br Conclusions br Acknowledgments This research

Conclusions
Acknowledgments This research was funded by the National Science Foundation Committee (NSFC) of China (Nos. 81171058 and 81471146) and Capital Development Scientific Research in China (Nos. 2011-5001-04 and 2014-4-5013). We are grateful for all of the participants who donated blood samples.
Introduction Thuringiensin is widely used as a biological insecticide in Europe, Asia and Africa Carlberg et al., 1995, Glare and O'Callaghan, 2000. The basic chemical structure of thuringiensin is similar to adenosine triphosphates (ATP). Thuringiensin is a heat-stable β-exotoxin (Fig. 1), and is produced by a number of strains of Bacillus thuringiensis (Bt) Levinson et al., 1990, Sebesta et al., 1981. Its toxicity to mammals was reported McClintock et al., 1995, Sebesta et al., 1981, Tsai et al., 2003. In our animal studies, thuringiensin was shown to be highly inflammatory and exhibited fibrotic properties in rat lungs (Tsai et al., 2003). Acting on concerns regarding mammalian toxicity, the WHO (1999) addressed the effect of non-specific thuringiensin from various strains of Bt on human health, and regulated Bt strains that produced thuringiensin. However, the potential influence of thuringiensin in regulating physiological reactions of mammals remains unclear. In this study, the effect of thuringiensin on adenylate cyclase was examined. Adenylate cyclase catalyzes the production of cyclic adenosine 3′5′-monophosphate (cAMP) from ATP. Cyclic AMP is an important mediator in the action of various physiological functions Ando et al., 1987, Levitzki, 1987. Cyclic AMP synthesis is influenced by numerous intracellular and extracellular regulators that either interact with the enzyme directly or control upstream activators or inhibitors. There are nine isoforms (ADCY1 to ADCY9) of adenylate cyclase identified in mammals Hanoune et al., 1997, Ludwig and Seuwen, 2002. Isoforms could be divided into four groups according to their common patterns of regulation, which, except ADCY9, the activity of adenylate cyclase could be stimulated by forskolin Hanoune and Defer, 2001, Vanvooren et al., 2000. Forskolin acted through its interaction with a site directly on the catalytic subunit of the enzyme Lemmer et al., 1991, Sano et al., 1983, Seamon et al., 1981. Additionally, the Fmoc-Cl MDL-12330A inhibited adenylate cyclase activity in different tissues, thus decreasing intracellular cAMP concentrations Gadea et al., 1999, Siegel and Wiech, 1976. In 1975, Grahame-Smith et al. demonstrated that thuringiensin inhibited fluoride-stimulated adenylate cyclase activity in an experiment involving partially purified rat brain enzyme preparations. The mechanism of thuringiensin on adenylate cyclase was presumably by competing with ATP as a substrate for adenylate cyclase, but the authors did not evaluate the effect of thuringiensin on basal adenylate cyclase activity in the rat brain. Thus, the effect of thuringiensin on adenylate cyclase activity remains poorly characterized. In this study, purified thuringiensin was used to investigate the influence of thuringiensin on adenylate cyclase activity in membrane preparations from rat cerebral cortex. Cyclic AMP concentrations were measured to elucidate the effect of thuringiensin on adenylate cyclase activity in rat brain.
Materials and methods
Results
Discussion In the present study, these results demonstrated that thuringiensin activated the basal adenylate cyclase activity in cerebral cortical membrane preparation of rats. Additionally, thuringiensin dose-dependently increased the production of cAMP in rat cerebral cortical membranes and in commercial E. coli adenylate cyclase. Thuringiensin concentrations required to activate basal adenylate cyclase activity were log-level different between rat brain membrane preparations and a commercial E. coli adenylate cyclase. In 1975, Grahame-Smith et al. demonstrated the ability of thuringiensin to compete with ATP as a substrate for adenylate cyclase, an ability that probably results from its structural similarity to ATP. However, Grahame-Smith et al. did not examine the effect of thuringiensin on the basal adenylate cyclase activity. In contrast, the present study examined the effect of thuringiensin on basal adenylate cyclase activity and found that thuringiensin alone did not stimulate the production of cAMP in cerebral cortical membrane preparation of rats without ATP (Fig. 2). Thuringiensin thus did not act directly as a substrate to increase cAMP production. Moreover, the addition of ATP did not reduce the cytotoxicity of thuringiensin in culture of primary monkey kidney cells (Laurent, 1978) or Chinese hamster lung fibroblast V79 cells (our unpublished data). These experimental results may indicate that the thuringiensin did not act on the ATP binding site of adenylate cyclase.