M. R. F. and Barracco, M. A. (2000) Lectins, as non-self-recognition factors, in crustaceans. Aquaculture. 191, 23-44
138 Vasta, G. R., Ahmed, H., Fink, N. E., Elola, M. T., Marsh, A. G., Snowden, A. and Odom, E. W. (1994) Animal Lectins as Self/Non-Self Recognition Molecules. Annals of the New York Academy of Sciences. 712, 55-73
139 Gowda, N. M., Goswami, U. and Khan, M. I. (2008) T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria. Journal of invertebrate pathology. 99, 141-145
140 Tunkijjanukij, S. and Olafsen, J. A. (1998) Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization. Developmental and comparative immunology. 22, 139-150
141 Mukherjee, S., Partch, C. L., Lehotzky, R. E., Whitham, C. V., Chu, H., Bevins, C. L., Gardner, K. H. and Hooper, L. V. (2009) Regulation of C-type lectin antimicrobial activity by a flexible N-terminal prosegment. The Journal of biological chemistry. 284, 4881-4888
142 Yokoyama, K., Doi, Y., Yamane, K., Kurokawa, H., Shibata, N., Shibayama, K., Yagi, T., Kato, H. and Arakawa, Y. (2003) Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet. 362, 1888-1893
143 Giedraitiene, A., Vitkauskiene, A., Naginiene, R. and Pavilonis, A. (2011) Antibiotic resistance mechanisms of clinically important bacteria. Medicina (Kaunas). 47, 137-146
144 Masecar, B. L., Celesk, R. A. and Robillard, N. J. (1990) Analysis of acquired ciprofloxacin resistance in a clinical strain of Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy. 34, 281-286
145 Sweadner, K. J., Forte, M. and Nelsen, L. L. (1977) Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation. Applied and environmental microbiology. 34, 382-385
146 Ogawa, N., Yamamoto, H., Katamine, T. and Tajima, H. (1975) Purification and some properties of urokinase. Thrombosis et diathesis haemorrhagica. 34, 194-209
147 Liu, S., Tobias, R., McClure, S., Styba, G., Shi, Q. and Jackowski, G. (1997) Removal of endotoxin from recombinant protein preparations. Clinical biochemistry. 30, 455-463
148 Liu, S., Tobias, R., McClure, S., Styba, G., Shi, Q. and Jackowski, G. (1997) Removal of endotoxin from recombinant protein preparations. Clin Biochem. 30, 455-463
149 Aida, Y. and Pabst, M. J. (1990) Removal of endotoxin from protein solutions by phase separation using Triton X-114. Journal of immunological methods. 132, 191-195
150 Watanabe, Y., Tateno, H., Nakamura-Tsuruta, S., Kominami, J., Hirabayashi, J., Nakamura, O., Watanabe, T., Kamiya, H., Naganuma, T., Ogawa, T., Naude, R. J. and Muramoto, K. (2009) The function of rhamnose-binding lectin in innate immunity by restricted binding to Gb3. Developmental and comparative immunology. 33, 187-197
151 Tateno, H., Ogawa, T., Muramoto, K., Kamiya, H. and Saneyoshi, M. (2002) Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid. Bioscience, biotechnology, and biochemistry. 66, 604-612
152 Tateno, H., Shibata, Y., Nagahama, Y., Hirai, T., Saneyoshi, M., Ogawa, T., Muramoto, K. and Kamiya, H. (2002) Tissue-specific expression of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss). Biosci Biotechnol Biochem. 66, 1427-1430
153 Shina, N., Tateno, H., Ogawa, T., Muramoto, K. ,Saneyoshi, M. and Kamiya, H. (2002) Isolation and characterization of L-rhamnose-binding lectins from chum salmon (Oncorhynchus keta) eggs. Fisheries Science. 68
154 Watanabe, Y., Shiina, N., Shinozaki, F., Yokoyama, H., Kominami, J., Nakamura-Tsuruta, S., Hirabayashi, J., Sugahara, K., Kamiya, H., Matsubara, H., Ogawa, T. and Muramoto, K. (2008) Isolation and characterization of l-rhamnose-binding lectin, which binds to microsporidian Glugea plecoglossi, from ayu (Plecoglossus altivelis) eggs. Dev Comp Immunol. 32, 487-499
155 Nitta, K., Kawano, T., Sugawara, S. and Hosono, M. (2007) [Regulation of globotriaosylceramide (Gb3)-mediated signal transduction by rhamnose-binding lectin]. Yakugaku Zasshi. 127, 553-561
156 Hosono, M., Kawauchi, H., Nitta, K., Takayanagi, Y., Shiokawa, H., Mineki, R. and Murayama, K. (1993) Purification and characterization of Silurus asotus (catfish) roe lectin. Biol Pharm Bull. 16, 1-5
157 Tateno, H., Saneyoshi, A., Ogawa, T., Muramoto, K., Kamiya, H. and Saneyoshi, M. (1998) Isolation and characterization of rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily. J Biol Chem. 273, 19190-19197
158 Tateno, H., Ogawa, T., Muramoto, K., Kamiya, H. and Saneyoshi, M. (2002) Distribution and molecular evolution of rhamnose-binding lectins in Salmonidae: isolation and characterization of two lectins from white-spotted Charr (Salvelinus leucomaenis) eggs. Biosci Biotechnol Biochem. 66, 1356-1365
159 Fallarino, A., Mavrangelos, C., Stroeher, U. H. and Manning, P. A. (1997) Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1. Journal of bacteriology. 179, 2147-2153
160 Knox, K. W. and Hall, E. A. (1965) The Isolation of Oligosaccharides from the Cell-Wall Polysaccharide of Lactobacillus Casei, Serological Group C. The Biochemical journal. 94, 525-533