Early asthmatic responses (EAR) and late asthmatic responses (LAR) to allergen are induced by the local release of a series of bronchoconstrictor mediators, including leukotrienes and histamine. Both anti-leukotrienes and other anti-asthma drugs, such as inhaled glucocorticoids, have been shown to reduce both EAR and LAR. The aim of the present study was to directly compare the effects of regular treatment with an oral anti-leukotriene, montelukast (Mont; 10 mg once daily, for 8 days), and an inhaled glucocorticoid [fluticasone propionate (FP) 250 microg twice daily for 8 days] on the EAR and LAR to an inhaled allergen challenge. Patients with a documented EAR and LAR at a screening visit were randomized to these treatments, or placebo, in a double-blind, double-dummy, crossover fashion. Allergen challenge at a dose causing both an EAR and LAR was given on the eighth day of treatment. The maximum fall in FEV1 during the EAR was 17.8% during placebo treatment, 8.3% during Mont and 16.3% during FP (P <0.05 for Mont vs placebo). The maximum fall during the EAR was 13.8% during placebo treatment, 11.8% during Mont and 2% during FP treatment (P <0.05 for FP vs placebo and FP vs Mont). PC20 methacholine was significantly higher 24 h after allergen challenge during FP-treatment compared with Mont (P <0.05). Both montelukast and fluticasone reduced the relative amount of sputum eosinophils after allergen compared with placebo treatment. This study shows that anti-leukotrienes are effective to attenuate the EAR, whereas inhaled glucocorticoids are more effective than anti-leukotrienes in attenuating the EARs and improves bronchial hyperresponsiveness to a greater extent. In conclusion, inhaled glucocorticoids have overall greater efficacy than oral anti-leukotrienes to attenuate allergen-induced airway responses in mild asthmatic patients.

Diphtheria toxin (DTx) provokes extensive internucleosomal degradation of DNA before cell lysis. The possibility that DNA cleavage stems from direct chromosomal attack by intracellular toxin molecules was tested by in vitro assays for a DTx-associated nuclease activity. DTx incubated with DNA in solution or in a DNA-gel assay showed Ca2+- and Mg2+-stimulated nuclease activity. This activity proved susceptible to inhibition by specific antitoxin and migrated with fragment A of the toxin. Assays in which supercoiled double-stranded DNA was used revealed rapid endonucleolytic attack. Discovery of a DTx-associated nuclease activity lends support to the model that DTx-induced cell lysis is not a simple consequence of protein synthesis inhibition.

The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g., cycloheximide). Here we show that endonucleolytic degradation of DNA is an intrinsic activity of DTx and also of the crossreactive mutant protein CRM197. Assays using DNA-impregnated gels as well as linear and supercoiled DNA in solution revealed not only that CRM197 has nuclease activity but also that its specific activity is actually significantiy greater than that' of the wild-type molecule. Since CRM197 contains a single amino acid substitution that renders it incapable of ADP-ribosylation, we propose that the active sites for ADP-ribosyltrasferase and nuclease activities are distinct.Diphtheria toxin (DTx) is a well-characterized toxic protein (1, 2). It has two functional domains, A and B, which can be resolved by limited proteolytic digestion and reduction of an interchain disulfide bond. Intoxication of cells by DTx involves endocytosis of receptor-bound toxin. Once the toxin is sequestered in endosomes, acid-triggered conformational changes promote the translocation of fragment A to the cytosol (3-5). Fragment A then proceeds to catalyze the transfer ofthe ADP-ribosyl group ofNAD to the diphthamide residue of translational elongation factor 2 (EF-2). Recently we have discovered that DTx also exhibits a cationdependent nuclease activity (6). In this report we explore the relationship between the toxin's ADP-ribosyltransferase and nuclease activities. CRM197, a mutant DTx with a Gly -* Glu52 substitution in its A domain (7), is unable to ADPribosylate EF-2 in vitro because of its extremely low affinity for NAD (8,9 Nuclease Activity Assay in DNA-Containing Gels. Samples of cleaved DTx monomer, cleaved CRM197, and "intact" CRM197 were solubilized in SDS reducing buffer (containing 0.01% 2-mercaptoethanol) and electrophoresed in a SDS/ 12.5% polyacrylamide gel (10) prepared with 32P-labeled DNA [0.2 ng/ml; 800,000 cpm/ng of sonicated salmon sperm DNA radiolabeled by the primer-extension method (11, 12), as described (6)]. The stacking gel was omitted. The gels were pre-run at 170 V for 90 min. For nondenaturing gels, SDS was omitted and samples were not boiled. After electrophoresis, the DTx lanes were separated from the CRM197 lanes and each gel piece was washed three times (40 min per wash) with 100 ml of 40 mM Tris/0.04% NaN3, pH 7.6, at 22TC. The gel pieces were then incubated with the same buffer containing 2 mM CaCl2 and 2 mM MgCl2 for 24 hr at 300C (for SDS gels) or for 12 hr at 370C (for nondenaturing gels), prior to autoradiography (-850C, Kodak XAR film, DuPont Cronex screen).Cleavage of Supercoiled and Linear Plasmid DNA. CRM197, DNase I, and -DTx were incubated in 10 Al of 10 mM Tris, pH 7.6/2.5 mM CaCl2/2.5 mM MgCl2 with 0.2 ,Ag of linear (EcoRI-cleaved) pBluescr...

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). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.Diphtheria toxin (DTx) is synthesized as a single polypeptide chain that can be "nicked" by limited proteolysis to generate A and B subunits (DTA and DTB, respectively) that remain connected by a cystine bridge (7,14,16). DTB is involved in receptor binding; following sequestration in endosomes and subsequent acidification of the endosomal compartment, DTA is transported to the cytosol (8,14,22). DTA is involved in the ADP ribosylation of elongation factor 2, which leads to the inhibition of protein synthesis (7,14,16). We and others have noted that extensive toxin-induced translation inhibition is not correlated with cell death in human K562 cells (4,19); these cells are lysis resistant to doses of DTx as high as 7.5 ,ug/ml despite the rapid and complete abrogation of protein synthesis (4). In addition, extensive inhibition of protein synthesis by unrelated treatments does not lead to the prelytic intemucleosomal DNA cleavage that we have observed in DTx-intoxicated cells or to cell lysis (4). These and other findings (5) led us to propose that DTx triggers the programmed cell death pathway (4). Testing this hypothesis resulted in the discovery of a nuclease activity intrinsic to the DTx molecule (3). Detection of this activity is highly reproducible (10), optimal reaction conditions have been established (13), and an ADP ribosyltransferase (ADPrT)-defective form of DTx (called CRM197) exhibits greater nuclease activity than DTx itself (2). Moreover, nuclease activity was found to comigrate with the DTA portions of both DTx and CRM197 during electrophoresis in DNA-containing sodium dodecyl sulfate (SDS) gels (2, 3, 10) and with whole DTx and CRM197 during native gel electrophoresis (2), even though each of these forms of DTx migrates with a distinctive mobility in each gel system. We now report that the nuclease activity exhibited by DTx is intrinsic to DTA. chromatography of DTx and trypsin-generated DTA (21); therefore, they proposed that the nuclease activity of their DTx preparations resides in an uncharacterized contaminating protein. Moreover, they showed that trypsin cleavage of intact DTx results in a severe decrease in total observed nuclease activity and a simultaneous increase in ADPrT activity (21). In contrast, we observed that endoproteinase ArgC-cleaved DTx remains fully active and that the amount of DTx-associated nuclease activity corresponds to the amount of DTA generated ...