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PmrAB, a Two-Component Regulatory System of Pseudomonas aeruginosa That Modulates Resistance to Cationic Antimicrobial Peptides and Addition of Aminoarabinose to Lipid A. Samuel M. Moskowitz, 2004.Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated . The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium . Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance . Thus, P . aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions . Antibacterial Activities of the Cathelicidins Prophenin (Residues 62 to 79) and LL-37 in the Presence of a Lung Surfactant Preparation. Yuqin Wang, 2004.The antibacterial activities of the cathelicidin peptides LL-37 and an 18-residue C-terminal fragment of prophenin, corresponding to positions 62 to 79 of native prophenin (PF-18), were analyzed in the presence of a modified surfactant preparation isolated from minced porcine lungs . At low micromolar concentrations, both LL-37 and PF-18 showed significant activities against different serotypes of group B streptococci, with LL-37 being more active on a molar basis . The surfactant preparation at a concentration of 10 mg/ml partly blocked the antibacterial activity of 9 µM LL-37 and completely blocked the antibacterial activity of 9 µM PF-18 . However, 10 mg of the surfactant preparation per ml had only minor inhibitory effects on LL-37 and PF-18 at 90 µM . Addition of up to 900 µM PF-18 did not affect the surface properties of the surfactant preparation . These data suggest that surfactant preparations containing antimicrobial peptides could be useful for the local treatment of pulmonary infections . Double-Layer Plaque Assay for Quantification of Enteroviruses. Laura Mocé-Llivina, 2004.We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay . The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses . The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay . We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay  suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters
most probable number of cytopathogenic units > monolayer plaque assay . Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers . Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method . In addition, the pretreatment of cells with 5-iodo-2'-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification .
Stimulation of Menaquinone-Dependent Electron Transfer in the Respiratory Chain of Bacillus subtilis by Membrane Energization. N. Azarkina, 2002.At a pH of  7, respiration of Bacillus subtilis cells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone [CCCP]) and a K+-ionophore (valinomycin) . The same effect was observed with cell spheroplasts lacking the cell wall . The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+-valinomycin was below 100 nM . Either CCCP or valinomycin alone was much less efficient than the combination of the two . The inhibitory effect was easily reversible and depended specifically on the H+ and K+ concentrations in the medium . Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol . Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling . The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores . We conclude that the menaquinone-dependent electron transfer in the B . subtilis respiratory chain is facilitated, thermodynamically or kinetically, by membrane energization . A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant of B . subtilis lacking succinate:quinone reductase as well as for substrates other than succinate . Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration in B . subtilis are discussed .
Genome-Wide Analyses Revealing a Signaling Network of the RcsC-YojN-RcsB Phosphorelay System in Escherichia coli. Daisuke Hagiwara, 2003. Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp . Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein. Ute Heinemann, 2003.The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp . strain PCC6803 . The gene was amplified by PCR and cloned into an expression vector . The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate . The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits . The purified nitrilase converted various aromatic and aliphatic nitriles . The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile . From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed . The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents .
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