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1.
Ion and pH gradients across the transport membrane of mitochondria following Mn ++ uptake in the presence of acetate 总被引:4,自引:0,他引:4
The electron paramagnetic resonance (EPR) spectrum of Mn++ loaded mitochondria is affected by the presence of the permeant anion acetate (Ac?) in the medium. The hyperfine sextet, shown earlier to have spectral characteristics like those expected of osmotically active Mn++ in the matrix space, grows in intensity with increasing [Ac?]. From estimates of mitochondrial water, the free internal [Mn++] can be calculated. The gradient of free [Mn++] across the inner mitochondrial membrane is believed to be at least 500:1 under conditions of high [Ac?]. Since Mn++ solubility is limited by [OH?], it is possible to place an upper limit on the pH in the matrix space. The variation of free internal [Mn++], as measured by EPR, with external pH indicates that the [H+] gradient is 1–1.5 pH units in the absence of permeant anions and considerably less in the presence of 100 mM acetate. 相似文献
2.
Guanylate cyclase from crude homogenates of vegetative has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn++ to Mg++ as divalent cation, requires Mn++ in excess of GTP for detectable activity, and is inhibited by high Mn++ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn++.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells. 相似文献
3.
Philippe P.R. Rosias Gertjan J.M. Den Hartog Charlotte M.H.H.T. Robroeks AALT Bast Raymond A.M.G. Donckerwolcke Jan W.C.M Heynens 《Free radical research》2013,47(9):901-909
Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy. 相似文献
4.
Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs 总被引:4,自引:0,他引:4
Mishra OP Zanelli S Ohnishi ST Delivoria-Papadopoulos M 《Neurochemical research》2000,25(12):1559-1565
Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca++-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO2 = 21%, n = 5) or hypoxic (FiO2 = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO4.7H2O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca2+- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury. 相似文献
5.
Measurement and characterization of postischemic free radical generation in the isolated perfused heart 总被引:10,自引:0,他引:10
J L Zweier P Kuppusamy R Williams B K Rayburn D Smith M L Weisfeldt J T Flaherty 《The Journal of biological chemistry》1989,264(32):18890-18895
Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed. 相似文献
6.
G. P. Butcher A. Raqabah M. J. Jackson J. Hoffman Jonathan M. Rhodes M. C. R. Symons 《Free radical research》1995,22(2):99-107
Electron paramagnetic resonance spectroscopy (EPR) was used to study free radicals and transition metal complexes in liver tissue taken from patients with liver disease. Samples were frozen to 77K directly following biopsy to prevent deterioration. Our major aim was to compare signals from patients suffering from alcohol abuse with those from patients having liver damage not induced by alcohol. Samples were obtained from 19 chronic alcohol abusers and 7 non-alcoholic liver disease patients. Of the 19 alcoholic patients, 18 had an increased fat content, 6 had Mallory's hyaline, 12 had an acute inflammatory response, 9 had increased stainable iron and 4 had evidence of fibrosis. A signal derived from free radicals with a spectroscopic splitting factor of g = 2.0045 was found in all samples. This signal in the alcoholic patients had a mean amplitude of 2.96 cm (± 1.42 SD), and in patients with non-alcoholic liver disease 2.12cm (±0.82) (p = 0.10NS), measured under identical instrument settings.
The molar proportion of diene conjugated linoleic acid (DCLA), a free radical marker, in the sera of alcoholic patients was 2.68% (±1.93), but did not correlate with the free radical signals obtained by EPR spectroscopy. Also, there was no correlation between the free radical derived EPR signal and fat content, Mallory's hyaline, inflammatory infiltrate, iron or fibrosis in the liver biopsy specimens. Similarly the concentrations of aspartate transaminase, albumin, and gamma-glutamyl transferase in serum samples showed no correlations with free radical concentrations.
The absence of any significant increase in the stable free radical signal in the presence of alcohol induced liver disease and the lack of correlation between the signal and either histological or serological evidence of liver damage, suggests that alcohol derived free radicals may not be involved in the pathogenesis of alcoholic liver disease.
Unusually large sextet features characteristic of MN(II) complexes were observed for all liver samples. Such signals are very rare in human tissue, showing that there is a strong accumulation of Mn (II) in the liver. However, no systematic trends were observed. In some samples signals characteristic of iron-sulphur cluster units were detected, but again no correlations could be discovered. 相似文献
The molar proportion of diene conjugated linoleic acid (DCLA), a free radical marker, in the sera of alcoholic patients was 2.68% (±1.93), but did not correlate with the free radical signals obtained by EPR spectroscopy. Also, there was no correlation between the free radical derived EPR signal and fat content, Mallory's hyaline, inflammatory infiltrate, iron or fibrosis in the liver biopsy specimens. Similarly the concentrations of aspartate transaminase, albumin, and gamma-glutamyl transferase in serum samples showed no correlations with free radical concentrations.
The absence of any significant increase in the stable free radical signal in the presence of alcohol induced liver disease and the lack of correlation between the signal and either histological or serological evidence of liver damage, suggests that alcohol derived free radicals may not be involved in the pathogenesis of alcoholic liver disease.
Unusually large sextet features characteristic of MN(II) complexes were observed for all liver samples. Such signals are very rare in human tissue, showing that there is a strong accumulation of Mn (II) in the liver. However, no systematic trends were observed. In some samples signals characteristic of iron-sulphur cluster units were detected, but again no correlations could be discovered. 相似文献
7.
Environmental Factors Influencing the Hyperfine Structure of Manganous Low-Temperature Electron Paramagnetic Resonance Spectra
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Hyperfine structure is observed in low temperature (T = -180°C) EPR (electron paramagnetic resonance) spectra of a number of solutions containing Mn++ ions 13, 15) which have characteristics in common with low temperature EPR spectra from biological substances such as mitochondria and microsomes (1-4). This investigation is an attempt to understand the features of these signals in terms of the molecular environment of the manganous ion, and a qualitative explanation for the observations reported here is advanced in terms of the amount of axial distortion of a manganese hydrate in different environments. 相似文献
8.
Iron-mediated formation of an oxidized adriamycin free radical 总被引:2,自引:0,他引:2
J L Zweier 《Biochimica et biophysica acta》1985,839(2):209-213
Electron paramagnetic resonance studies are reported which demonstrate that the reduction of Fe3+ to Fe2+ by adriamycin results in the formation of an oxidized adriamycin free radical with an EPR signal at g = 2.004. A transient iron-adriamycin free radical complex is also observed at g = 2.34. The free radical is quantitated and its aerobic stability is determined. Observation of the oxidized adriamycin free radical signal confirms that adriamycin donates an electron to the bound Fe3+. In the presence of glutathione the drug-mediated reduction of Fe3+ to Fe2+ is bypassed, and the oxidized adriamycin radical signal is not observed. The oxidized adriamycin radicals and reduced oxygen radicals which are formed are two different mediators, whose relative concentrations could modulate the therapeutic and toxic effects of adriamycin. 相似文献
9.
We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product. 相似文献
10.
When young intact forespores of Bacillus megaterium were incubated with either Mn++ or the ionophore X-537A, the pool of 3-phosphoglyceric acid (3-PGA) was stable. However, incubation of forespores with Mn++ plus the ionophore X-537A resulted in rapid and complete utilization of the 3-PGA. This effect was not seen with Ca++ or Mg++, and was also not observed with older forespores or fresh dormant spores. Since the phosphoglycerate mutase of has an absolute and specific requirement for Mn++, it is possible that phosphoglycerate mutase in developing forespores may be inactive because of a low intrasporal level of free Mn++. 相似文献
11.
《Free radical research》2013,47(4):269-280
The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO*) and nonperoxyl (P*) free radicals were similar, we concluded that ROO* radicals are not formed from P* radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10-3 s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used. 相似文献
12.
《Free radical research》2013,47(3-6):337-342
The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (aN = 16.1G, aHβ = 24.0G) and PBN (aN = 15.7G. aHβ = 4.4G and aN = 17.0G, aHβ = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent. 相似文献
13.
Manganese is essential for normal development and activity of the nervous tissue. Mn2+ ions are involved in protein synthesis and may prevent free radical damage. Since it is now established that alcohol degradation may produce free radicals, we studied the effect of Mn2+ on ethanol induced alterations using cultured nerve cells as an experimental model of the central nervous system. Neurons and glial cells were cultured from rat brain cortex; a tumoral rat glial cell line (C6) was also examined. We measured enzymatic markers of nerve cell maturation (enolase, glutamine synthetase) and superoxide dismutase, a scavenger of free radicals; all these enzymes being activated by Mn2+ ions. Only for the glial cell types an alcohol antagonizing effect was found when Mn2+ was combined with ethanol. Neurons were not sensitive to that Mn2+ effect. 相似文献
14.
Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels. 相似文献
15.
Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels. 相似文献
16.
Chlorophyll fluorescence measurements were performed on attached leaves of wheat plants (Triticum aestivum L. cv. Nandu) that were exposed to ambient air and to air supplemented with 80 and 120nmol mol-1 ozone. Decreases in the “current photochemical capacity” were observed that were dependent on both the ozone concentration and duration of exposure. Electron paramagnetic resonance (EPR) spectra on freeze-dried samples from the same batches of plants showed the presence of an unidentified stable free radical, whose spectra had similarities to that of the ubisemiquinone radical. The intensity of this radical signal increased with the duration of ozone exposure in leaves that received an additional 120nmol mol-1 ozone. In contrast, with exposure to air with 80nmol mol-1 added ozone, there was little if any change in free radical signal intensity over the 4 week period of the experiment. The increase in intensity of the EPR signal occurred later than the chlorophyll fluorescence changes, which suggests that it is associated with permanent leaf damage. 相似文献
17.
It was shown that gamma-radiation (8-16 Gy) did not influence the number of paramagnetic centers of hemal (g = 2.42, g = 2.25) and nonhemal (g = 1.94) iron of rat tissues: free radicals were significantly reduced 5 min after 16 Gy irradiation. The combined effect of gamma-radiation (8 Gy) and ubiquinone-9 decreased the number of the above-mentioned ESR signals after 48 h. The number of free radicals in the rat liver, kidneys and spleen was almost twice reduced 28 days following castration. The administration of methyl testosterone increased the free radical amount in the heart and was ineffective in other rat organs. 相似文献
18.
The roasting of coffee beans generates stable radicals within melanoidins produced by non-enzymatic browning. Roasting coffee beans has further been suggested to increase the antioxidant (AO) capacity of coffee brews. Herein, we have characterized the radical content and AO capacity of brews prepared from Coffea arabica beans sourced directly from an industrial roasting plant. In-tact beans exhibited electron paramagnetic resonance signals arising from Fe3+, Mn2+ and at least three distinct stable radicals as a function of roasting time, whose intensity changed upon grinding and ageing. In coffee brews, the roasting-induced radicals were harboured within the high molecular weight (> 3 kD) melanoidin-containing fraction at a concentration of 15 nM and was associated with aromatic groups within the melanoidins. The low molecular weight (< 3 kD) fraction exhibited the highest AO capacity using DPPH as an oxidant. The AO activity was not mediated by the stable radicals or by metal complexes within the brew. While other non-AO functions of the roasting-induced radical and metal complexes may be possible in vivo, we confirm that the in vitro antiradical activity of brewed coffee is dominated by low molecular weight phenolic compounds. 相似文献
19.
Haddy A 《Photosynthesis research》2007,92(3):357-368
Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment
of the Mn4Ca cluster of photosystem II. Since the discovery of the multiline signal from the S2 state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals
from nearly all of the S-states of the Mn4Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various
EPR signals obtained from the Mn4Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical. 相似文献
20.
Deborah P. Rondanini Maria del Pilar Vilariño Marcos E. Roberts Marina A. Polosa Javier F. Botto 《Physiologia plantarum》2014,152(4):784-794
Early shade signals promote the shade avoidance syndrome (SAS) which causes, among others, petiole and shoot elongation and upward leaf position. In spite of its relevance, these photomorphogenic responses have not been deeply studied in rapeseed (Brassica napus). In contrast to other crops like maize and wheat, rapeseed has a complex developmental phenotypic pattern as it evolves from an initial rosette to the main stem elongation and an indeterminate growth of floral raceme. In this work, we analyzed (1) morphological and physiological responses at individual level due to low red/far‐red (R/FR) ratio during plant development, and (2) changes in biomass allocation, grain yield and composition at crop level in response to high R/FR ratio and low irradiance in two modern spring rapeseed genotypes. We carried out pot and field experiments modifying R/FR ratios and irradiance at vegetative or reproductive stages. In pot experiments, low R/FR ratio increased the petiole and lamina length, upward leaf position and also accelerated leaf senescence. Furthermore, low R/FR ratio reduced main floral raceme and increased floral branching with higher remobilization of soluble carbohydrates from the stems. In field experiments, low irradiance during post‐flowering reduced grain yield, harvest index and grain oil content, and high R/FR ratio reaching the crop partially alleviated such effects. We conclude that photomorphogenic signals are integrated early during the vegetative growth, and irradiance has stronger effects than R/FR signals at rapeseed crop level. 相似文献