Structural Factors Controlling the Spin–Spin Exchange Coupling: EPR Spectroscopic Studies of Highly Asymmetric Trityl–Nitroxide Biradicals

Highly asymmetric exchange-coupled biradicals, e.g., the trityl–nitroxides (TNs), possess particular magnetic properties that have opened new possibilities for their application in biophysical, physicochemical, and biological studies. In the present work, we investigated the effect of the linker length on the spin–spin coupling interaction (<i>J</i>) in TN biradicals using the newly synthesized biradicals CT02-GT, CT02-AT, CT02-VT, and CT02-PPT as well as the previously reported biradicals TNN14 and TN1. The results show that the magnitude of <i>J</i> can be easily tuned from ∼4 G (conformer 1 in CT02-PPT) to >1200 G (in TNN14) by varying the linker separating the two radical moieties and changing the temperature. Computer simulations of EPR spectra were carried out to estimate <i>J</i> values of the TN biradicals directly. In addition to the spin–spin coupling interaction of TN biradicals, their <i>g</i>, hyperfine-splitting, and zero-field-splitting interactions were explored at low temperature (220 K). Our present study clearly shows that varying the spin–spin interaction as a function of linker distance and temperature provides an effective strategy for the development of new TN biradicals that can find wide applications in relevant fields

Esterified Dendritic TAM Radicals with Very High Stability and Enhanced Oxygen Sensitivity

In this work, we have developed a new class of dendritic TAM radicals (TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O₂ sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03. Like the previous dendritic TAM radicals, these new compounds also show extremely high stability toward various reactive species owing to the dendritic encapsulation. The highly charged nature of these molecules resulting from nine carboxylate groups prevents concentration-dependent EPR line broadening at physiological pH. Furthermore, we demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the nine carboxylate groups and the resulting PEGylated analogue dTdG–PEG completely inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These new dendritic TAM radicals show great potential for in vivo EPR oximetric applications and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications

Measurement and Characterization of Superoxide Generation from Xanthine Dehydrogenase: A Redox-Regulated Pathway of Radical Generation in Ischemic Tissues

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, ^•O₂^–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of ^•O₂^–, experiments were performed to measure and characterize ^•O₂^– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-<i>N</i>-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of ^•O₂^–. NAD⁺ and NADH inhibited the generation of ^•O₂^– from XDH in a dose-dependent manner, with NAD⁺ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD⁺ and NADH, which occur during and following tissue ischemia, enhanced the generation of ^•O₂^– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca²⁺-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD⁺ and NADH. Thus, XDH can be an important redox-regulated source of ^•O₂^– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury

Pulsed ESR Dipolar Spectroscopy for Distance Measurements in Immobilized Spin Labeled Proteins in Liquid Solution

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron–electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized

Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O₂‑Dependent Superoxide Production

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV–vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O₂) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O₂ by the trityl carbanions was shown to be superoxide radical (O₂^•–) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (<i>k</i>) depend on the types of both trityl radicals and thiols with the order of <i>k</i>_ET‑03/Cys (0.336 M^–1 s^–1) > <i>k</i>_ET‑03/GSH (0.070 M^–1 s^–1) > <i>k</i>_AT‑03/Cys (0.032 M^–1 s^–1) > <i>k</i>_AT‑03/GSH (0.027 M^–1 s^–1). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the p<i>K</i>_a of the thiols and is further reflected by the rate of O₂^•– production and consumptions of O₂ and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

<div><p>Background</p><p>Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.</p><p>Results</p><p>Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.</p><p>Conclusions</p><p>Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.</p></div

miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.

<p>(<b>A</b>,<b>B</b>) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (<b>C</b>,<b>D</b>) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).</p

Dicer deletion in the adult heart leads to oxidative stress.

<p>(<b>A</b>) Representative EPR imaging of nitroxide radical decay in (A) Dicer^+/+ and (B) Dicer^−/− hearts. (<b>B</b>) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer^+/+ and line with grey circles represent Dicer^−/−. (<b>C</b>) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (<b>D</b>) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer^+/+ and Dicer^−/− hearts and was significantly higher in the later. (<b>E</b>) Total GSSG to GSH ratio in Dicer^+/+ mice heart compared to Dicer^−/− heart. (<b>F</b>) Lactate levels measured in Dicer^+/+ and Dicer^−/− mice hearts.</p

Suppression of induced miRNA-15b attenuates loss of Pim-1 and improves mitochondrial integrity.

<p>Immunohistochemical comparison of (<b>A</b>) Dicer (<b>B</b>) ANF, (<b>C</b>) Pim-1(<b>D</b>) ANT-1 (<b>E</b>) Comparison of cardiac histopathology sections stained with hematoxylin and eosin (H&E) (<b>F</b>) Bar graph showing quantification of IHC images. Image analysis software (Axiovision 4.3, Zeiss, Germany) was used to quantify fluorescence intensity (fluorescent pixels) and analyzed as a percent change in relative fluorescence unit (RFU). (n = 3) (<b>G</b>) Comparison between % of total mitochondria belonging to Class I/II or Class III/IV. (<b>H</b>) Representative TEM images of mitochondria from Dicer depleted heart tissue treated with anti-miR-15b. <i>Bar graphs represent equal SD on both sides of the mean.</i></p

Arterial Levels of Oxygen Stimulate Intimal Hyperplasia in Human Saphenous Veins via a ROS-Dependent Mechanism

<div><p>Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO₂), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO₂. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO₂ using an established organ culture model. Saphenous veins cultured with arterial pO₂ developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO₂ or internal mammary arteries cultured at arterial pO₂ did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO₂ is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO₂ in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO₂. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO₂ relative to those cultured at venous pO₂. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO₂ stimulates IH via a ROS-dependent pathway.</p></div