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Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.

Pseudomonas aeruginosa and Acinetobacter baumannii are frequently isolated in hospital outbreaks of nosocomial infections. In our hospital, among 1018 strains isolated one year in an intensive care unit, 84 strains (8.3%) of P. aeruginosa and 155 strains (15.2%) of A. baumannii were considered responsible for infections. The major problem related to these bacteria is their multiresistant characteristic which confers great difficulty in treating infections. We carried out a 24 h time-kill study to assess the bactericidal effect of three beta-lactams [imipenem (IPM), ticarcillin+clavulanic acid (TCC), piperacillin+tazobactam (PTB)] in combination with each other and with sulbactam (SUL) and amikacin (AKN) against 8 P. aeruginosa strains and 8 A. baumannii strains. The initial inoculum was 10(6) cfu/ml. Antibiotics were tested at clinically achievable concentrations: TCC (112 mg/l), PTB (100 mg/l), IPM (25 mg/l) and AKN (15 mg/l). The results showed: IMP + TCC + AKN = PTB + SUL + AKN = PTB + TCC + AKN > > IMP + SUL + AKN against P. aeruginosa; and PTB + SUL + AKN = PTB + TCC + AKN > IMP + SUL + AKN or IMP + TCC + AKN against A. baumannii. When infection due to these multiresistant strains was suspected, PTB + AKN combined with either TCC or SUL was bactericidal against both strains. These combinations appeared to be an alternative therapy in the treatment of undocumented nosocomial infections in intensive care units. These in vitro results are being evaluated in patients and seem to give good results for the moment.