For this reason it is very likely that the protein could not function well with an unpaired cysteine residue in PI-II. Relative stability of the protein would have been regained only when the counterpart cysteine residue of the pair was also lost. It is known that pseudo genes usually do not react to selection and will likely rapidly accumulate mutations, except occasionally some segments may be picked up into functional genes through recombination. Most of the successive versions of the gene during the generation of Pi6C or Pi7C must have had somewhat beneficial functions for the plants during this mutation process in order for selection to occur and to avoid disruption of the open reading frames. In other words, they were rarely pseudo genes. The ratio of non-synonymous substitutions to the rate of synonymous substitutions can be used as an indicator of selective LDK378 ALK inhibitor pressure acting on a protein-coding gene. A Ka/Ks ratio greater than 1.0 is usually indicative of positive selection pressure. The evolution from the ancestor to Pi6C and Pi7C clearly occurred under positive selection with Ka/Ks ratios much greater than 1.0. As the emergence of Pi7C and Pi6C genes was clearly under positive selection, their intermediate gene versions are very likely to have been functional. The functional-to-functional evolution inferred from the analysis of these novel genes in the PI-II gene Vorinostat 149647-78-9 family may provide insights into the evolutionary process of many other genes. Increasing multidrug resistance in clinical isolates is currently a major problem in infection control. In particular, the resistance of multidrug resistant Pseudomonas aeruginosa to major antipseudomonal agents, such as carbapenems, quinolones, and aminoglycosides, has been demonstrated and is known to cause nosocomial outbreaks in Japan. P. aeruginosa has natural intrinsic resistance tendencies, and MDRPs have complex resistance mechanisms. In particular, multidrug efflux pumps, especially resistance-nodulation-cell division family pumps, can decrease the sensitivity of P. aeruginosa to various types of compounds. Twelve intrinsic efflux systems belonging to the RND family have been characterized from the genome sequence of P. aeruginosa, and in particular MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY efflux systems are known to have important roles in multidrug resistance. These systems can increase their resistance levels by acquiring additional resistance factors. During this period of new antibacterial agent scarcity, RND pump inhibitors appear useful for treating MDRP infections. The enhancing effects of an experimentally available efflux pump inhibitor, Phe-Arg-bnaphthylamide, on antibacterial activities of compounds in combination with several antibiotics have been published, although no clinically useful inhibitor is known. Recently, 3D structures of MexB and cocrystal structures of AcrB with various substrates have been resolved, and some information regarding their mechanisms of efflux is available. At present, rational approaches are being employed to develop potent efflux pump inhibitors. However, no 
satisfactory method to determine the efflux inhibitory activities of candidate compounds directly is available. Several fluorometric methods evaluating efflux pump inhibitors have been published using substrates of these pumps such as alanine b-naphthylamide, N-phenylnaphthylamine, ethidium bromide, and pyronin Y. Lomovskaya et al. used a related compound MC-002,595 instead of PAbN in the methods using alanine b-naphthylamide or N-phenylnaphthylamine.