Whereas previous studies have shown correlations between volatile sulphur compounds (VSC) and bad breath levels, it is probable that other compounds found in the oral cavity may contribute to oral malodor. In the present investigation, the possibility that diamines (cadaverine and putrescine) are associated with oral malodor parameters was assessed. Saliva samples from 52 subjects were analyzed for cadaverine and putrescine by HPLC. Oral malodor of whole mouth, tongue, and saliva of the subjects was recorded by an experienced judge on a continuous 10-cm scale; peak and steady-state VSC intraoral levels were measured by the Interscan 1170 sulphide monitor. Log-transformed VSC and diamine levels were compared with odor judge measurements by Pearson analysis and stepwise forward multiple regression. Putrescine scores were not significantly associated with odor judge parameters or with VSC levels (p > 0.1). However, highly significant correlations (p < or = 0.003) were found between cadaverine levels and all three odor judge assessments. In contrast, associations between cadaverine and VSC measurements were non-significant. In an attempt to correlate odor judge results in terms of both VSC and diamines, we carried out stepwise forward multiple regression. Results showed that VSC and cadaverine both factor significantly in explaining each of the odor judge measurements, with multiple r values ranging from 0.545 (p = 0.0002) to 0.604 (p < 0.0001). The results suggest that cadaverine levels are associated with oral malodor, and that this association may be independent of VSC.

The purpose of the present investigation was to test the association between the BANA test (Perioscan, Oral-B), and oral malodor parameters. The subject population consisted of 52 Israeli adults, 43 of whom complained of oral malodor. Oral malodor measurements consisted of peak and steady-state volatile sulphide measurement by a portable sulphide monitor (Interscan Corp., model 1170), as well as organoleptic measurements of malodor from whole mouth, tongue, and saliva. Samples for the BANA test were obtained from four loci (shallow pocket, deep pocket, tongue dorsum, saliva); results were scored as negative (0), weak (1), or strong (2). BANA scores were significantly associated with odor-judge ratings, with the highest association obtained when BANA saliva scores and odor-judge saliva assessment were compared (r = 0.500; p < 0.001). BANA tests from the different loci were not significantly associated with sulphide monitor levels. Stepwise multiple-regression analysis of odor-judge measurements in terms of sulphide levels and average BANA scores showed that both log peak sulphide levels as well as BANA scores were significantly factored into the equations, yielding, in all cases, highly significant correlations (multiple r = 0.57, 0.50, and 0.59, respectively, with significance levels of 0.0001, 0.001, and < 0.0001, for whole mouth, tongue, and saliva malodor, respectively). The results suggest that the BANA scores are associated with a component of oral malodor which is independent of volatile sulphide measurements and suggest its use as an adjunct test to volatile sulphide measurement.

Structural details on the interacting faces of toxins and sodium channels (Navs), and particularly identification of elements that confer specificity for insects, are difficult to approach and require suitable experimental systems. Therefore, natural toxins capable of differential recognition of insect and mammalian Navs are valuable leads for design of selective compounds in insect control. We have characterized several scorpion toxins that vary in preference for insect and mammalian Navs, and identified residues important for their action. However, despite many efforts worldwide, only little is known about the receptor sites of these toxins, and particularly on differences between these sites on insect and mammalian Navs. Another problem arises from the massive overuse of chemical insecticides, which increases resistance buildup among various insect pests. A possible solution to this problem is to combine different insecticidal compounds, especially those that provide synergic effects. Our recent finding that combinations of insecticidal receptor site-3 toxins (sea anemone and scorpion alpha) with scorpion beta toxins or their truncated derivatives are synergic in toxicity to insects is therefore timely and strongly supports this approach. Our ability to produce toxins and various Navs in recombinant forms, enable thorough analysis and structural manipulations of both toxins and receptors. On this basis we propose to (1) restrict by mutagenesis the activity of insecticidal scorpion -toxins and sea anemone toxins to insects, and clarify the molecular basis of their synergic toxicity with antiinsect selective -toxins; (2) identify Nav elements that interact with scorpion alpha and sea anemone toxins and those that determine toxin selectivity to insects; (3) determine toxin-channel pairwise side-chain interactions by thermodynamic mutant cycle analysis using our large collection of mutant -toxins and Nav mutants identified in aim 2; (4) clarify the mode of interaction of truncated -toxins with insect Navs, and elucidate how they enhance the activity of insecticidal site-3 toxins. This research may lead to rational design of novel anti-insect peptidomimetics with minimal impact on human health and the environment, and will establish the grounds for a new strategy in insect pest control, whereby a combination of allosterically interacting compounds increase insecticidal action and reduce risks of resistance buildup.

Motivated by the high risks to the environment and human health imposed by the current overuse of chemical insecticides we offer an alternative approach for the design of highly active insect-selective compounds that will be based on the ability of natural toxins to differentiate between insect and mammalian targets. We wish to unravel the interacting surfaces of insect selective toxins with their receptor sites on voltage-gated sodium channels. In this proposal we put forward two recent observations that may expedite the development of a new generation of insect killers that mimic the highly selective insecticidal toxins: (i) A small (27aa) highly insecticidal sea anemone toxin, Av3, whose toxicity to mammals is negligible; (ii) The prominent positive cooperativity between distinct channel ligands, such as the strong enhancement of pyrethroids effects by anti-insect selective scorpion depressant toxins. We possess a repertoire of insecticidal toxins and sodium channel subtypes all available in recombinant form for mutagenesis followed by analysis of various pharmacological, electrophysiological, and structural methods. Our recent success to express Av3 provides for the first time a selective toxin for receptor site-3 on insect sodium channels. In parallel, our recent success to determine the structures and bioactive surfaces of insecticidal site-3 and site-4 toxins establishes a suitable system for elucidation of toxin-receptor interacting faces. This is corroborated by our recent identification of channel residues involved with these two receptor sites. Our specific aims in this proposal are to (i) Determine the bioactive surface of Av3 toward insect Na-channels; (ii) Identify channel residues involved in binding or activity of the insecticidal toxins Av3 and LqhaIT, which differ substantially in their potency on mammals; (iii) Illuminate channel residues involved in recognition by the anti-insect depressant toxins; (iv) Determine the face of interaction of both site-3 (Av3) and site-4 (LqhIT2) toxins with insect sodium channels using thermodynamic mutant cycle analysis; and, (v) Examine whether Av3, LqhIT2, pyrethroids, and indoxacarb (belongs to a new generation of insecticides), enhance allosterically the action of one another on the fruit fly and cockroach paraNa-channels and on their kdr and super-kdr mutants. This research establishes the grounds for rational design of novel anti-insect peptidomimetics with minimal impact on human health, and offers a new approach in insect pest control, whereby a combination of allosterically interacting compounds increases insecticidal action and reduces risks of resistance buildup.

Voltage-gated sodium channels (Navs) play a pivotal role in excitability and are a prime target of insecticides like pyrethroids. Yet, these insecticides are non-specific due to conservation of Navs in animals, raising risks to the environment and humans. Moreover, insecticide overuse leads to resistance buildup among insect pests, which increases misuse and risks. This sad reality demands novel, more selective, insect killers whose alternative use would avoid or reduce this pressure. As highly selective insect toxins exist in venomous animals, why not exploit this gift of nature and harness them in insect pest control? Many of these peptide toxins target Navs, and since their direct use via transformed crop plants or mediator microorganisms is problematic in public opinion, we focus on the elucidation of their receptor binding sites with the incentive of raising knowledge for design of toxin peptide mimetics. This approach is preferred nowadays by agro-industries in terms of future production expenses and public concern. However, characterization of a non-continuous epitope, that is the channel receptor binding site for such toxins, requires a suitable experimental system. We have established such a system within more than a decade and reached the stage where we employ a number of different insect-selective toxins for the identification of their receptor sites on Navs. Among these toxins we wish to focus on those that bind at receptor site-3 and inhibit Nav inactivation because: (1) We established efficient experimental systems for production and manipulation of site-3 toxins from scorpions and sea anemones. These peptides vary in size and structure but compete for site-3 on insect Navs. Moreover, these toxins exhibit synergism with pyrethroids and with other channel ligands; (2) We determined their bioactive surfaces towards insect and mammalian receptors (see list of publications); (3) We found that despite the similar mode of action on channel inactivation, the preference of the toxins for insect and mammalian channel subtypes varies greatly, which can direct us to structural features in the basis of selectivity; (4) We have identified by channel loop swapping and point mutagenesis extracellular segments of the Navinvolved with receptor site-3. On this basis and using channel scanning mutagenesis, neurotoxin binding, electrophysiological analyses, and structural data we offer: (i) To identify the residues that form receptor site-3 at insect and mammalian Navs; (ii) To identify by comparative analysis differences at site-3 that dictate selectivity toward various Navs; (iii) To exploit the known toxin structures and bioactive surfaces for modeling their docking at the insect and mammalian channel receptors. The results of this study will enable rational design of novel anti-insect peptide mimetics with minimized risks to human health and to the environment. We anticipate that the release of receptor site-3 molecular details would initiate a worldwide effort to design peptide mimetics for that site. This will establish new strategies in insect pest control using alternative insecticides and the combined use of compounds that interact allosterically leading to increased efficiency and reduced risks to humans or resistance buildup among insect pests.