Insect adipokinetic hormones (AKHs) are pleiotropic hormones known to play a protective role in response to oxidative stress (OS). However, the precise signaling pathways are unclear. We present evidence that AKH may primarily employ the Forkhead box class O transcription factor (FoxO) to exert this effect. The impact of knocking down AKH synthesis or its over-expression in its response to OS was studied in Drosophila melanogaster. AKH knockdown (AKH-RNAi) as well as AKH overexpression (AKH-oex) was achieved using the Gal-4/UAS system and controls were w(1118) (+/+), AKH-Gal4/+, UAS-AKH/+ and UAS-AKH-RNAi/+. Exposure to 80 μM hydrogen peroxide (HP) revealed that AKH-RNAi flies showed significantly higher mortality than AKH-oex or the respective control lines. This susceptibility was evidenced by significantly enhanced levels of protein carbonyls - a biomarker of OS, in AKH-RNAi flies compared to controls and AKH-oex flies. Interestingly, AKH-oex flies had the least amount of protein carbonyls. AKH-RNAi flies had significantly less dFoxO transcript and translated protein compared to control and AKH-oex flies in un-challenged condition as well as when challenged with HP. Sestrin - a major antioxidant defense protein and one of the targets of dFoxO - was also significantly down-regulated (both at mRNA and protein level) in AKH-RNAi flies (both unchallenged and challenged with HP) compared to control flies and flies with over-expressed AKH. These findings imply that dFoxO may act downstream of AKH as a transcription factor to mediate response to OS in D. melanogaster.

• MeSH
• biologické markery metabolismus   MeSH
• Drosophila melanogaster genetika   fyziologie   MeSH
• forkhead transkripční faktory genetika   metabolismus   MeSH
• geneticky modifikovaná zvířata MeSH
• hmyzí hormony antagonisté a inhibitory   genetika   metabolismus   MeSH
• karbonylace proteinů účinky léků   MeSH
• křížení genetické MeSH
• kyselina pyrrolidonkarboxylová analogy a deriváty   antagonisté a inhibitory   metabolismus   MeSH
• léková rezistence MeSH
• MAP kinasový signální systém účinky léků   MeSH
• messenger RNA metabolismus   MeSH
• oligopeptidy antagonisté a inhibitory   genetika   metabolismus   MeSH
• oxidační stres * MeSH
• oxidancia toxicita   MeSH
• peroxid vodíku toxicita   MeSH
• proteiny Drosophily antagonisté a inhibitory   genetika   metabolismus   MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• regulace genové exprese * účinky léků   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.

• MeSH
• aktivace transkripce účinky léků   MeSH
• genetické inženýrství metody   MeSH
• knihovny malých molekul analýza   MeSH
• lidé MeSH
• luciferasy genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• objevování léků metody   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• reportérové geny MeSH
• rychlé screeningové testy metody   MeSH
• sekvenční delece MeSH
• steroidní receptory agonisté   antagonisté a inhibitory   genetika   metabolismus   MeSH
• steroidy farmakologie   MeSH
• transfekce MeSH
• transkripční faktory genetika   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Genetické modifikace (GM) hmyzu zahrnují řadu metod vedoucích k odstranění (umlčení) určitého genu (nejčastěji pomocí RNAi, nebo knockouť genu) nebo vnesení cizorodého genu (hlavně pomocí transpozonů, z nichž je nejznámější systém P-elementů). V obou případech se úspěšnost GM sleduje pomocí exprese „marker" genů (dnes většinou „green fluorescent protein"). Pro drozofilu jsou dostupné další techniky a zejména velké banky různých mutací. Systém cis elementu UAS („Upstream Activation Sequence") a jeho aktivátoru Gal4 umožňuje exprimovat vnesený gen v určitých buňkách či orgánech. Genetické modifikace u drozofily i dalších modelových druhů hmyzu se používají jako prostředek pro objasnění vývojových a fyziologických regulací, včetně poruch, které napodobují lidská onemocnění. GM mohou také omezit rozmnožování, nebo způsobit úhyn škodlivého hmyzu. Tato technika by však vyžadovala vypouštění GM hmyzu do přírody a to je v současnosti nemožné. Prakticky významné jsou však GM využívané v chovech hmyzu, který je po sterilizaci vypouštěn do přírody, aby svým přečíslením podstatně snížil rozmnožování přírodní populace (tzv. „sterile insect technice", SIT).

Genetic modifications of insects include diverse methods of gene silencing, such as gene knockdown or RNAi, and methods of transgenesis when a foreign gene is introduced into insect genome, usually with the aid of transponsons such as the P elements. The success of GM is typically visualized through expression of a gene encoding the green fluorescent protein or another marker. Additional techniques and also large banks of diverse mutations are available for drosophila. The system of UAS (Upstream Activation Sequence) and its transcription activator Gal4 allows in this species transgene expression in certain cells and organs. Genetic modifications of drosophila and other model insect species provide efficient tool for elucidating developmental and physiological regulations, including defects mimicking human diseases. GM can also be used to curb insect reproduction or survival. Practical deployment of this approach, however, would require release of GM insects into the environment and this would hardly be permitted. On the other hand, GMs restricted to insect cultures are practically exploited in the sterile insect technique (SIT), when sterilized, non-GM insects are released into the field and due to their high numbers and random mating suppress the growth of the native wild population.

• Klíčová slova
• umlčení genů, transgeneze, P-elementy, Piggy bag, GFP, UAS/Gal4, vypouštění sterilních samců,
• MeSH
• financování organizované MeSH
• geneticky modifikovaná zvířata genetika   MeSH
• hmyz genetika   MeSH
• regulace genové exprese genetika   MeSH
• RNA interference MeSH
• technika přenosu genů MeSH
• transgeny fyziologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH