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In Vitro Testing of Antimicrobial Activity of Bone Cement. Volker Alt, 2004.The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples . Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin . After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts . The medium was monitored in real time by recording a time proliferation curve for each well . Microplate testing revealed no antibacterial effect of plain bone cement . The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains . The effect of high-porosity silver was dose dependent . Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver . The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility . In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver . Chill Induction of the SigB-Dependent General Stress Response in Bacillus subtilis and Its Contribution to Low-Temperature Adaptation. Matthias Brigulla, 2003.A variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB of Bacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes . This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance . By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15°C) . Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of the sigB operon and the SigB regulon, respectively, in cells continuously exposed to low temperatures . In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress in B . subtilis . The physiological importance of the induction of the general stress response for the adaptation of B . subtilis to low temperatures is emphasized by the observation that growth of a sigB mutant is drastically impaired at 15°C . Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of the sigB mutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by which B . subtilis cells cope with chill stress . Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking. Karine Seyer, 2003.Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens . For a given process lethality (F7010 of 1, 3, and 5 min), the intracellular concentration of DnaK in E . coli varied with the heating temperature (50 or 55°C) . In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time . Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process . Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress . Indeed, E . coli cells that had undergone a sublethal heat shock (105 min at 55°C, F7010 = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F7010 = 5 min) . Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment . Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris. Mônica C. Triches Damaso, 2003.Highly efficient production of a Thermomyces lanuginosus IOC-4145 ß-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter . P . pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures . Furthermore, an optimization of enzyme production was carried out with a 23 factorial design . The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter . A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved . Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior . Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus . The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical . For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C . The high level of fully active recombinant xylanase obtained in P . pastoris makes this expression system attractive for fermentor growth and industrial applications .
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