ABSTRACT
Metabolic profiling of macrophage metabolic response upon exposure to 4-hydroxynonenal (HNE) demonstrates that HNE does not simply inactivate superoxide-generating enzymes but also could be responsible for the impairment of downfield signaling pathways. Multianalyte microphysiometry (MAMP) was employed to simultaneously measure perturbations in extracellular acidification, lactate production, and oxygen consumption for the examination of aerobic and anaerobic pathways. Combining the activation of oxidative burst with phorbol myristate acetate (PMA) and the immunosuppression with HNE, the complex nature of HNE toxicity was determined to be concentration- and time-dependent. Further analysis was utilized to assess the temporal effect of HNE on reactive oxygen species (ROS) production and on protein kinase C (PKC). Increased levels of HNE with decreasing PKC activity suggest that PKC is a target for HNE adductation prior to oxidative burst. Additionally, localization of PKC to the cell membrane was prevented with the introduction of HNE, demonstrating a consequence of HNE adductation on NADPH activation. The impairment of ROS by HNE suggests that HNE has a greater role in foam cell formation and tissue damage than is already known. Although work has been performed to understand the effect of HNE's regulation of specific signaling pathways, details regarding its involvement in cellular metabolism as a whole are generally unknown. This study examines the impact of HNE on macrophage oxidative burst and identifies PKC as a key protein for HNE suppression and eventual metabolic response.
Subject(s)
Aldehydes/metabolism , Aldehydes/chemistry , Aldehydes/toxicity , Animals , Cell Line , Electrochemical Techniques , Electrodes , Luminol/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Microscopy, Confocal , NADP/chemistry , Protein Kinase C/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
A strategy is presented for the live cell imaging of messenger RNA using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNase degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.
Subject(s)
Colloids , DNA/metabolism , Gold , RNA, Messenger/genetics , Cell Line , DNA/chemistry , Flow Cytometry , Fluorescence , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Respiratory Syncytial Viruses/geneticsABSTRACT
Respiratory viruses are a constant concern for all demographics. Examples include established viruses such as respiratory syncytial virus (RSV), the leading cause of respiratory infection in infants and young children, and emerging viruses such as severe acute respiratory syndrome (SARS), which reached near pandemic levels in 2003, or H1N1 (swine) influenza. Despite this prevalence, traditional methods of virus detection are typically labor intensive and require several days to successfully confirm infection. Recently, however, nanoparticle-based detection strategies have been employed in an effort to develop detection assays that are both sensitive and expedient. Each of these platforms capitalizes on the unique properties of nanoparticles for the detection of respiratory viruses. In this article, several nanoparticle-based scaffolds are discussed. Gold nanoparticles (AuNPs) have been functionalized with virus specific antibodies or oligonucleotides. In each of these constructs, AuNPs act as both an easily conjugated scaffolding system for biological molecules and a powerful fluorescence quencher. AuNPs have also been immobilized and used as electrochemical transducers. They efficiently serve as a conducting interface of electrocatalyic activity making them a powerful tool in this application. Quantum dots (QDs) posses unique fluorescence properties that have also been explored for their application to virus detection when combined with direct antibody conjugation or streptavidin-biotin binding systems. QDs have an advantage over many traditional fluorophores because their fluorescence properties can be finely tuned and they are resistant to photobleaching. The development of these nanoparticle-based detection strategies holds the potential to be a powerful method to quickly and easily confirm respiratory virus infection.
