ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.
Subject(s)
Biosensing Techniques/methods , Green Fluorescent Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Motor Activity/drug effects , Muscles/drug effects , Mutant Chimeric Proteins/genetics , Animals , Animals, Genetically Modified , Azinphosmethyl/analysis , Azinphosmethyl/toxicity , Dose-Response Relationship, Drug , Founder Effect , Galantamine/analysis , Galantamine/toxicity , Gene Expression Regulation , Green Fluorescent Proteins/agonists , Green Fluorescent Proteins/antagonists & inhibitors , Green Fluorescent Proteins/metabolism , Intracellular Signaling Peptides and Proteins/agonists , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Muscles/metabolism , Mutant Chimeric Proteins/agonists , Mutant Chimeric Proteins/antagonists & inhibitors , Mutant Chimeric Proteins/metabolism , Notochord/drug effects , Notochord/metabolism , Pesticides/analysis , Pesticides/toxicity , Promoter Regions, Genetic , Propoxur/analysis , Propoxur/toxicity , ZebrafishABSTRACT
The stoichiometry of the ß2-adrenergic receptor (ß2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that ß2AR mainly existed as monomers under physiological conditions and exhibited ß-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of ß2AR dimerization with biased signalling is revealed.
Subject(s)
Carbazoles/pharmacology , Optical Imaging , Propanolamines/pharmacology , Receptors, Adrenergic, beta-2/metabolism , Carbazoles/chemistry , Carvedilol , Green Fluorescent Proteins/agonists , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Isoproterenol/chemistry , Isoproterenol/pharmacology , Ligands , Microscopy, Fluorescence , Propanolamines/chemistry , Propranolol/chemistry , Propranolol/pharmacology , StereoisomerismABSTRACT
Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals.
Subject(s)
Fish Proteins/genetics , Green Fluorescent Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Metals, Heavy/toxicity , Oryzias/genetics , Water Pollutants, Chemical/toxicity , Animals , Animals, Genetically Modified , Benzhydryl Compounds/toxicity , Female , Fish Proteins/agonists , Fish Proteins/metabolism , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/agonists , Green Fluorescent Proteins/metabolism , HSP70 Heat-Shock Proteins/agonists , HSP70 Heat-Shock Proteins/metabolism , Hexachlorocyclohexane/toxicity , Intestinal Mucosa/metabolism , Intestines/drug effects , Kidney/drug effects , Kidney/metabolism , Liver/drug effects , Liver/metabolism , Male , Nitrophenols/toxicity , Oryzias/metabolism , Phenols/toxicity , Polychlorinated Dibenzodioxins/toxicity , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Skin/drug effects , Skin/metabolismABSTRACT
The melanocortin 4 receptor is important in the regulation of satiety. In this study we have investigated the propensity of the MC4 receptor to homodimerize. MC4 receptors with either a modified green fluorescent protein (GFP(2)) or Renilla luciferase (RLuc) at their C-terminus were constructed. These receptors showed equivalent binding and functional properties to the wild-type MC4 receptor. Bioluminescence resonance energy transfer readings indicated that the MC4 receptor exists as a constitutive homodimer, which was not regulated by peptide interaction. The efficiency of MC4 receptor to form homodimers was greatly enhanced compared to its ability to heterodimerize with the kappa opioid receptor.
