We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3h,5h-cyclic nucleotide phosphodiesterases (type IV PDEs ; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5h-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis that is inadequately treated with currently available drugs (1). One potential strategy to better treat this disease is to reduce the influx of leukocytes into the joint, since recent studies have shown that the extent of neutrophil infiltration into the joints of RA patients precedes clinical signs of inflammation and is predictive of pain (2). There are a number of mediators of the inflammatory response that could potentially be responsible for neutrophil accumulation, but leukotriene B4 (LTB4) is an attractive target since it is a potent chemotactic agent for human neutrophils (3), is produced in large amounts by these cells, and is found in the synovial fluid of patients with RA (4).In this paper, we describe experiments to assess the role of LTB4 in a murine model of RA, collagen-induced arthritis. The immunological and histological features of this model resemble those seen in RA patients. The strategy we employed was to use a LTB4 receptor antagonist to block the biological effects of endogenously produced LTB4. Several potent and selective LTB4 receptor antagonists, with a variety of structural types, have been reported (reviewed in ref. 5). However, there are no data on the efficacy of these agents in models of arthritis. CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxychroman-7-yl]cyclopentane carboxylic acid, is a newly discovered LTB4 receptor antagonist that has a high affinity OH for human and mouse LTB4 receptors and has a long plasma half-life in the mouse, which allows the maintenance of pharmacologically relevant concentrations of the drug with a once daily dosing protocol. Here we report the use of CP-105,696 to demonstrate the importance of LTB4 as a critical mediator in the pathogenesis of murine collagen-induced arthritis. MATERIALS AND METHODSIn Vitro LTB4 Receptor Ligand Binding Assays. The procedure for [3H]LTB4 binding was adapted from the method of Cheng and co-workers (6). Binding was performed in 150 ,ul in a buffer containing 50 mM Tris HCl (pH 7.3), 10 mM MgCl2, 9% methanol, 0.7 nM [3H]LTB4 (5920-7400 GBq/ mmol; New England Nuclear), and either 0.83 mg (murine spleen) or 0.13 mg (human neutrophil) of membrane per ml. Unlabeled LTB4 was added at a concentration of 5 ,uM to determine nonspecific binding. Incubations were carried out in microtiter plates at 4°C for 30 min and the bound ligand was separated from the free ligand with a Betaplate apparatus (Pharmacia LKB) with double-thickness glass fiber filter mats.In Vitro Chemotaxis Assay. Chemotaxis assays were performed as described by Harvath and co-workers (7). Neutrophils were isolated according to the procedure of Ferrante and Thong (8...

Several quinolones and antitumor compounds were tested as inhibitors of purified calf thymus topoisomerase II in unknotting, catenation, radiolabeled DNA cleavage, and quantitative nonradiolabeled cleavage assays. The antitumor agents and ellipticine demonstrated drug-enhanced topoisomerase II DNA cleavage (the concentration of drug that induced 50% of the maximal DNA cleavage in the test system [CC50]) at levels of s5 ,ug/ml. Nalidixic acid, norfloxacin, and oxolinic acid did not induce significant topoisomerase II DNA cleavage, whereas ciprofloxacin did induce some cleavage above background levels. CP-67,015, a new 6,8-difluoro-7-pyridyl 4-quinolone which possesses potent antibacterial activity, inhibited bacterial DNA gyrase at 0.125 ,ug/ml in a nonradioactive DNA cleavage assay. Unlike other quinolones characterized to date, CP-67,015 was shown to strongly enhance topoisomerase II-induced radiolabeled DNA cleavage with a CC50 of 33 ,ug/ml and demonstrated cleavage in a nonradiolabeled DNA cleavage assay with a CC50 of 73 ,ug/ml. The topoisomerase 1I-mediated cleavage of DNA by CP-67,015 is consistent with its reported clastogenic effect on DNA in cell culture and its positive mutagenic response in mouse lymphoma cells. In vitro topoisomerase II catalytic and cleavage assays are useful for gaining preliminary information concerning the possible interaction(s) of some quinolones with eucaryotic topoisomerase II which may relate directly to their safety (mutagenicity, clastogenicity, or both) in human and veterinary medicinal usage.In vitro DNA cleavage mediated by mammalian topoisomerase II has been reported to occur (in the absence of drug) at high enzyme to DNA ratios (17,19) and to be enhanced by antitumor compounds such as 4'-(9-acridinylamino)methanesulfon-m-aniside (m-AMSA), ellipticine, 4,22,24,29,30,39). At the same time, these compounds have shown variable inhibitory effects on the catalytic activity of the enzyme (2, 4, 29). Since this class of drugs has also been associated with DNA breakage observed in cell culture, it has been suggested that enhanced topoisomerase II-mediated DNA cleavage is an important mechanism for the antitumor effects of these agents (4,6,20,21,24). Quinolone antibacterial agents are thought to exert their effects by a similar interference with bacterial DNA gyrase (6,14,23,33), the bacterial homolog of eucaryotic topoisomerase II (15,18,21,37). This class of antibacterial agents has recently been introduced into clinical use (1, 38). Since interaction with topoisomerase II is central to the activity of both quinolones and antitumor agents, it is of interest from a safety standpoint (5, 12, 13) to determine the effects of quinolone antibacterial agents on mammalian topoisomerase II, especially in light of the recent controversy over the mechanism of action of 4-quinolone antibacterial agents as to whether quinolones bind to DNA, gyrase, or the ternary complex of DNA gyrase (7,11,13,32,36).Several different assays have been developed by others (16-19, 21, 22, 27-29, 37, 40) ...

Strains of Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae that were resistant to ceftazidime (MIC > 16 ,ug/ml) but susceptible to BMY 28142 (MIC < 4 ,ug/ml) were found to contain higher levels of j-lactamase activity (50-to 3,340-fold) than control strains of the corresponding species. Ceftazidime was at least as resistant as BMY 28142 to hydrolysis by these enzymes. However, the apparent K, of BMY 28142 for each enzyme was larger (8-to >20-fold) than that of ceftazidime; i.e., the affinity of these enzymes for BMY 28142 appeared to be lower than that for ceftazidime. Thus, BMY 28142 was affected less than ceftazidime by a mechanism of resistance that depends, at least in part, on the relative affinities of cephalosporins for the j3-lactamases of these species. These results indicate that the affinity between a jI-lactamase and a cephalosporin may be a distinguishing factor in the evaluation of ,-lactamase-resistant cephalosporins and suggest that affinity can play a major role in susceptibility to highly ,(-lactamase-resistant cephalosporins.During routine evaluation of a new cephalosporin, BMY 28142 (5), occasional clinical isolates of Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae, as well as the P-99 strain of E. cloacae, were found to be resistant (MIC > 16 pug/ml) to ceftazidime but susceptible to BMY 28142 (MIC < 4 ,ug/ml). Resistance to cephalosporins that are intrinsically stable to P-lactamase hydrolysis has been correlated with high levels of ,B-lactamase activity (1, 2, 9-11, 14). The concept of trapping or nonhydrolytic interference was proposed to explain this apparently paradoxical phenomenon (8,10,11,14,15). Criticism of this concept, based on the premise that for this mechanism to operate the total number of P-lactamase molecules must exceed the number of P-lactam molecules (9), does not take into consideration the presence of the outer membrane barrier, as discussed by Vu and Nikaido (13). Their analysis, based on outer membrane permeability, kinetic parameters of the enzyme, affinity toward penicillin-binding proteins, and MIC, suggests that hydrolysis may be more important than nonhydrolytic binding for ,-lactams such as cefoperazone, cefoxitin, and cefotaxime (13). However, the results of their calculations for ceftazidime, which is much more resistant to hydrolysis than the other three cephalosporins, were less conclusive with respect to hydrolysis versus nonhydrolytic interference as the major determinant of resistance in the single E. cloacae strain that they examined.We therefore investigated the contribution of P-lactamaserelated factors to the difference in the relative activity of ceftazidime and BMY 28142 against some of these ceftazidime-resistant isolates. For each species one to three strains resistant to ceftazidime but susceptible to BMY 28142 were compared with a control strain for which MICs of BMY 28142 and ceftazidime were similar to the respective MIC50s * Corresponding author. t Present address:

No abstract