Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study

Experimental allergic encephalomyelitis (EAE) is a demyelinating autoimmune disorder that can be induced in susceptible mice by T lymphocytes sensitized to central nervous system (CNS) myelin components and is a prime animal model for the human CNS demyelinating disorder, multiple sclerosis (MS). Although CNS inflammation in which T lymphocytes and activated macrophages are the predominant cell types is observed in mice with EAE and in humans with MS, the exact mechanisms underlying the CNS damage and demyelination are not understood. Nitric oxide (NO), a gaseous free radical, has recently been shown to be a cytolytic product of activated macrophages. Using electron paramagnetic resonance spectroscopy, the nitric oxide free radical complexed with iron-sulfur proteins has been identified in affected spinal cords of mice with EAE, concurrent with the diminution of iron-sulfur proteins. These results indicate NO may play a role in the disease process of EAE, and perhaps MS.     

An application of electron paramagnetic resonance to evaluate nitric oxide and its quenchers

Electron paramagnetic resonance (EPR) spectrometry has been used for detection of free radicals, including nitric oxide (NO). The method was applied to evaluate if lazaroids, nonspecific scavengers of oxygen-derived free radicals, can reduce another radical species, namely, NO. When solutions containing 0 to 10 microM lazaroids (U78517F, U83836E, and U74500A) were mixed with NO (approximately 8.0 microM), and with a spin-trap agent specific to NO (10 microM carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide, c-PTIO), the spectrum of NO-c-PTIO determined by the EPR decreased with increasing concentrations of lazaroids. An identical study with 1 to 1000 microM methylprednisolone or dexamethasone, which are prototypes of lazaroids, failed to reduce NO. In a separate study, rat red blood cells preloaded with U78517F were exposed to NO. EPR spectrometry demonstrated that hemoglobin in these cells were less nitrosylated compared with the cells not preloaded with U78517F. An ability of lazaroids to quench NO in vivo was evaluated by an intravenous injection of sodium nitrite (4 mumol/kg body weight) into rats pretreated with 10 mg/kg lazaroids (U78517F and U74500A). The nitrosylation of RBC hemoglobin was markedly attenuated in rats pretreated with lazaroids compared with those with vehicle. Thus, these lipophilic compounds protected cells from NO-induced nitrosylation both in vitro and in vivo. Lazaroids may be applicable as anti-NO agents.     

Binding of nitric oxide to intact human erythrocytes as monitored by electron paramagnetic resonance

Human blood was diluted in isotonic saline at pH 7.4 and deoxygenated. After gentle exposure to nitric oxide gas (NO), the red blood cells (erythrocytes) remained intact. Increasing the cell volume fraction allowed detection of strong electron paramagnetic (EPR) signals, even at ambient temperature (293 K). These spectra were compared to those recorded at 77 K. With maximal NO exposure, a relatively featureless and stable spectrum was recorded. Reduced exposure produced a spectrum, which gradually transformed into the final one, with more structure. The spectral features with unfrozen samples reflected the degree of resolution of the hyperfine triplet component observed at 77 K. This hyperfine coupling was independent of the temperature. The spectra reveal rearrangement of the NO ligand between the subunits of hemoglobin. At subsaturation levels, binding to the alpha subunit, with its iron pentacoordinated in the T-state, dominates.     

Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.     

Multipolar laminated electromagnet for low-field magnetic resonance imaging and electron paramagnetic resonance imaging

A cylindrical 16-pole electromagnet (EM) for electron paramagnetic resonance imaging (EPRI) and low-field magnetic resonance imaging (MRI) has been designed by means of two-dimensional and three-dimensional (3-D) finite element analysis (FEA). The use of an automatic procedure that combines FEA with a minimization routine allowed the optimization of the design, in order to improve the homogeneity along the axis of the EM. A prototype has been built by using electrical steel sheets that were cut by laser; this solution reduced significantly the manufacturing cost. The EM operates with a maximum flux density, in the bore, of 0.08 T and has a homogeneity along the axis of about 40 parts per million (ppm) in a spherical region 10 cm in diameter. It generates the main field and two of the three field gradients required in the 3-D image reconstruction. Good agreement was found between the results of simulation and the measured values.     

Nitric oxide synthase. Structural studies using anti-peptide antibodies

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681-4690]. Overall, they allow prediction of whether each peptide region will be an alpha-helix, a beta-strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies. These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (approximately 50%). Most of the selected anti-peptide antibodies were not able to cross-react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross-reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric oxide prevents oxidative damage produced by tert-butyl hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron. Electron paramagnetic resonance evidence

We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-iu m-1, 2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2, 2'-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.     

Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance

Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.     

Why spin = 1, 2 species have no electron paramagnetic resonance signal under normal conditions: possible detection by electron paramagnetic resonance at frequency close to D value?

A universal EPR simulation program has been created by the author, which is based on the following spin Hamiltonian equation: [equation: see text] where D and E are the axial and rhombic zero-field splitting parameters, respectively. The program can be used for simulation of EPR spectra with half-integer electronic spin (S = n/2, n = 3, 5, 7, 9) systems. In this article, the integer spin (S = n/2, n = 2, 4) systems are also considered. The EPR simulation results show that when D > frequency, no EPR signal can be seen from EPR simulation; when D approximately frequency, whichever X/Q/W-band is used, the EPR signal can be seen on the basis of the simulated EPR results presented.     

Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation

The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate. Reactions were monitored through the use of freeze-quench EPR spectroscopy. Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1. Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment. Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b. Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly. However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen. In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus. Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism.     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.     

Iron catalyzes both decomposition and synthesis of S-nitrosothiols: optical and electron paramagnetic resonance studies

This paper discusses the issues of providing an empirically validated, manual-based-treatment when viewed from the perspective of a practicing clinical psychologist. The trend for empirically proven treatment is reviewed briefly, and initial data are provided illustrating a manual-based-treatment for post-traumatic stress disorder (PTSD) following a motor vehicle accident. The relatively brief (9-12 session) psychological treatment was effective in reducing PTSD symptoms as measured on the Clinician Administered PTSD Scale, for all 10 subjects. The results are discussed form the practicing clinician's perspective: generalization to a clinical population, ethical concerns of limited treatment goals, individually tailored vs standardized treatments in clinical practice, concerns for co-morbid conditions, and how this type of study might impact on practice in an era of managed health care. Issues of incorporating manual based treatments into clinical practice are discussed, with consideration of gains, the limits and the constraints this would bring to the practice of psychology.