Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to a family of structurally related peptides which have numerous functions in both neural and endocrine signaling. By homology screening, we cloned a novel gene sharing the highest homology with the NPY Y1 receptor gene from humans, rabbits, and several other species. This novel gene of rabbit encodes a functional NPY/PYY receptor, designated Y2b, which prefers NPY 13-36 rather than [Leu 31 ,Pro 34 ]NPY despite its higher identity with the Y1 receptor. Although, at low levels, mRNA was detected in the tissues and brain regions, including hypothalamus. Further, sequence data revealed that this gene is the orthologue of the recently cloned mouse novel NPY receptor, Y5. However, our study demonstrates that the receptor function of this gene has been inactivated in primates by a frameshift mutation occurring early in primate evolution. This novel NPY receptor represents the first neurotransmitter receptor identified that has universally lost its receptor function in primate species. Interestingly, despite its inactivation in humans, the transcripts were abundantly detected in the heart and skeletal muscle, suggesting a novel function of the human gene.Neuropeptide Y (NPY), 1 peptide YY (PYY), and pancreatic polypeptide (PP) belong to a family of structurally related 36-amino acid peptides which have functions in both neural and endocrine signaling. These peptides exert their actions via receptors on the targeted neurons and peripheral cells. Several receptor subtypes have been defined by their ability to bind NPY, PYY, PP, and derivatives of these peptides. Earlier studies classified this receptor family into at least three receptor subtypes, Y1, Y2, and Y3 (1). The existence of additional receptors were also proposed, "atypical" Y1 receptor (mediating the feeding response of NPY in hypothalamus), PP-preferring receptor (exerting PP activity), and PYY-preferring receptor (2, 3). Molecular cloning studies have revealed the structure of Y1, Y2, and Y4/PP1 receptors (4 -9). These are all heptahelix (seven-transmembrane regions) receptors which couple to G-proteins. The Y4/PP1 receptor has higher affinity for PP than PYY and NPY, suggesting this receptor to be a member of PP receptors.As yet unknown members of this NPY receptor family are expected to be identified by molecular cloning. Very recently, during the course of the present work, the cloning and characterization of mouse Y5 (10) and rat Y5 (11) receptors was reported. The mouse Y5 receptor has significant homology with the Y1 receptor, but the rat Y5 receptor has little identity with the previously cloned NPY receptors, showing that these receptors are not species orthologues, despite having the same name, Y5. In the present report, we describe the cloning of a novel gene sharing the highest homology with the Y1 receptor from humans, rabbits, and several other species. Our study revealed that this novel gene is the orthologue of the mouse Y5 receptor gene (10) and encodes functional ...

Inhibitory effects of five quinolones against DNA gyrases purified from four quinolone-resistant clinical isolates of Pseudomonas aeruginosa and the quinolone-susceptible strain PAO1 were examined. All of the quinolone-resistant strains tested were found to be DNA gyrase mutants. The 50% inhibitory concentrations (IC 50 s) of the quinolones for these DNA gyrases roughly correlated with their MICs. Interestingly, gyrase inhibition by DU-6859a was found to be significantly less affected by these mutations than inhibition by other currently available quinolones. To assess the enhanced activity shown by DU-6859a, the effects of quinolones with altered substituents at the N-1, C-7, and C-8 positions of the quinolone ring of DU-6859a were tested. Measurement of MICs for four DNA gyrase mutants and IC 50 s for their purified DNA gyrases showed that removal of the C-8 chlorine of DU-6859a significantly increased MICs and IC 50 s for DNA gyrase mutants. However, no deleterious effects were observed when either the fluorine on the cyclopropyl substituent at the N-1 position or the cyclopropyl ring at the C-7 substituent was removed. Moreover, removal of the C-8 chlorine also increased the MIC for 19 of 20 quinolone-resistant clinical isolates. Our results led to the conclusion that DU-6859a is much more active against quinolone-resistant clinical isolates of P. aeruginosa than other currently available quinolones, probably because of its strong inhibitory effects against mutant quinoloneresistant DNA gyrases, and that the C-8 chlorine is necessary for these potent effects., is a new fluoroquinolone with antibacterial activity significantly better than that of any currently available quinolone (13, 21). One of its unique characteristics is its activity against quinolone-resistant clinical isolates of Pseudomonas aeruginosa (21). Sato et al. reported that the activity of DU-6859a was roughly comparable to that of ciprofloxacin (CPFX) at the level of the MIC at which 50% of the isolates were inhibited, but its activity against ofloxacin-resistant strains was more than four times greater than that of CPFX (21). Since strains of P. aeruginosa are often isolated from clinical specimens and since the frequency of quinoloneresistant P. aeruginosa strains has increased (7), this high level of potency of DU-6859a has attracted considerable interest. It has been reported that the quinolone resistance mechanisms in P. aeruginosa involve chromosomal mutations, affecting drug permeability (3,6,11,(18)(19)(20) or the drug sensitivity of DNA gyrase (4), the main target of the quinolones (10). DNA gyrase is thought to be a tetramer composed of two subunits, A2 and B2 (10). Inoue et al. reported that alterations in the A subunit render P. aeruginosa resistant to quinolones (10). Several studies have indicated that the majority of quinolone-resistant clinical isolates of P. aeruginosa alter DNA gyrase (30, 31). In light of the predominant gyrase mutations found in quinolone-resistant clinical isolates, we postulated that the potent activity...

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In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, the subunit A and B proteins of DNA gyrase were separately purified from a crude extract of S. aureus FDA 209-P. The reconstituted enzyme exhibited ATP-dependent DNA supercoiling activity. The inhibitory effects of quinolones on the supercoiling activity of the purified enzyme were measured by the quantitative electrophoresis method (17), using plasmid DNA, pBR322 or pUB110, as substrates and expressed as the 50% inhibitory concentrations (IC50s). The IC50s of ofioxacin, DR-3355 (1-ofloxacin), ciprofloxacin, tosufloxacin, sparfloxacin, and DS-4524, a new quinolone derivative, for pBR322 were 63.0, 37.8, 30.5, 46.0, 28.5, and 3.2 ,ug/mI, respectively. These values were closely correlated with antibacterial activity (MIC), with correlation coefficients of 0.953 for pBR322 and 0.938 for pUB110. These results indicate that, in S. aureus, as in gram-negative bacteria, DNA gyrase is likely to be a major target enzyme of quinolones.The DNA gyrase, which catalyzes the ATP-dependent supercoiling activity of relaxed covalently closed circular DNA (4, 5), of gram-negative rods is known to be the major target for quinolones. There is a parallel relationship between the antibacterial activities of new quinolones and their inhibitory actions against DNA gyrase from the bacteria (1, 8,10,11,14,17). However, it is inconclusive whether quinolones inhibit the supercoiling activities of DNA gyrases of gram-positive cocci, e.g., Staphylococcus aureus and Micrococcus luteus (2,13,20, 22). Zweerink and Edison (22) reported that the inhibitory potencies of quinolones against DNA gyrase did not always correlate with their antimicrobial potencies against M. luteus. Fu et al. (2) indicated that quinolones scarcely inhibited the supercoiling activity of M. luteus DNA gyrase, while novobiocin and coumermycin Al fully inhibited this activity. Georgopapadakou and Dix (6) found that the supercoiling activity of the purified DNA gyrase of S. aureus ATCC 25923 was scarcely inhibited by norfloxacin, ofloxacin, or ciprofloxacin. In contrast, Takahata and Nishino (20) showed that the DNA gyrase from S. aureus was strongly inhibited by ofloxacin and ciprofloxacin at concentrations similar to their MICs.In this report, we describe the correlation of the antibacterial activities of quinolones, including those of a newly introduced compound, with their inhibitory activities against purified DNA gyrase of S. aureus FDA 209-P.Ofloxacin (18), DR-3355 (1-ofloxacin) (11), DR-3354 (dofloxacin) (11), DS-4524 {(-)-7-[3-(1-aminoethyl)-1-pyrrolidinyl]-6,8-difluoro-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid [12]}, ciprofloxacin (21), tosufloxacin (3), lomefloxacin (7), and sparfloxacin (15) were synthesized at Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. The MICs, the lowest concentrations of antimicrobial agents that inhibited visible growth after 18 h at 37°C, were determined by the agar dilution method with heart infusion agar (Nissui Seiyaku Co., Tokyo...

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