Section on Oxidative Stress Tissue Injury, Laboratory of Physiologic Studies, National Institutes of Health, National Institute of Alcohol Abuse and Alcoholism, Bethesda, Maryland; Linus Pauling ...Institute, Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon; and Department of Intensive Care Medicine, University Hospital, Lausanne, Switzerland
The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.
•Model of coupled reaction–diffusion into cells encapsulated in protective polymer coating.•Dimensionless model over realistic range of physical parameters.•Impact of glucose on oxygen concentration ...less important than relevant oxygen parameters.•Correlated change between oxygen and glucose shown as a function of varying parameters.
The coupled reaction–diffusion transport of glucose and oxygen into a core–shell geometry is modeled using dimensionless parameterization. The consumption of oxygen and glucose are coupled by a metabolic modulation function. The results are fit to a Bayesian-Ridge model that can be easily applied to generate large data sets spanning the parameter space. The non-linear correlation between the oxygen and glucose concentrations are presented and the effect of system parameters is explored. The maximum radius of a viable system is shown to decrease by up to 13.8% with high glucose saturation.
The insulation around the electrical cabling in nuclear power plants is frequently made of ethylene-propylene rubber and crosslinked polyethylene that is subjected to low levels of environmental ...stressors and radiation over the duration of their decades long service life. For the purpose of maintenance and reactor recertification, it is necessary to develop a non-destructive approach to determine the degree of damage the insulation has sustained. Accelerated aging experiments are used to develop these methods, although it is unclear how to relate these specimens to in situ aged ones. Here a kinetic rate model is used to investigate the impact of radiation dose rate and total dose on the crosslinking and chain scission of polyethylene. Analytical expressions for the concentration of crosslinking and scission sites as a function of time and dose rate, both during and post irradiation, are presented. During irradiation the concentration of crosslinking sites increases linearly with time, and when the dosing ends the crosslinking reaction terminates. The scission reaction begins slower, due to the necessity of forming intermediate species, but increases in rate, and eventually the concentration of scission sites overtakes crosslinking. However, at low dose rates, less than 10 Gy/hr, scission damage is always the primary form of damage. Unlike the crosslinking reaction, scission continues for months and years due to the slow decomposition of the intermediate species.
CuATSM is a PET-imaging agent that has recently received attention for its success in extending the lifespan in animals in several neurodegenerative disease models. In the SOD1G93A model of ALS, ...CuATSM prolonged mouse longevity far longer than any previously tested therapeutic agents. The mechanism underlying this outcome has not been fully understood, but studies suggest that this copper complex contributes to maintaining copper homeostasis in mitochondria. More specifically for the SOD1 model, the molecule supplies copper back to the SOD1 protein. Additionally, CuATSM demonstrated similar protective effects in various in vivo Parkinson's disease mouse models. In the current pilot study, we utilized a neurodegenerative mouse model of motor neuron degeneration induced by the neurotoxin β-sitosterol β-D-glucoside. In this model, slow but distinct and progressive features of sporadic ALS occur. Treatment with CuATSM kept animal behavioural performance on par with the controls and prevented the extensive motor neuron degeneration and microglia activation seen in the untreated animals. These outcomes support a broader neuroprotective role for CuATSM beyond mutant SOD models of ALS.
•Plant-derived neurotoxin, BSSG, when fed to mice produces features of ALS-PDC•CuATSM prevented locomotor deficit and motor neuron degeneration caused by BSSG•CuATSM also prevented motor neuron degeneration caused by DMSO•CuATSAM reduced activated microglia levels caused by BSSG neurotoxin
Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian‐like degeneration ...characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100β, GFAP, isolectin and/or p75NTR immunoreactivity. Different subsets of reactive SCs expressed colony‐stimulating factor‐1 (CSF1) and Interleukin‐34 (IL‐34) and closely interacted with numerous endoneurial CSF‐1R‐expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c‐Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c‐Kit in the sciatic nerves of SOD1G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c‐Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF‐1R and c‐Kit with masitinib in SOD1G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.
Schwann cells expressing CSF1, IL‐34, and SCF accumulate in the sciatic nerves from ALS subjects.
Schwann cells interact with myeloid and mast cells expressing CSF‐1R and c‐Kit receptors, respectively.
Pharmacological inhibition of CSF‐1R and c‐Kit ameliorates sciatic nerve pathology.
A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine (HE) or its mitochondrially targeted ...derivative Mito-HE (MitoSOX Red). Two different excitation wavelengths are used to distinguish the superoxide-dependent hydroxylation of Mito-HE (385-405 nm) from the nonspecific formation of ethidium (480-520 nm). Furthermore, the dual wavelength imaging in live cells can be combined with immunocolocalization, which allows superoxide formation to be compared simultaneously in cocultures of two types of genetically manipulated cells in the same microscopic field. The combination of these approaches can greatly improve the specificity for imaging superoxide formation in cultured cells and tissues.
Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due ...to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.
The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium ...cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycinstimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4 x 10⁶ M⁻‘·s⁻¹, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.
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