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Lyhne Hedegaard posted an update 6 months, 3 weeks ago
Mitochondria are targets of newly synthesized drugs and being tested for the treatment of various diseases caused or accompanied by disruption of cellular bioenergetics. In drug development, it is necessary to test for drug-induced changes in mitochondrial enzyme activity that may be related to therapeutic or adverse drug effects. Measurement of drug effect on mitochondrial oxygen consumption kinetics and/or protective effects of drugs against calcium-induced inhibition of the mitochondrial respiration can be used for the study mitochondrial toxicity and neuroprotective effects of drugs. Supposing that the drug-induced inhibition of the mitochondrial respiratory rate and/or individual mitochondrial complexes is associated with adverse drug effects, the effects of drugs on mitochondrial respiration in isolated mitochondria allow selection of novel molecules that are relatively safe for mitochondrial toxicity.Mitochondrial dysfunction is regarded as a key factor involved in the pathogenesis of septic disorders, leading to a decline in energy supply. The influence of short- and medium-chain fatty acids (SCFA/MCFA) on mitochondrial respiration under inflammatory conditions has thus far not been investigated. In the following protocol we describe the assessment of mitochondrial respiration using high-resolution respirometry under inflammatory and baseline conditions. For this approach, human endothelial cells and monocytes were pretreated with TNF-α to mimic inflammation followed by incubation with SCFA/MCFA and then subjected to high-resolution respirometry. Mitochondrial DNA content was assessed by PCR .This chapter describes the complementary experimental techniques Electron Transmission Spectroscopy and Dissociative Electron Attachment Spectroscopy, two of the most suitable means for investigating interactions between electrons and gas-phase molecules, resonance formation of temporary molecular negative ions, and their possible decay through the dissociative electron attachment (DEA) mechanism. The latter can be seen as the gas-phase counterpart of the transfer of a solvated electron in solution, accompanied by dissociation of the molecular anion, referred to as dissociative electron transfer (DET). DET takes place in vivo under reductive conditions, for instance, in the intermembrane space of mitochondria under interaction of xenobiotic molecules possessing high electron affinity with electrons “leaked” from the mitochondrial respiratory chain. A likely mechanism of the toxic activity of dichlorodiphenyltrichloroethane based on its DEA properties is briefly outlined, and compared with the well-established harmful effects of the model toxicant carbon tetrachloride ascribed to reductive dechlorination in a cellular ambient. A possible mechanism of the antioxidant activity of polyphenolic compounds present near the main site of superoxide anion production in mitochondria is also briefly discussed.Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA , respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. selleckchem The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAs into the mitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and “in solution” chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ0) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.
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