Nucleotide sequence analysis of the gyrA genes of 10 spontaneous quinolone-resistant gyrA mutants of Escherichia coli KL16, including four mutants examined previously, disclosed that quinolone resistance was caused by a point mutation within the region between amino acids 67 and 106, especially in the vicinity of amino acid 83, of the GyrA protein.Quinolones are considered to exert antibacterial activity by inhibiting DNA gyrase (EC 5.99.1.3), which catalyzes topological changes of DNA (4, 11). DNA gyrase of Escherichia coli consists of subunits A and B, which are the products of the gyrA and gyrB genes, respectively. Mutations in either gene can cause quinolone resistance (4,(15)(16)(17) were determined by dideoxy-chain termination (9) with phage M13mpl8 and M13mpl9 vectors. Table 1 shows the sites and types of mutations and the levels of resistance to quinolones of 10 quinolone-resistant gyrA mutants of E. coli KL16. Four mutants (N-51, P-18, P-10, and N-89) were analyzed previously (17). All 10 point mutations were considered to be solely responsible for quinolone resistance, because replacement of the 0.6-kilobase Sacl-SmaI fragment containing the mutations by the corresponding fragment from wild-type gyrA gene resulted in complete loss of quinolone resistance (data not shown). Sequencing of the 0.6-kilobase SacI-SmaI fragments of the mutant gyrA genes revealed that these mutations were located within a relatively small region (amino acids 67 through 106) of the A subunit, which we call a quinolone resistance-determining region. There were no other mutations in all of the sequenced fragments. Eight of the 10 mutations were in a limited area (amino acids 81 through 87) of the region; surprisingly, five mutations were situated at the same site of amino acid 83. The levels of resistance to quinolones seemed to be related to the mutation sites, because quinolone MICs were high in the decreasing order of MICs for mutants with mutations at amino acids 83, 87, 81, 84, 67, and 106. This result suggests the importance of an area around amino acid 83 of the gyrase A subunit for determining quinolone resistance.Amino acid changes detected at amino acid 83 were Ser to

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The The increase in methicillin-resistant Staphylococcus aureus is a serious problem because only a few effective agents are clinically available. Some quinolones have been used for the treatment of methicillin-resistant S. aureus infections, but the emergence of quinolone resistance has been reported elsewhere (32). Unlike the mechanism underlying the quinolone resistance of gram-negative bacteria such as Escherichia coli (2,7,9,11,12,15,27,31,(36)(37)(38)(39) and Pseudomonas aeruginosa (4,13,16,29,30,36,40)

Thirteen spontaneous quinolone-resistant gyrB mutants of Escherichia coli KL16, including two that were examined previously, were divided into two types according to their quinolone resistance patterns. Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones and were hypersusceptible to amphoteric quinolones. Nucleotide sequence analysis disclosed that all nine type 1 mutants had a point mutation from aspartic acid to asparagine at amino acid 426 and that all four type 2 mutants had a point mutation from lysine to glutamic acid at amino acid 447. Quinolones are a group of antibacterial agents whose target is DNA gyrase (EC 5.99.1.3), an enzyme that catalyzes topological changes of DNA (4). The DNA gyrase of Escherichia coli consists of two A and two B subunits, which are the products of the gyrA (48 min) and gyrB (83 min) genes, respectively (3,7,11,21,29). Mutations in the gyrA gene are as frequent as those in the gyrB gene in spontaneous quinolone-resistant mutants of E. coli KL16, although the majority of quinolone-resistant clinical E. coli isolates have gyrA mutations (23). In the gyrA gene, quinolone resistance is caused by a point mutation within the relatively narrow region of amino acids 67 to 106, which is called the quinolone resistance-determining region (34, 35). In the gyrB gene, two quinolone resistance-determining sites (amino acids 426 and 447) have been found (32,33). To obtain more information on the region responsible for quinolone resistance in the gyrB gene, 11 additional quinolone-resistant gyrB mutants of E. coli KL16 were analyzed.MATERIALS AND METHODS Strains. Quinolone-resistant mutants of E. coli KL16 were isolated by plating the organism on LB agar (18) containing nalidixic acid or enoxacin at four times the MIC, and gyrB mutants were identified by transformation with the wild-type gyrB gene as described previously (23).Reagents, plasmids, and phages. Nalidixic acid (14) Cloning and sequencing of the E. coli gyrB genes. HindIII DNA fragments of about 13 kb in size containing the gyrB gene were cloned from quinolone-resistant gyrB mutants of E. coli KL16 as described previously (33). Nucleotide sequences were determined by the dideoxy-chain termination method (17) by using phage M13mpl8 and M13mpl9 vectors. RESULTS AND DISCUSSIONThe levels of resistance or hypersusceptibility (the increase or decrease in MIC compared with that for E. coli KL16) to various quinolones of 13 quinolone-resistant gyrB mutants of E. coli KL16 are given in Table 1. The MICs of some quinolones for N-24 and N-31 were not identical to those reported previously (23, 32) but were within experimental fluctuations. All the mutants could be divided into two types with respect to their quinolone resistance. Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones, such as nalidixic acid, oxolinic acid, cinoxacin, piromidic acid, and flumequine but were hypersusceptible to amphoteric quinolones, ...

Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.

We characterized the absorption and short-term translocation of cadmium (Cd) in rice (Oryza sativa 'Nipponbare') quantitatively using serial images observed with a positron-emitting tracer imaging system. We fed a positron-emitting 107 Cd (halflife of 6.5 h) tracer to the hydroponic culture solution and noninvasively obtained serial images of Cd distribution in intact rice plants at the vegetative stage and at the grain-filling stage every 4 min for 36 h. The rates of absorption of Cd by the root were proportional to Cd concentrations in the culture solution within the tested range of 0.05 to 100 nM. It was estimated that the radial transport from the culture to the xylem in the root tissue was completed in less than 10 min. Cd moved up through the shoot organs with velocities of a few centimeters per hour at both stages, which was obviously slower than the bulk flow in the xylem. Finally, Cd arrived at the panicles 7 h after feeding and accumulated there constantly, although no Cd was observed in the leaf blades within the initial 36 h. The nodes exhibited the most intensive Cd accumulation in the shoot at both stages, and Cd transport from the basal nodes to crown root tips was observed at the vegetative stage. We conclude that the nodes are the central organ where xylem-to-phloem transfer takes place and play a pivotal role in the half-day travel of Cd from the soil to the grains at the grain-filling stage.