Studies on Nicotinoids as an Insecticide

In search for the mechanism of insecticidal action of nicotinoids, the kinetics of house fly head cholinesterase inhibition by nicotine were studied to determine the type of inhibition. The pH dependency of inhibition was interpreted in terms of protonation of nitrogen atom in the molecule and the inhibition was shown to be the mixed type closing to competitive type. The Michaelis constants are 3.5 × 10^?4 M and 4.1 × 10^?4 M, while the apparent inhibition constants obtained are 1.0 × 10^?3 M and 2.3 × 10^?3 M at pH 7.4 and 8.4, respectively. The type of the inhibition by nicotine monomethiodide carring univalent cation was competitive and the apparent inhibition constant is 1.5 × 10^?4 M. These data indicated that the cationic head of nicotinium ion interacts with the anionic site in the active center of cholinesterase.     

Studies on Nicotinoids as an Insecticide

3-Pyridylmethylamines, such as 3-pyridylmethyl-N-diethylamine, N-(3-pyridylmethyl)-piperi-dine and 3-pyridylmethyl-N-dimethylamine provided with the essential moiety of nicotinoids molecule, show comparable toxicity to nicotine against house flies.

3-Pyridylmethylamine without substituent on amino-nitrogen, N-(3-pyridylmethyl)-mor-pholine having low basic nitrogen, 4-pyridylmethyl-N-diethylamine not provided with proper N-N distance and some nicotine-methiodides having quaternary nitrogen show low or no toxicity.

These data support our proposition that the essential moiety of nicotinoids molecule responsible for high toxicity is 3-pyridylmethylamine grouping.     

Studies on Nicotinoids1) as an Insecticide

Toxicities of some nicotinoids as an insecticide were determined. 5′-methylnornicotine, a new synthetic isomer of nicotine, shows similar toxicity to nicotine. The essential moiety in nicotinoids molecule responsible for high toxicity may be 3-pyridylmethylamine group, the amino nitrogen of which must have high basicity (p^K_a′: 7.4~9.0). All nicotinoids of high toxicity are estimated to be largely as monocation at physiological pH of 7.     

On Some Structural Analogies between Acetylcholinesterase and the Macromolecular Receptor of Acetylcholine

Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins.     

Anionic center of porcine pancreatic phospholipase A2

The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions.     

The fasciculin-acetylcholinesterase interaction

Acetylcholinesterase (AChE) terminates the action of the neurotransmitter acetylcholine at cholinergic synapses in the central and peripheral nervous systems. Fasciculins, which belong to the family of "three-fingered" snake toxins, selectively inhibit mammalian AChEs with Ki values in the picomolar range. In solution, the cationic fasciculin appears to bind to the enzyme's peripheral anionic site, located near the mouth of the gorge leading to the active center, to inhibit catalysis either allosterically or by creating an electrostatic barrier at the gorge entry (or both). Yet the crystal structure of the fasciculin-mouse AChE complex, which shows that the central loop of fasciculin fits snugly at the entrance of the gorge, suggests that the mode of action of fasciculin is steric occlusion of substrate access to the active center. Mutagenesis of the fasciculin molecule, undertaken to establish a functional map of the binding surfaces, identified determinants common to those identified by the structural approach and revealed that only a few of the many fasciculin residues residing at the complex interface provide the strong contacts required for high affinity binding and enzyme inhibition. However, it did not reconcile the disparity between the kinetic and structural data. Finally, the crystal structure of mouse AChE without bound fasciculin shows a tetrameric assembly of subunits; within the tetramer, a short loop at the surface of a subunit associates with the peripheral site of a facing subunit and sterically occludes the entrance of the active center gorge. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of fasciculin bound to AChE. This suggests not only that the peripheral site of AChE is a site for association of heterologous proteins with interactive surface loops, but also that endogenous peptidic ligands of AChE sharing structural features with the fasciculin molecule might exist.     

Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain

Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [³H]QNB reversibly with a K _d of 260 PM and B_max of 1 pmol/g of heads from direct binding measurements. The K_d calculated from the ratio of the dissociation rate constant (2 × 10⁻⁴ sec⁻¹) to the association rate constant (2.5 × 10⁶ M⁻¹ Sec⁻¹) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.     

Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations

The toxicity of spinosad was determined in one susceptible and five insecticide-resistant laboratory strains of house fly, Musca domestica L. Spinosad was relatively slow-acting, but highly toxic to house flies. In a feeding bioassay, spinosad LC50 at 72 h was 0.51 microg of spinosad per gram of sugar, making it 6.3- and 3.5-fold more toxic to house flies compared with azamethiphos and methomyl, respectively. In topical application bioassay, the LD50 at 48 h of spinosad in susceptible house flies was 40 ng per 20 mg of house fly, making spinosad less toxic than the pyrethroid bioresmethrin synergized by piperonyl butoxide and the organophosphate dimethoate. The insecticide-resistant laboratory strains had resistance factors to spinosad at LC50 in feeding bioassay from 1.5 to 5.5 and at LD50 in topical application bioassay from 2.5 to 4.7, indicating that in house fly cross-resistance to the major insecticide classes will not initially be of major concern for the use of spinosad for house fly control. The toxicity of spinosad was also evaluated against 31 field populations of house flies collected from livestock farms across Denmark. The field populations were 2.2- to 7.5-fold resistant to spinosad at 72 h in feeding bioassay, but based on steep slopes in the bioassay and the limited variation of spinosad toxicity against the various field populations, we consider the field populations to be spinosad-susceptible. We propose a diagnostic dose of 12 microg of spinosad per gram of sugar in feeding bioassay with impregnated sugar for determination of resistant house flies, which is 10x the LC95 of the susceptible strain WHO and approximately = 2x the LD95 of the field populations. Spinosad showed no substantial cross-resistance to the pyrethroid bioresmethrin synergized by piperonyl butoxide, the anticholinesterases dimethoate, azamethiphos, methomyl, and spinosad in house fly field populations.     

α-Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax

Abstract: House fly heads contain a site that binds α-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α-bungarotoxin (labeled with [³H]pyridoxamine phosphate) reversibly with a K _d of 6 nM. The effects of 48 drugs have been compared on the α-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-β-methylcholine, but lower affinities for nicotine, atropine, and dihydro-β-erythroidine.     

Modelling Bioaccumulation and Toxicity of Metal Mixtures

Bioaccumulation of metals in mixtures may demonstrate competitive, anticompetitive, or non-competitive inhibition, as well as various combinations of these and/or enhancement of metal uptake. These can be distinguished by plotting (metal in water)/(metal in tissue) against metal in water and comparison to equivalent plots for single-metal exposure. For the special case of pure competitive inhibition where only one site of uptake is involved, inhibition of metal accumulation in any metal mixture can be predicted from bioaccumulation of the metals when present singly. This is consistent with the commonly used Biotic Ligand Model (BLM) but does not explain bioaccumulation of metals in Hyalella azteca. Options for modelling toxicity of metal mixtures include concentration or response addition based on metal concentrations in either water or tissues. If the site of toxic action is on the surface of the organism, if this is the same as the site of metal interaction for bioaccumulation, if there is only one such type of site, and if metal bioaccumulation interactions are purely competitive (as in the BLM), then metal toxicity should be concentration additive and predictable from metal concentrations in either water or tissues. This is the simplest toxicity interaction to model but represents only one of many possibilities. The BLM should, therefore, be used with caution when attempting to model metal interactions, and other possibilities must also be considered.     

AMI and PM3 study of a low molecular weight structural mimic of hydrogen exchange within the catalytic center of aspartic proteases

Based on recent X-ray studies, a low molecular weight model of the active center of aspartic proteases is proposed. The model is small enough to enable unattended geometry optimizations (including search for saddle-points) by molecular orbital methods. It consists of two malonic acid molecules and a water molecule; there is a carboxylic dimer at one end and the water molecule is located between the carboxylate and the carboxyl group at the other. The latter structure reproduces the geometry of the catalytic center of the native enzyme penicillopepsin with a root-mean-square deviation of 0.46 Å for five O--O distances. The AMI and PM3 molecular orbital methods were used to study the H-bond exchange within the model. Both methods lead consistently to the following conclusions: Among 2 pairs of symmetry-equivalent stationary states of the catalytic center there are at least 4 symmetry-independent hydrogen-exchange pathways, and many more when including symmetry of the center. Energetics and geometry of all identified pathways are presented. In summary, they result in juggling all three active center protons (COON and HOH) among all five active center oxygens (COO^–, COOH and H₂O) providing the center with a high delocalisation with respect to the actual position of its anionic site and/or its protonation status. The relevance of the delocalisation of the acidic proton to the mechanism of enzymatic action is briefly discussed. Correspondence to: J. Ciarkowski     

Tityus serrulatus toxin VII bears pharmacological properties of both beta-toxin and insect toxin from scorpion venoms

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.     

The dosage-mortality relationship for two strains of house flies exposed to vapor of di-isopropylphosphorofluoridate

The dosage-mortality relationship for normally susceptible and di-isopropylphosphorofluoridate (DFP)-resistantMusca domestica L. was determined by means of vapor exposures of varied durations and concentrations.With reference to concentration, males of either strain were approximately 1.5 × as sensitive as the respective females, and the strains differed by a factor of about 10. Probit kill at each concentration was proportional to log exposure time for all four groups, and the log of time required to kill half a given sex or strain (log Lt₅₀) was proportional to the log of DFP concentration. For all four groups the apparent order of toxic reaction was the same, — namely slightly more than 2.5.Since the actual toxic process probably is a bimolecular reaction between DFP and nervous system cholinesterase (ChE), the observations indicate that an appreciable portion of the entering DFP was diverted to nontoxic pathways. The data contain no suggestion that the sexes and strains are distinguished by any qualitative differences in these pathways. The differences in tolerance are significant but unexplained.With all four groups, like percentages of kill were accompanied on the average by like percentages of inhibition of head ChE despite considerable intergroup differences in the dosages required. Per cent inhibition was directly proportional to kill. The overall correlation observed corroborates the view that inhibition of central nervous system ChE is related causally to toxicity. In general, flies whose head ChE was inhibited less than about 50% did not succumb, while those with more than 50% inhibition died irrespective of their source and the dose of DFP needed.Although the relationship favors the conclusion that ChE inhibition is instrumental in the toxicity of DFP, it does not indicate that inhibition of head ChE is more than an indirect index of events at the as yet unknown true sites of action, nor that an acetylcholine-ChE mechanism of synaptic transmission is vitally involved in the functioning of insect nerve.

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Die Dosis-Mortalitäts-Benziehungen zweier Stubenfliegen-Stämme, die di-isopropulphosphorfluoridat-Dämpfen Ausgesetzt wurden

Zusammenfassung Wenn die Geschlechter zweier Stubenfliegen-Stämme (ein normal empfindlicher und ein DFP-resistenter) getrennt DFP-Dämpfen ausgesetzt wurden, waren die entsprechenden Reihen der log Lt₅₀ zwar verschieden, wurden jedoch in jedem Falle linear abhängig von der logarithmischen DFP-Konzentration und einander parallel befunden.Für alle vier Gruppen betrugen die erkennnbaren Werte der toxischen Reaktion etwas mehr als 2,5. Da die tatsächliche toxische Reaktion bimolokularer Kinetik zu gehorchen scheint, wird geschlossen, daß ein beträchtlicher Anteil des von den Fliegen absorbierten DFP in nicht-toxische Prozesse abgeleitet wird.Die Ergebnisse liefern keinerlei neue Erklärung der Toleranzunterschiede zwischen den Geschlechtern und Stämmen, und infolgedessen bleiben die Ursachen für diese Unterschiede unbekannt.Im Durchschnitt war die Hemmung der Cholinesterase des Kopfes direkt proportional zum Abtötungsprozentsatz, trotz des Vorliegens einer beträchtlichen Variabilität, welche zufallsmäßig war und offensichtlich auf inneren Faktoren beruht. In dieser Hinsicht bestanden keine signifikanten Unterschiede zwischen den Geschlechtern und Stämmen.Das beobachtete Verhältnis wird als strenger Beweis für ein kausale Beziehung zwischen Cholinesterase und Giftwirkung angesehen, jedoch wird der Grad der Kopf-Cholinesterase-Hemmung nur als Index für ein unbekanntes Ausmaß der Hemmung an anderen hypothetisch vitalen Punkten betrachtet. Die Befunde liefern weder Anhaltspunkte für noch wider die Auffassung, daß ein Acetylcholin-Cholinesterase-Mechanismus der synaptischen Übertragung eine vitale Funktion im Insekt besitzt.

     

Epinigericin toxicity towards Tetrahymena pyriformis GL; changes in cell volume and intracellular pH

A study of the toxicity of epinigericin, an antibiotic ionophor, towards the ciliate Tetrahymena pyriformis showed that this molecule stopped cell division, increased cell volume and led to a more basic intracellular pH. The action of epinigericin was probably linked to its function as an ionophor. The ionic selectivity of this molecule is still not known. The raising of the intracellular pH of ciliates by this antibiotic may be linked to its toxic action and its ion-transport mechanism in Tetrahymena. *** DIRECT SUPPORT *** AG903066 00009     

Homology modeling of human α1β2γ2 and house fly β3 GABA receptor channels and Surflex-docking of fipronil

To further explore the mechanism of selective binding of the representative γ-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human α1β2γ2 and house fly β3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human α1β2γ2 and house fly β3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly β3 GABAR than with human α1β2γ2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human α1β2γ2 and house fly β3 GABARs may result in differential selectivity for fipronil.     

Kinetic evaluation of multiple initial rate data by simultaneous analysis with two equations

During the acetylcholinesterase catalytic process, the acetylcholine substrate attaches to the peripheral anionic site and then slides to the catalytic site situated in the center of the enzyme, at the bottom of a deep and narrow active gorge. Before the first catalytic cycle is complete, a second substrate molecule approaches and modulates the reaction. An inhibitor interferes with all steps and further complicates the situation. The reaction can be studied separately in the presence of two substrates, one good and one poor, and it can also be conducted simultaneously using a poor substrate as an inhibitor of the hydrolysis of a good substrate. Here, we have performed such an analysis, reducing the number of unknowns to those for the two substrates, while gaining additional information from the inhibition measurements without introducing new unknowns. To lower the number of realistic global minima in the analysis, we coupled the specific rate equation of the model to the rational polynomial of the corresponding degree. In contrast to the good substrate, the acetylation step by the poor substrate is found to be enhanced by the binding of the second substrate molecule to the peripheral anionic site. We attribute this to the different rate-limiting steps during the acetylation process: enhanced accommodation of the substrate paranitrophenylacetate is still slower than the hindered exit of the product paranitrophenol.     

A model system for receptor cell studies with the taste modifier, hodulcin

Hodulcin (from Hovenia dulcis leaves) is known to suppress selectivelysweet taste perception in humans. The effects of hodulcin onPhormia regina behavioral and taste receptor cell action potentialresponses to sucrose and receptor cell responses to NaCl weretested to determine the suitability of this fly for neurophysiologicalstudies with hodulcin. The same hodulcin concentration and temporalparameters that had been used in the human studies were usedfor the fly experiments, so that the human perceptual, fly behavioraland fly neurophysiological inhibition times and hodulcin selectivitycould be compared. Hodulcin reversibly suppressed fly behavioraland receptor cell responses to sucrose with an inhibition timesimilar to the inhibition time in humans. The effect in flieswas selective: hodulcin did not suppress receptor cell responsesto NaCl. These data indicate that P.regina is an appropriatemodel for receptor cell studies with hodulcin.     

Cholinesterase inhibition in the bulb miteRhizoglyphus echinopus (Acari: Acaridae) in relation to the acaricidal action of organophosphates and carbamates

The 5000-g supernatant fraction of whole-bulb-mite homogenates was shown to possess a cholinesterase (ChE) that hydrolyzed acylcholine esters in the order acetyl>propionyl>butyryl. Acetyl--methylcholine, but not benzoylcholine, also was hydrolyzed as were acetylthiocholine and acetyl--methylthiocholine. No inhibition by excess substrate was observed at cholinester concentrations as high as 30mM. Cholinesterase activity was markedly insensitive to eserine and to certain other carbamates and organophosphates. Only organophosphates of the dimethylphosphate type generally were active ChE inhibitors. It was concluded that the inability of carbamates such as eserine, and organophosphates such as those with alkyl groups larger than dimethyl, to inhibit the bulb-mite ChE was probably a consequence of the nature of the esteratic site. The data suggested that ChE inhibition was likely involved in the toxicity to bulb mites of some of the toxic carbamates and organophosphates, but that it might not be the only mechanism involved, at least with several of the compounds.Contribution from the Missouri Agricultural Experiment Station, Columbia, MO, Journal Series No. 10 924.     

Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector

Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.     

The biological properties of lysine-derived surfactants

Summary. We examine the effects of aquatic toxicity on Daphnia magna, the antimicrobial activity of new anionic lysine-derivative surfactants, and the influence of different-sized counterions associated with the surfactants. Surfactants with Tris and Lithium had less of a toxic effect on Daphnia, while all surfactants proved highly active against yeasts and the gram-negative bacteria Bordetella bronchiseptica. Counterion size was found to have no effect on aquatic toxicity or antimicrobial activity.