4.1.4. Effects on Enzymatic Pathways

Since the finding that the injection of Jaburetox diminished the enzyme activity of Nitric Oxide Synthase (NOS) in the central nervous system of *T. infestans* [84], a series of approaches were undertaken in order to try to understand the basis of this alteration. Besides its function in nitrinergic signaling in the central nervous system, NO participates in the immune response of insects due to its capacity of inducing oxidation of heme groups and nitrosylation of amino acid residues in proteins of pathogens [92]. The diminution of NOS activity upon Jaburetox injection was not related to the protein levels, since no differences in band intensities were seen in Western blots of brain homogenates of vehicle-injected and Jaburetox-injected insects [84]. The NOS activity also decreased when the homogenates were incubated with the peptide in vitro, suggesting a direct effect of the Jaburetox on the enzyme [84]. Similar results were obtained in the central nervous system of the related triatome *R. prolixus*, with both in vivo and in vitro Jaburetox treatments leading to a decrease in NOS enzyme activity without affecting its gene expression. Nevertheless, the effect of Jaburetox was different on the activity of NOS in the salivary glands and hemocytes, where the expression of its gene was increased, indicating an organ-specific

effect [78]. Jaburetox-induced alterations of NOS is not restricted to triatomines, since the peptide also induced a decrease in NOS enzyme activity in the central nervous system of the cockroach *N. cinerea*, upon in vivo or in vitro treatments, without affecting the protein expression [86]. The regulation of NO production mediated by Jaburetox is complex and could involve more than one level, for example, affecting directly the enzyme as the in vitro assays pointed out and/or, indirectly, through modifications on the expression of its gene, or even altering the membrane properties of target cells [77].

Considering that Jaburetox interacted with UAP in the central nervous system of *T. infestans*, the effect of the peptide on this enzyme was also tested. It was observed that the UAP enzyme was affected either after in vivo or in vitro treatments with Jaburetox, in this case causing a significant increase in activity without modifying the expression of its corresponding gene. Again, an organ-specific effect was demonstrated, with different responses of UAP in the central nervous system as compared to the salivary glands and hemocytes [77]. When a recombinant version of the *R. prolixus*' UAP was incubated in vitro with Jaburetox, no modification of the enzyme activity was observed, suggesting that other factor(s) present in the tissue homogenates are probably required for the peptide to exert its regulatory effect [78].

Taking into account the various functions of the main product of UAP, UDP-*N*-acetylglucosamine, several physiological processes can be influenced by the toxin. One of such process is chitin synthesis, catalyzed by chitin synthase and serving UDP-*N*-acetylglucosamine as substrate. The effect of Jaburetox on the expression of chitin synthase gene was then explored. It was found that Jaburetox treatment led to a diminution of chitin synthase expression in the central nervous system, salivary glands, anterior midgut, Malpighian tubules and fat body, but not in the hemocytes or the posterior midgut of *R. prolixus* [77,78]. The profile of UAP modulation by the Jaburetox in *N. cinerea* was different, since its activity was only affected 18 h after injection. This distinct response when compared to the triatomines could be related to the fact that this cockroach is so far the only species found to be resistant to the acute lethal effect of Jaburetox [86]. As the activity of acetylcholinesterase, an enzyme involved in the resistance of insects to organic pesticides, increased upon treatment of the cockroach with Jaburetox, the lack of lethality could be a reflex of an analogous mechanism(s) disabling the toxic effects of the peptide [86]. However, Jaburetox is far from being innocuous to *N. cinerea*, as the paralyzing effect and alterations in behavior caused by the peptide could lead to death as well, due to inability to hide and avoid danger, or to find food.
