Probing the redox activity of T-lymphocytes deposited at electrode surfaces with voltammetric methods

Background: Reactive oxygen species and redox signaling play an important role in the regulation of many vital biological processes. However, they are also tightly connected with many pathological conditions. The detection and evaluation of these signaling events are very often accompanied with great difficulties. In this article, we describe the development of a novel electrochemically-based technique for monitoring the cellular redox state. Methods and results: T-cells were attached on the surface of a working electrode, which was modified with 2-palmitoylhydroquinone as a redox mediator. Using cyclic voltammetry, we were able to indirectly (via the redox mediator) monitor an electron transport from the cells towards the working electrode, which enabled us to evaluate the redox activity of the cells. Conclusions: This new technique is rather simple and sensitive and may be used in the future as a valid diagnostic procedure in various branches of biomedical science

Protein film voltammetry: electrochemical enzymatic spectroscopy. A review on recent progress

This review is focused on the basic principles, the main applications, and the theoretical models developed for various redox mechanisms in protein film voltammetry, with a special emphasis to square-wave voltammetry as a working technique. Special attention is paid to the thermodynamic and kinetic parameters of relevant enzymes studied in the last decade at various modified electrodes, and their use as a platform for the detection of reactive oxygen species is also discussed. A set of recurrent formulas for simulations of different redox mechanisms of lipophilic enzymes is supplied together with representative simulated voltammograms that illustrate the most relevant voltammetric features of proteins studied under conditions of square-wave voltammetry

Redox Chemistry of Ca-Transporter 2-Palmitoylhydroquinone in an Artificial Thin Organic Film Membrane

The redox chemistry of 2-palmitoylhydroquinone (H2Q), a recently introduced synthetic transmembrane Ca2+ transporter, was studied with cyclic and square-wave voltammetry in an artificial thin organic-film membrane sandwiched between a pyrolytic graphite electrode and an aqueous solution. The membrane has a micrometer dimension and consists of the water immiscible organic solvent nitrobenzene, which contains suitable electrolyte and H2Q as a redox active compound. The potential drop at the electrode/membrane interface is controlled by the potentiostat, whereas the potential drop at the membrane/water interface is dependent on the ClO4 - concentration, which is present in a large excess in both liquid phases. The redox transformation of H2Q at the electrode/membrane interface is accompanied by a corresponding ion-transfer reaction at the other side of the membrane. Proton transfer at the membrane/water interface is critical for the redox transformation of H2Q in the interior of the membrane, as a strong dependence of the voltammetric response on the pH of the aqueous medium was observed. H2Q undergoes two oxidation processes due to existence of two distinctive redox forms of H2Q. The electrochemical mechanism can be explained with two tautomer forms of H2Q formed by migration of a proton between the 1-hydroxyl group and the adjacent carbonyl group of the palmitoyl residue. Both tautomers undergo 2e/2H+ distinctive redox transformations to form the quinone form of the studied compound. In the presence of Ca2+ in the aqueous phase, voltammetric experiments confirmed the capability of both tautomers to form 1:1 complexes with Ca2+ and to extract it into the organic membrane. Upon the oxidation of the complexes, Ca2+ is expelled back to the aqueous phase. The studied compound exhibits very similar complexing affinity toward Mg2+, implying that it is not highly selective for transmembrane Ca2+ transport

Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM

STIM-Orai Channels and Reactive Oxygen Species in the Tumor Microenvironment

The tumor microenvironment (TME) is shaped by cancer and noncancerous cells, the extracellular matrix, soluble factors, and blood vessels. Interactions between the cells, matrix, soluble factors, and blood vessels generate this complex heterogeneous microenvironment. The TME may be metabolically beneficial or unbeneficial for tumor growth, it may favor or not favor a productive immune response against tumor cells, or it may even favor conditions suited to hijacking the immune system for benefitting tumor growth. Soluble factors relevant for TME include oxygen, reactive oxygen species (ROS), ATP, Ca2+, H+, growth factors, or cytokines. Ca2+ plays a prominent role in the TME because its concentration is directly linked to cancer cell proliferation, apoptosis, or migration but also to immune cell function. Stromal-interaction molecules (STIM)-activated Orai channels are major Ca2+ entry channels in cancer cells and immune cells, they are upregulated in many tumors, and they are strongly regulated by ROS. Thus, STIM and Orai are interesting candidates to regulate cancer cell fate in the TME. In this review, we summarize the current knowledge about the function of ROS and STIM/Orai in cancer cells; discuss their interdependencies; and propose new hypotheses how TME, ROS, and Orai channels influence each other

Redox regulation of calcium ion channels: Chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca2+ concentration are key triggers for diverse cellular functions. Ca2+ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca2+ permeable ion channels such as voltage-gated (CaV) Ca2+ channels, transient receptor potential (TRP) channels and Orai channels

Calcium Binding and Transport by Coenzyme Q

Coenzyme Q10 (CoQ10) is one of the essential components of the mitochondrial electron-transport chain (ETC) with the primary function to transfer electrons along and protons across the inner mitochondrial membrane (IMM). The concomitant proton gradient across the IMM is essential for the process of oxidative phosphorylation and consequently ATP production. Cytochrome P450 (CYP450) monoxygenase enzymes are known to induce structural changes in a variety of compounds and are expressed in the IMM. However, it is unknown if CYP450 interacts with CoQ10 and how such an interaction would affect mitochondrial function. Using voltammetry, UV�vis spectrometry, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), fluorescence microscopy and high performance liquid chromatography�mass spectrometry (HPLC�MS), we show that both CoQ10 and its analogue CoQ1, when exposed to CYP450 or alkaline media, undergo structural changes through a complex reaction pathway and form quinone structures with distinct properties. Hereby, one or both methoxy groups at positions 2 and 3 on the quinone ring are replaced by hydroxyl groups in a time-dependent manner. In comparison with the native forms, the electrochemically reduced forms of the new hydroxylated CoQs have higher antioxidative potential and are also now able to bind and transport Ca2þ across artificial biomimetic membranes. Our results open new perspectives on the physiological importance of CoQ10 and its analogues, not only as electron and proton transporters, but also as potential regulators of mitochondrial Ca2þ and redox homeostasis

Cytotoxic Efficiency of Human CD8+ T Cell Memory Subtypes

Immunological memory is important to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are well defined, single TEM and TCM cell cytotoxicity has not been quantified. To quantify cytotoxic efficiency of TEM and TCM, we developed a FRET-based single cell fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis, secondary necrosis following apoptosis, and primary necrosis after TEM- or TCM-target cell contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic efficiency of TEM compared to TCM, as quantified by target cell apoptosis and secondary necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in TEM compared to TCM. Higher perforin levels (likely in combination with higher granzyme levels) mediate higher cytotoxic efficiency of TEM compared to TCM. Both, TEM and TCM need the same time to find their targets, however contact time between CTL and target, time to induce apoptosis, and time to induce secondary necrosis are all shorter for TEM. In addition, immune synapse formation in TEM appears to be slightly more efficient than in TCM. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanisms is important to optimize future subset-based immune therapies