Bacterial degradation of N,N-diethyl-m-toluamide (DEET): cloning and heterologous expression of DEET hydrolase

Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.     

Bacterial Degradation of N,N-Diethyl-m-Toluamide (DEET): Cloning and Heterologous Expression of DEET Hydrolase

Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.     

Neurotoxicity and Mode of Action of N,N-Diethyl-Meta-Toluamide (DEET)

Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD₅₀ ca. 1.5 µg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC₅₀ values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6–12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC₅₀: 120 µM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na⁺ and K⁺ channels in patch clamped rat cortical neurons, with IC₅₀ values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans.     

Chemical structure, juvenile hormone activity and persistence within the insect of juvenile hormone mimics for Rhodnius prolixus.

41 chemicals were tested for juvenile hormone (JH) activity for Rhodnius prolizus by two methods of application. Several different classes of chemical were represented, viz. long chain terpenoid compounds, aryl terpenoid compounds, aryl terpenoid amines and aryl terpenoid ethers.All classes of chemical contained members which showed at least moderate activity. The most active compound was N-(2,5-dichlorophenyl)-3,7-dimethyl-2,6-octadienylamine; 0.0024 μg induced the production of an insect which was halfway between a normal adult and a complete supernumerary larva. Since several compounds with widely differing chemical groupings showed high JH activity, it is considered that overall molecular size and shape are important considerations in determining JH activity, although they do not by themselves provide a complete explanation. Some chemicals tested are specific in their JH activity to relatively few insects. JH mimic specificity is discussed and it is considered that molecular shape can explain some aspects of this phenomenon.The persistence within the insect of the JH mimics varied greatly. In general, compounds with a methyl ester group at one end and an epoxide group at the other had least persistence. The absence of one or more of these features tends to induce greater persistence. Within most classes of chemical, the members with highest JH activity tended to have least persistence and it is considered that the major factor affecting persistence with the insect is the rate of enzymic degradation of the JH mimic.     

Molecular modeling, design, synthesis, and biological evaluation of novel 3',4'-dicamphanoyl-(+)-cis-khellactone (DCK) analogs as potent anti-HIV agents

Our current studies aimed at developing new potential anti-AIDS drug candidates have focused on the design and synthesis of new DCK analogs with improved molecular water solubility. Based on the structures and biodata of previous DCK analogs, 3D-QSAR studies have been performed which resulted in two reliable computational models, CoMFA and CoMSIA, with r(2) values of 0.995 and 0.987, and q(2) values of 0.662 and 0.657, respectively. In accord with these 3D-QSAR models, 15 new DCK analogs with polar functional groups at the 3-position were subsequently designed, synthesized, and evaluated against HIV-1 replication in H9 and MT4 cell lines. New DCK analogs 3b, 3c, 4b, 4c, 6a, 7c, and 9a showed promising potency with EC(50) values ranging from 0.09 to 0.0002 microM in both assays. Meanwhile, these promising compounds also showed a wide range of predicted logP values from 0.90 to 5.19, which increased the probability of identifying anti-HIV drug candidates from this class of compounds for clinical trials. Furthermore, both experimental and predicted values matched well, corroborating the reliability of the established 3D-QSAR models.     

Growth, lipid class and fatty acid composition in juvenile mud crabs (Rhithropanopeus harrisii ) following larval exposure to Fenoxycarb, insect juvenile hormone analog

This study examines the effects of Fenoxycarb on larval growth, and lipid class and fatty acid composition in first crabs of the mud crab Rhithropanopeus harrisii reared through total larval development in nominal water concentrations from 1 to 100 microg/l. In first crabs of R. harrisii, dry weight (microg) decreased significantly (P < 0.05) from 228.8+/-38.2 microg (n = 9) in the controls to 131.8+/-10.1 microg (n = 4) in animals exposed throughout larval development to 100 microg/l. A significant (P < 0.05) reduction was found between total lipid content in the controls and first crabs reared at concentrations greater than 50 microg/l. In relative terms (% dry weight), different lipid classes predominated in the controls and the various fenoxycarb exposure concentrations. There were no significant (P > 0.05) differences among the treatment groups in phospholipid level, while the triglyceride content was significantly lower in crabs exposed to 10 and 100 microg/l. No significant differences in the percent of free fatty acids were found in crabs exposed to 1-10 microg/l and the controls. Free sterols in crabs exposed to concentrations higher than 10 microg/l were below the detection limit. Control animal fatty acid profiles were dominated by palmitic, stearic, and oleic acid, accounting for 48% of total fatty acids (TFA). The fatty acid composition of crabs exposed to 100 microg/l significantly (P < 0.05) differed from the controls. The results suggest that fenoxycarb has substantial effects on growth, lipid class and fatty acid composition in developing larvae of R. harrisii at water concentrations greater than 10 microg/l.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.     

Effects of juvenile hormone analogs on new reproductives and colony growth of Pharaoh ant (Hymenoptera: Formicidae)

Two juvenile hormone analogs (JHAs), pyriproxyfen and S-methoprene, were impregnated into dried tuna fish and fed to colonies of Monomorium pharaonis (L.) at very low concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 microg/ml). Its effects on the production of sexuals and colonial growth were observed. Colonies treated with pyriproxyfen yielded sexuals with physical abnormalities. Both female and male sexuals developed bulbous wings, decreased melanization, and died shortly after emergence. Sexuals emerged from colonies treated with S-methoprene did not possess anomalous characteristics. Both pyriproxyfen and S-methoprene did not have significant effects on colonial growth because of the low concentrations of the baits. A commercial bait containing 0.3% S-methoprene (Bioprene-BM) also was evaluated for its efficacy on Pharaoh's ant colonies. Results showed that Pharaoh's ant colonies succumbed to the lethal effects of S-methoprene. Colony members were reduced significantly. Production of queens also decreased significantly in treated colonies and treated queens were unable to lay eggs. JHAs are slow acting and eliminate ant colonies at a relatively slow rate. At low concentrations, pyriproxyfen recorded baffling results, i.e., bulbous wings and demelanized exoskeleton, and it is vital that further studies are initiated to solidify these findings.     

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.     

Synergism of toxicity of N,N-diethyl-m-toluamide to German cockroaches (Orthoptera: Blattellidae) by hydrolytic enzyme inhibitors

Various compounds were tested for effects on the toxicity of the insect repellent N, N-diethyl-m-toluamide (DEET) in German cockroaches, Blattella germanica (L.). Organophosphate and carbamate acetylcholinesterase inhibitors carbaryl, DEF, eserine (physostigmine, malathion and pyridostigmine bromide synergized DEET toxicity also synergized the toxicity of the formamidine pesticides. Amitraz and chlordimeform. Results suggest that DEET may have some toxic actions that are similar to those of formamidine pesticides. DEET synergized the toxicity of some acetylcholinesterase inhibitors but not others. Results further suggest that some mechanism other than acetylcholinesterase inhibition was responsible for the toxic interactions observed between DEET and the acetylcholinesterase inhibitors.     

Ethyl (E)-3,5-ethano-7,11-dimethyl-2,4-dodecadienoate,A new insect growth regulator with potent juvenile hormone activity

A number of analogs of ethyl (2E,4E)-3,7,11-trimethyl-2,4-dodecadienoate were prepared and bioassayed for juvenile hormone activity on the yellow-fever mosquito (Aedes aegypti), the greater wax moth (Galleria mellonella), the yellow mealworm (Tenebrio molitor), the house fly (Musca domestica), and the tobacco budworm (Heliothis virescens). The analog ethyl (E)-3,5-ethanol-7,11-dimethyl-2,4-dodecadienoate (VI), containing a cyclopentene ring, showed remarkable potency on the above insect species. Since this compound possesses a fixed 3-s-trans-diene conformation it may provide some insight into the active conformation of bound 2,4-dienoate analogs.     

Binding of copper(II) to thyrotropin-releasing hormone (TRH) and its analogs

Spectroscopy (UV-Vis, ¹H NMR, ESR) and electrochemistry revealed details of the structure of the Cu(II)-TRH (pyroglutamyl-histidyl-prolyl amide) complex. The ¹H NMR spectrum of TRH has been assigned. NMR spectra of TRH in the presence of Cu(II) showed that Cu(II) initially binds TRH through the imidazole. TRH analogs, pGlu-His-Pro-OH, pGlu-(1-Me)His-Pro-amide, pGlu-His-(3,4-dehydro)Pro-amide, pGlu-His-OH, pGlu-Glu-Pro-amide, and pGlu-Phe-Pro-amide provided comparison data. The stoichiometry of the major Cu(II)-TRH complex at pH 7.45 and greater is 1:1. The conditional formation constant (in pH 9.84 borate with 12.0 mM tartrate) for the formation of the complex is above 10⁵ M⁻¹. The coordination starts from the 1-N of the histidyl imidazole, and then proceeds along the backbone involving the deprotonated pGlu-His amide and the lactam nitrogen of the pGlu residue. The fourth equatorial donor is an oxygen donor from water. Hydroxide begins to replace the water before the pH reaches 11. Minority species with stoichiometry of Cu-(TRH)_x (x = 2-4) probably exist at pH lower than 8.0. In non-buffered aqueous solutions, TRH acts as a monodentate ligand and forms a Cu(II)-(TRH)₄ complex through imidazole nitrogens. All the His-containing analogs behave like TRH in terms of the above properties.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

A novel, mild, specific and indirect maleimido-based radioiodolabeling method. Radiolabeling of analogs derived from parathyroid hormone (PTH) and PTH-related protein (PTHrP).

In an effort to design a mild, non-oxidative and site-specific means of radiolabeling bioactive molecules we have employed maleimido-sulfhydryl chemistry to produce bioactive hormone radioligands. We have prepared two novel radioiodolabeled reagents, 3'-maleimidopropanoyl-3-125I-tyramide and its retro analog, N-maleoyl-N'-3-(4-hydroxy-3-125I-phenyl)propanoyl ethylenediamide, by either oxidative radioiodination of the precursors or radiolabeling of the phenolic component prior to its incorporation into the radiolabeling reagents. These reagents were then used to radiolabel analogs of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) in an efficient way, yielding reaction mixtures which were easily purified. The radioligands obtained are stable upon storage and bind in a reversible manner to a single population of binding sites displaying affinity in the low nanomolar range. The potencies of these analogs are comparable to the non-modified PTH and PTHrP analogs. This study demonstrates the utility of the novel maleimido-based indirect radioiodination approach and highlights some of its advantages over either direct oxidative procedures or acylation using the Bolton-Hunter reagent.