Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers: Preparation, Characterization, and Use
DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998, they have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Potential areas of application involve mitochondrial gene therapy, antioxidant therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Analysis of Mitochondrial Morphology and Function Under Conditions of Mitofusin 2 Deficiency
Recent discoveries linking mitochondrial dynamics to various pathologies have generated interest in the design of robust methods to screen proteins involved in mitochondrial dynamics and small molecules that modulate mitochondrial dynamics. Here, we describe functional screening protocols to analyze mitochondrial parameters such as mitochondrial morphology, reactive oxygen species (ROS) levels, mitochondrial calcium, and oxygen consumption rate in cultured cells. Novel proteins that participate in the regulation of mitochondrial dynamics and function can be identified using such an approach. (Source: Springer protocols fee...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Yeast as a Tool to Study Mitochondrial Retrograde Pathway En Route to Cell Stress Response
Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, ...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Cell Energy Budget Platform for Assessment of Cell Metabolism
We describe a simple methodology for high-throughput multiparametric assessment of cell bioenergetics, called cell energy budget (CEB) platform, and demonstrate its practical use with cell models. The CEB relies on a standard multi-label reader with time-resolved fluorescence capabilities, the lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA) extrusion, the phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), the bioluminescent total ATP assay, and absorbance-based total protein assay. This approach ca...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
In Vivo Visualization and Quantification of Mitochondrial Morphology in C. elegans
Caenorhabditis elegans is a highly malleable model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here, we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Mitochondrial DNA as a Biosensor of UV Exposure in Human Skin
Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Assessment of Short- and Medium-Chain Fatty Acids on Mitochondrial Function in Severe Inflammation
Mitochondrial dysfunction is regarded as one 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 subj...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Atomic Force Microscopy-Based Shape Analysis of Heart Mitochondria
Atomic force microscopy (AFM) has become an important medical and biological tool for the noninvasive imaging of cells and biomaterials in medical, biological, and biophysical research. The major advantages of AFM over conventional optical and electron microscopes for bio-imaging include the facts that no special coating is required and that imaging can be done in all environments—air, vacuum, or aqueous conditions. In addition, it can also precisely determine pico–nano Newton force interactions between the probe tip and the sample surface from force–distance curve measurements. (Source: Springer protocol...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Enzymatic Assays for Probing Mitochondrial Apoptosis
Isolated mitochondria are an invaluable analytical tool to probe mitochondrial function and evaluate apoptosis induction via the so-called mitochondrial pathway. Irrespective of their tissue origin (e.g., heart, liver, muscle, brain), these organelles participate actively to cell and life decision by producing energy for cell metabolism, but also by undergoing a lethal and irreversible mitochondrial membrane permeabilization (MMP) in stress and pathological conditions. MMP consequences consist, at least in part, in loss of mitochondrial transmembrane potential (ΔΨm), matrix swelling, arrest of respiration and ATP...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Predicting Mitochondrial Targeting by Small Molecule Xenobiotics Within Living Cells Using QSAR Models
Whether small molecule xenobiotics (biocides, drugs, probes, toxins) will target mitochondria in living cells can be predicted using an algorithm derived from QSAR modeling. Application of the algorithm requires the chemical structures of all ionic species of the xenobiotic compound in question to be defined, and for certain numerical structure parameters (AI, CBN, log P, pKa, and Z) to be obtained for all such species. How the chemical structures are specified, how the structure parameters are obtained or estimated, and how the algorithm is used are described in an explicit protocol. (Source: Springer protocols feed by Ph...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
A Cybrid Cell Model for the Assessment of the Link Between Mitochondrial Deficits and Sporadic Parkinson’s Disease
Parkinson’s disease (PD) is a multifactorial and clinically complex age-related movement disorder. The cause of its most common form (sporadic PD, sPD) is unknown, but one prominent causal factor is mitochondrial dysfunction. Although several genetic- and toxin-based models have been developed along the last decades to mimic the pathological cascade of PD, cellular models that reliably recapitulate the pathological features of the neurons that degenerate in PD are scarce. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Analysis of Mitochondrial Network by Imaging: Proof of Technique in Schizophrenia
Mitochondria, similar to living cells and organelles, have negative membrane potential and can therefore accumulate permeable lipophilic cations. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of the lipophilic cation 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Targeting Mitochondria with Small Molecules: The Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide
Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen p...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
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Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Synthesis of Triphenylphosphonium Phospholipid Conjugates for the Preparation of Mitochondriotropic Liposomes
We describe here a simple protocol to conjugate a triphenylphosphonium group to several commercially available functionalized phospholipids. The resulting triphenylphosphonium conjugated lipids can be used to prepare liposomes that preferentially associate with mitochondria when exposed to live mammalian cells in culture. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
