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.     

Response variability of juvenile hormone and juvenile hormone analogs in workers of the Formosan subterranean termite (Coptotermes formosanus Shiraki)

Juvenile hormone (JH) or juvenile hormone analog (JHA) can induce soldier formation in termites. However, different studies have yielded inconsistent results on the effects of JHA on soldier production in Coptotermes formosanus Shiraki. Using filter paper as the testing substrate, the effects of JH III, pyriproxyfen and methoprene on the intact filter paper left, worker mortality and presoldier formation were tested on six colonies. Our results showed that pyriproxyfen and methoprene were more repellent than JH III. No significant difference in toxicity was observed among methoprene, pyriproxyfen and JH III. JH III and pyriproxyfen did not cause workers to differentiate into presoldiers, but methoprene can induce formation of presoldiers. Colony response variability to methoprene was observed. The confirmation of the effects of JH/JHA on C. formosanus establishes the foundation for molecular studies of soldier differentiation in this species.     

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.     

Lethal and behavioural effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti mosquitoes in laboratory assays

The knock-down, mortality and 'irritancy' effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti (L) (Diptera: Culicidae) were evaluated in the laboratory in the absence of animal bait. Filter paper tests were carried out to assess the knock-down effect (KDt(50) and KDt(95)) and mortality (LC(50) and LC(95)) induced by each repellent. 'Irritancy' tests were carried out to compare the flight response (time to first take-off, or FT) to increasing concentrations of repellents (2-7%) and at five distances from the treated surface (0-40 mm). DEET had an insecticidal effect (KDt(50) = 9.7 min at 7%; CL(50)= 1165 mg/m(2)), whereas IR3535 and KBR 3023 did not. Relative to an untreated control, IR3535 was an irritant (relative irritancy or RI > 1) at doses of 5% and 7% (RI = 17.7 and 9.9, respectively), whereas DEET was an irritant at lower concentrations (RI = 12.3 at 2% DEET). KBR 3023 was the weakest irritant over the same range of concentrations (RI(max) = 3.6 at 6%). DEET was more of an irritant (RI(20) = 9.4) than IR3535 (RI(20) = 2.9) over a range of distances (0-20 mm), and KBR 3023 was not an irritant unless mosquitoes made contact with the treated surface. All three repellents had a significant effect on mosquitoes, but DEET exhibited a more complex mode of action than the others due to its insecticidal properties. The repellents do not behave as a single class of compounds with a common mode of action, but most probably affect different physiological systems in insects. The physiological and molecular mechanisms of repellents, especially DEET, should be investigated to ensure a better use of these molecules for skin applications and/or for treating materials against mosquitoes.     

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.     

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.     

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.     

小菜蛾保幼激素受体基因PxMet-1和PxMet-2的分子特性、表达模式与功能分析

【目的】本研究旨在明确小菜蛾Plutella xylostella保幼激素受体基因Met的分子特性与表达模式,分析其生殖调控作用,为筛选有效控制小菜蛾的新靶标奠定基础。【方法】根据本课题组已有的小菜蛾基因组数据库,采用PCR技术克隆小菜蛾两个Met基因的cDNA全长序列;利用qPCR测定其在小菜蛾不同发育阶段及成虫不同组织中的表达模式;基于RNAi解析其在小菜蛾雌成虫生殖发育中的作用。【结果】克隆获得小菜蛾PxMet-1(GenBank登录号: MK697672)与PxMet-2(GenBank登录号: MK697673)的cDNA序列,开放阅读框(ORF)全长分别为1 575和2 100 bp,预计分别编码524和699个氨基酸,理论分子质量分别为60.5和70.7 kD,预测等电点分别为6.73和5.50。PxMet-1和PxMet-2都具有4个保守结构域,即1个helix-loop-helix结构域(bHLH)、2个PAS保守结构域及1个PAC保守基序。系统发育树分析表明,小菜蛾PxMet-1和PxMet-2聚为不同的两支,但两者均与鳞翅目昆虫Met聚在一起。表达模式分析表明,小菜蛾PxMet 1与PxMet-2在蛹期(化蛹后1-3 d)与雌成虫期(羽化后0-72 h)均有表达;PxMet-1的表达量在蛹期(化蛹后1-3 d)无明显差异,但均显著高于雌成虫期(羽化后0-48 h),在羽化后72 h达到高峰;而PxMet-2在雌成虫期(羽化后0-48 h)的表达量呈先上升后下降的趋势,在羽化后12 h出现表达高峰,且成虫期(羽化后0-36 h)的表达量显著高于蛹期。PxMet-1与PxMet-2在成虫脂肪体中的表达量显著高于其他组织。注射PxMet-1+PxMet-2 dsRNA 24 h后,小菜蛾PxMet-1与PxMet-2的表达量均受到显著抑制;同时干扰PxMet-1和PxMet-2后,小菜蛾成熟卵子数目显著减少,羽化后3 d内单雌产卵量显著下降。【结论】抑制Met基因表达能够显著降低小菜蛾雌虫的卵子形成与产卵量。本研究为探索保幼激素的生殖调控机理奠定了基础,在实践上有助于筛选小菜蛾种群遗传调控的潜在靶标。     

Amphiphilic growth hormone releasing factor (GRF) analogs: peptide design and biological activity in vivo

The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.     

保幼激素类似物对烟蚜茧蜂生长发育、寄生率和蜕皮相关酶活性的影响

【目的】烟蚜Myzus persicae是烟草上重要的害虫之一,烟蚜茧蜂Aphidius gifuensis是烟蚜的一种优势寄生蜂。本研究旨在筛选出可延迟烟蚜茧蜂羽化时间的最佳保幼激素,解决规模化繁殖过程中出现的烟蚜茧蜂羽化不一致、不整齐,生产上急需烟蚜茧蜂防控烟蚜时所需烟蚜茧蜂数量不足而影响防蚜效果等严重问题。【方法】利用液浸法测定了不同浓度(5 000, 1 000, 200, 40和8 ng/μL)的5种保幼激素类似物包括稀虫乙酯(ZR-512)、稀虫炔酯(ZR-777)、稀虫酯(ZR-515)、苯氧威［(对苯氧乙基)氨基甲酸乙酯)］和保幼激素Ⅲ(2,6-壬二烯酸)处理后对烟蚜茧蜂羽化率、羽化时间、成蜂寿命、雌蜂比例和寄生率的影响;通过生物化学方法测定1 000 ng/μL这5种保幼激素类似物处理后烟蚜茧蜂蛹内与蜕皮相关酶含量和活性,筛选能延迟烟蚜茧蜂羽化时间的最佳保幼激素类似物。【结果】测试的不同浓 度的5种保幼激素类似物中200 ng/μL ZR-777和1 000 ng/μL ZR-512处理后能显著延迟烟蚜茧蜂羽化时间,分别比对照(10%丙酮处理)延迟了44.00和56.00 h;不同浓度的ZR-515和苯氧威处理后烟蚜茧蜂羽化率较对照组显著降低。与对照组相比,测试的不同浓度的5种保幼激素类似物对成蜂寿命均没有显著影响;1 000和40 ng/μL ZR-777处理显著降低了雌蜂比例。200 ng/μL ZR-777, 1 000 ng/μL ZR-512和5 000 ng/μL ZR-512处理对烟蚜茧蜂的寄生率无显著影响。5种保幼激素类似物以1 000 ng/μL浓度处理时,ZR-512处理组中烟蚜茧蜂蛹内酚氧化酶含量和活性以及几丁质酶活性均最低。【结论】生产上可用1 000 ng/μL ZR-512和200 ng/μL ZR-777处理烟蚜茧蜂,以达到调控烟蚜茧蜂羽化时间,取得足量、一致的烟蚜茧蜂。结果为烟蚜茧蜂规模化繁殖提供了理论依据。     

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.