Dihydrooxadiazines: Octopaminergic system as a potential site of insecticidal action

Dihydrooxadiazines are structural analogs of octopamine and were compared with octopamine for their ability to compete with [³H]dihydroergocryptine ([³H]DHE) for binding sites on DHE-sensitive receptors, to stimulate adenylate cyclase activity in nervous system homogenates of Periplaneta americana L., and to modulate the action of the peptide proctolin on the oviducal muscles of Locusta migratoria L. [³H]DHE binding was inhibited by low concentrations (μM range) of octopamine, phentolamine, N-demethylchlordimeform (DCDM) and several dihydrooxadiazines. The tested dihydrooxadiazines acted as aminergic agonists in stimulating cyclic AMP production in cockroach nervous system homogenates and did not show additive effects with octopamine, whereas additivity was observed with 5-hydroxytryptamine. The relative potency of octopaminergic antagonists, including mianserin, cyproheptadine, phentolamine, and gramine, to block octopamine-mediated elevation of cyclic AMP production was similar to the rank-order potency of the same antagonists to inhibit dihydrooxadiazine-mediated elevation of cyclic AMP production. Octopamine, 2-(4-bromophenyl)-5,6-dihydro-4H-1,3,4-oxadiazine (4-Br-PDHO), and 8-Br-cyclic AMP caused increased phosphorylation of proteins that are phosphorylated by exogenously added cyclic AMP-dependent protein kinase. These results indicate that the dihydrooxadiazine-induced rise in cyclic AMP levels in homogenates of the cockroach nervous system results directly in activation of an endogenous cyclic AMP-dependent protein kinase. 4-Br-PDHO behaved similarly to octopamine in modulating the action of proctolin-induced contractions in locust oviducal muscles. These observations suggest that dihydrooxadiazines act as octopamine agonists and have an octopaminergic action in modulating the action of proctolin. Thus, it is proposed that dihydrooxadiazines exert at least part of their insecticidal and miticidal actions through interaction with the octopaminergic system.     

章鱼胺对营养性肥胖小鼠减肥作用的研究

采用高脂饲料建立小鼠肥胖模型,以小鼠体重、Lee's指数、脂肪湿重、脂肪系数、血清总胆固醇(TC)、甘油三脂(TC)及高密度脂蛋白(HDL-C)等指标的变化,研究章鱼胺对小鼠的减肥作用.结果表明,肥胖对照组小鼠较普通对照组小鼠的体重、脂肪湿重、脂肪系数、血清总胆固醇(TC)、甘油三脂(TG)及高密度脂蛋白(HDL-C)含量均显著增加(P<0.05),表明营养肥胖模型构建成功;章鱼胺各剂量组及阳性对照组中小鼠的体重、脂肪湿重、脂肪系数、血清总胆固醇(TC)与模型组存在显著性差异(P<0.05),其中Lee's指数和甘油三脂(TG)的中、高剂量组与模型组存在显著性差异(P<0.05);章鱼胺对营养性肥胖小鼠有一定的减肥降脂作用.     

赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆、表达及功能

【目的】章鱼胺信号系统在调节昆虫行为和生理过程中具有至关重要的作用。赤拟谷盗(Tribolium castaneum)作为一种模式昆虫,被广泛用于解析昆虫生长发育及生理等调控机制的研究工作。本研究以赤拟谷盗为对象,旨在明确章鱼胺受体在调节赤拟谷盗行为和生理方面的功能。【方法】根据Gen Bank登录的相关序列信息(XP_008198078),利用RT-PCR技术克隆赤拟谷盗章鱼胺受体基因Tc OctβR3的c DNA序列。利用在线生物信息学分析软件预测该基因的开放阅读框、编码的氨基酸序列以及跨膜结构域等信息,基于邻接法构建该基因与其他昆虫相关序列的系统发育树,明确系统进化关系。分别提取赤拟谷盗各发育阶段(卵、幼虫、蛹和成虫)、不同组织(中枢神经系统、脂肪体、中肠、后肠、马氏管、精巢和卵巢)以及饥饿胁迫后的RNA,以赤拟谷盗核糖体蛋白S3(Tc RPS3)为内参基因,采用实时定量PCR技术分析该基因在赤拟谷盗不同发育阶段、不同组织以及在饥饿胁迫下的表达模式。运用哺乳动物异源表达系统在人胚胎肾细胞HEK293中瞬时表达Tc OctβR3,进而利用第二信使c AMP含量测定技术分析Tc OctβR3与配体的结合能力。最后,通过体外合成赤拟谷盗Tc OctβR3的双链RNA,利用RNA干扰以及轨迹球行为分析等技术探究该基因的生理功能。【结果】序列分析结果表明,赤拟谷盗Tc OctβR3开放阅读框全长1 305 bp,编码434个氨基酸,序列中含有G蛋白偶联受体典型的7个跨膜结构域。基于邻接法构建的系统发育树表明,该基因编码的蛋白质与小蜂甲(Aethina tumida)的OctβR3亲缘关系最近。实时定量PCR分析结果表明,Tc OctβR3在赤拟谷盗各发育阶段均有表达,尤其在低龄幼虫期转录水平最高,而在其他发育阶段表达量无显著差异;在赤拟谷盗不同组织中,Tc OctβR3在中枢神经系统的表达量显著高于其他组织;赤拟谷盗幼虫在经饥饿处理24 h的过程中,Tc OctβR3的表达量呈先下降后上升的趋势,且在处理6 h的表达量最低,为对照的0.47倍,在16 h表达量最高,为对照的1.80倍,最后恢复到正常水平。通过HEK293细胞异源表达Tc OctβR3后,c AMP测定结果表明章鱼胺(OA)呈浓度依赖性地激活Tc OctβR3,其EC50为8.68×10-7 mol·L-1,萘甲唑啉(NA)的激动活性最高,其有效中浓度EC50为8.56×10-8 mol·L-1。4种供试生物胺激动剂活性强弱为:萘甲唑啉酪胺(TA)章鱼胺多巴胺(DA)。进一步采用RNA干扰技术的分析结果表明,注射ds RNA能有效抑制Tc OctβR3的表达,沉默效率高达61.5%,但干扰该基因表达后不会影响赤拟谷盗成虫的爬行速度和产卵量。【结论】Tc OctβR3在赤拟谷盗中枢神经系统可能发挥重要作用,能调节幼虫对饥饿胁迫的响应。明确了Tc OctβR3的分子生物学特性,可为将来以其为靶标筛选高效激动剂和抑制剂提供理论依据。     

Identification of novel inhibitors of calling and in vitro [14C]acetate incorporation by pheromone glands of Plodia interpunctella

Some octopamine agonists were found to suppress in vitro biosynthesis of the calling pheromone of the Indian meal moth, Plodia interpunctella. Isolated pheromone-gland preparations incorporated sodium [14C]acetate at a linear rate for 3 h when incubated with the pheromone biosynthesis activating neuropeptide (PBAN). This incorporation was dependent on the dose of PBAN (up to 0.5 microM). Thin-layer chromatography of a pheromone-gland extract revealed quantitative incorporation of radioactivity into a product exhibiting the same mobility as (Z,E)-9,12-tetradecadienyl acetate, the main component of the calling pheromone of P interpunctella. Twenty-seven octopamine agonists were initially screened using a calling behaviour bioassay of female P interpunctella. Four derivatives with activity in the nanomolar range were identified which were, in order of decreasing pheromonostatic activity: 2-(2,6-diethylphenylimino)thiazolidine > 2-(2,6-diethylphenylimino)oxazolidine > 2-(2,6-dimethylphenylimino)thiazolidine > 2-(2-ethylphenylimino)oxazolidine. These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis. The results of the present study indicate that these derivatives could provide useful information in the characterization and differentiation of octopaminergic receptor types and subtypes.     

Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 3. Modification of N-tert-butylhydrazine moiety

Nineteen analogues were synthesized by modifying the tert-butylhydrazine moieties of N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methyl-2,3-dihydro-1,4-benzodioxine-6-carbohydrazide and N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methylchromane-6-carbohydrazide (chromafenozide), and the synthesized analogues were evaluated for their insecticidal activity against Spodoptera litura F. While all of the synthesized analogues had insecticidal activity inferior to those of the lead compounds, several of the analogues nonetheless showed high insecticidal activity. Chromafenozide has shown very high selectivity toward lepidopteran species.     

Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords

To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.     

Baclofen intoxication in a dog successfully treated with hemodialysis and hemoperfusion coupled with intensive supportive care

Objective: To describe a case of confirmed baclofen intoxication in a dog that was successfully treated with hemodialysis and hemoperfusion (HD/HP) and to report the serum baclofen kinetics. Case summary: A 2.5‐year‐old, 23 kg, spayed female Brittany Spaniel‐mix was treated after ingesting 21‐52 mg/kg of baclofen. The dog was comatose and was receiving manual ventilation at the time of presentation. Extracorporeal HD/HP was started 10 hours after admission. Within 3 hours of starting HD/HP the dog began initiating breaths and was extubated 18 hours after admission. Serial serum samples that were obtained during the first 24 hours of hospitalization were later analyzed for baclofen concentrations. The dog had elevated creatine phosphokinase and liver enzymes that correlated with an agitated recovery period. The dog had thrombocytopenia that resolved by 10 days after presentation. New or unique information provided: HD/HP shortened the baclofen serum elimination half‐life from 5 to 1.5 hours in the initial 2 hours of treatment. The intrinsic elimination rate constant (K_intr) for this dog was 0.138/hour and the total elimination rate constant (K_tot) during the first 2 hours of HD/HP treatment was 0.458/hour. In this dog, HD/HP was an effective method for rapidly decreasing serum baclofen concentration after an acute overdose.     

Effect of chronic administration of a gonadotropin‐releasing agonist on luteal function and pregnancy rates in dairy cattle

Increased embryonic losses may be associated with inadequate progesterone (P4) concentrations in high‐producing lactating dairy cattle. The objectives of the present studies were to determine if chronic administration of a gonadotropin‐releasing hormone (GnRH) agonist, Deslorelin, would increase circulating P4 concentrations and subsequently increase pregnancy rates in dairy cattle. Administration of Deslorelin for 12 days increased (p < .05) luteal volume and circulating P4 concentrations in primiparous lactating dairy cows, but increased only luteal volumes in multiparous cows. Treatment with Deslorelin increased Day 45 pregnancy rates in cows as compared to untreated controls. Chronic treatment with Deslorelin in dairy cattle; (a) increased luteal volume of the primary CL, (b) induced accessory CL, (c) increased circulating P4 concentration in primiparous cows only, (d) did not lengthen the estrous cycle upon removal of treatment, and (e) increased pregnancy rates. Although luteal volume was increased in multiparous cows and circulating P4 concentrations were not with Deslorelin treatment, there was an apparent effect on pregnancy rates. This hormonal strategy may represent a suitable model to address local effects of P4 and GnRH/luteinizing hormone on uterine environment and subsequent embryonic survival.