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In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy. Samir Jawhara, 2004.A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed . This study was divided into three steps: (i) construction of a plasmid to transform Escherichia coli into a bioluminescent variant, (ii) study of the bioluminescent E . coli in vitro as a function of temperature and pH, and (iii) determination of the MIC and the minimal bactericidal concentration of sulfamethoxazole-trimethoprim (SMX-TMP) . Finally, the efficacy of SMX-TMP was monitored in vivo in a cutaneous wound model (hairless rat) infected with this bioluminescent bacterium by using a bioluminescence imaging system . E . coli was transformed by electroporation with a shuttle vector (pRB474) containing the firefly (Photinus pyralis) luciferase gene, resulting in a bioluminescent phenotype . It was found that pH 5.0 was optimal for incorporation of the susbstrate D-luciferin for the luciferase reaction . In vitro, when the agar dilution method, standard turbidity assays, and the bioluminescence imaging system were used, E . coli(pRB474) proved to be susceptible to SMX-TMP . In vivo, at 4 h, SMX-TMP treatment was already efficient compared to no treatment (P = 0.034) . At 48 h, no bioluminescence was detected in the wound, demonstrating the susceptibility of E . coli to SMX-TMP . In conclusion, this study points out the advantage of using bioluminescence imaging to evaluate the effects of antibiotics for the treatment of acute infections in vivo in a nondestructive and noninvasive manner . Online Tool for Analysis of Denaturing Gradient Gel Electrophoresis Profiles. Florian Huber, 2004. Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin. Athena Sudom, 2003.The 1.8-Å resolution structure of the ATP-Mg2+-Ca2+-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg2+-Mn2+-pyruvate-PCK, except for the Ca2+ and Mn2+ binding sites . Ca2+ was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511) . This report found that Ca2+ bound only at the active site, indicating that there is likely no surface allosteric site . 45Ca2+ bound to PCK with a Kd of 85 µM and n of 0.92 . Glu508Gln Glu511Gln mutant PCK had normal activation by Ca2+ . Separate roles of Mg2+, which binds the nucleotide, and Ca2+, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca2+-bound structure . Partial trypsin digestion abolishes Ca2+ activation (desensitizes PCK) . N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396 . Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization . C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized . Phe409 and Phe413 interact with residues in the Ca2+ binding site, probably stabilizing the C terminus . Phe409Ala,  Phe409, Phe413Ala,
397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca2+ site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes .
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