RESUMEN
Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.
Asunto(s)
Cardenólidos/metabolismo , Erysimum/genética , Erysimum/metabolismo , Herbivoria/efectos de los fármacos , Repelentes de Insectos/metabolismo , Animales , Áfidos/fisiología , Brassica/metabolismo , Cardenólidos/química , Digitoxigenina/química , Digitoxigenina/metabolismo , Expresión Génica , Glucosinolatos/química , Glucosinolatos/metabolismo , Repelentes de Insectos/química , Mariposas Nocturnas/metabolismo , Mutación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estrofantidina/química , Estrofantidina/metabolismoRESUMEN
Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.
Asunto(s)
Digoxigenina/toxicidad , Hepatocitos/efectos de los fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Ouabaína/análogos & derivados , Estrofantidina/toxicidad , Células Th17/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Digoxigenina/química , Relación Dosis-Respuesta a Droga , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Ouabaína/química , Ouabaína/toxicidad , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Estrofantidina/química , Relación Estructura-Actividad , Células Th17/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na(+)/K(+) ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency. FROM THE CLINICAL EDITOR: In this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency.
Asunto(s)
Lípidos/administración & dosificación , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/genética , Estrofantidina/administración & dosificación , Animales , Endocitosis/genética , Silenciador del Gen/efectos de los fármacos , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Ligandos , Lípidos/química , Nanopartículas/química , ARN Interferente Pequeño/química , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estrofantidina/químicaRESUMEN
Toxicosis of Adonis aestivalis is well documented in horses, but little is known of its toxicity in cattle. A. aestivalis (summer pheasant's eye) was collected over multiple years, under different growing conditions, and at various stages of maturity, dried, and administered to calves to evaluate the toxicity of A. aestivalis in cattle. Four 300-lb Holstein, and 2 90-lb, preruminating Jersey calves were administered 1% body weight of ground A. aestivalis via a stomach tube and monitored for clinical signs for 2 weeks and 1 week, respectively. The Holstein calves were then fed 0.2 to 1% body weight A. aestivalis daily for 4 to 5 weeks. The Holstein calves had transient, mild cardiac abnormalities during the feeding trial. Mild, transient gastrointestinal and cardiac signs were noted in the preruminating calves. No gross or microscopic lesions were seen on necropsies performed at the end of the study. Based on the results of this study, cattle do not appear to be as susceptible to toxicosis from A. aestivalis as other species, such as horses and pigs.