1 Chronic inflammation is a central feature of asthma. The inflammatory cytokine tumour necrosis factor a (TNFa) has been implicated in this disease, and is known to alter airway smooth muscle functionally. 2 The aim of this study was to investigate the influence of TNFa on tachykinin-induced airway relaxation. Mouse tracheae were cultured in the absence and presence of TNFa for 1 or 4 days. 3 In the absence of TNFa, substance P (SP) and neurokinin A (NKA) induced comparable levels of relaxation in fresh and cultured segments. Functional studies with selective antagonists/inhibitors indicated that the relaxation was mediated by the NK 1 receptor coupled to cyclooxygenase (COX)-2 activation and subsequent release of prostaglandin E 2 (PGE 2 ). TNFa attenuated SP-and NKAinduced relaxation in a time-and concentration-dependent manner, decreasing the ability of PGE 2 to relax tissues. 4 Further studies indicated that TNFa elevated COX-2 activity and that concomitant inhibition of COX-2 reversed TNFa-attenuated PGE 2 relaxation. Culture with PGE 2 decreased SP-and PGE 2 -mediated relaxation, further implicating the activity of COX-2 in the attenuation of tachykinin signalling. 5 Gene expression analysis demonstrated that TNFa increased the expression of smooth muscle COX-2, PGE 2 synthase and EP 2 receptor mRNA, and decreased the expression of the EP 4 receptor. 6 Overall, these results show that NK 1 receptor-mediated relaxation induced by PGE 2 is attenuated by prolonged TNFa stimulation. Increased COX-2 activity induced by TNFa appears to be central to this process.

The adenovirus type 2 major late upstream element factor (UEF) recognizes two similar elements that lie between the major late promoter (MLP) and IVa2 promoter cap sites (the previously characterized MLP-UE from nucleotides-49 to-67 and the IVa2-UE from nucleotides-98 to-122). DNase I footprinting and gel retention assays showed that the UEF has a lower affinity for the IVa2-UE than for the MLP-UE. In vitro transcription experiments demonstrated first that the IVa2 promoter, which lacks a consensus TATA box, may work, as does the MLP, in the absence of its proximal upstream element and second that the IVa2-UE stimulated IVa2 transcription two-to threefold, as MLP-UE did for the MLP. In addition, we demonstrated that the more distal upstream element has a weak stimulatory effect on transcription of both promoters.

Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type. ( Journal of Biomolecular Screening 2004:44-51)