Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation. Radiation effect on nitroxyl decay of a lipophilic spin probe in the head region

X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood-brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

Local pharmacokinetic analysis of a stable spin probe in mice by in vivo L-band ESR with surface-coil-type resonators

In vivo ESR spectroscopy using a low frequency microwave of approximately 1 GHz has been developed to measure non-invasive ESR spectra in animals given paramagnetic compounds, in which a loop-gap-type resonator was used and ESR spectra were measured at the animal's head or abdomen. Therefore, the concentrations of paramagnetic species in both the blood and organs were compositely contributed to the spectra. When we understand the kinetics of paramagnetic species in detail, it is essentially important to know how these kinetics are expressed in each organ. For this purpose, a surface-coil-type resonator, which enabled local ESR measurement in specific organs, has been developed. By using this method, we studied the real-time pharmacokinetics of spin clearance curves detected in the organs of mice given 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) intravenously (i.v.), by monitoring the inferior vena cava, liver and kidney. Quantified clearance curves in the organs were analyzed on the basis of a two-compartment model, and pharmacokinetic parameters were estimated based on the curve-fitting. The obtained pharmacokinetic parameters were found to depend on the measurement site, and the distribution and elimination processes of the spin probe were successfully separated between the blood and organs of mice.     

Repair of damage caused by UV- and X-irradiation in the amoeboflagellateNaegleria gruberi

Summary Cysts ofNaegleria gruberi have a normal UV- and an extremely high X-ray resistance compared to other protozoans. Caffeine and 3-aminobenzamide applied to excysting amoeba after irradiation in the encysted state (UV and X-rays) by feeding with drug-containing bacteria increased lethality, while fractionated irradiation (UV) and liquid-holding (UV and X-rays) increased survival. Illumination with visible light after UV-irradiation restored almost 100% viability. The results are discussed in regard to the activity of repair mechanisms.     

In vivo effect of asparagine in the hydrophobic region of the signal sequence

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.     

The effect of a myelin apoprotein on lipid structure: a spin probe study.

Spin probes, stable free radical derivatives of stearic acid and cholestanone, were used to observe the effects of the "Folch-Lees" protein isolated from the white matter of bovine brain on the organization and motion of lipid molecules. The incorporation of the organic solvent soluble form of this protein decreased the tendency of a variety of lipid molecules with zero, positive or negative net charges to arrange themselves close to the normal to the lipid bilayer. The aqueous form of the protein also had a profound chaotropic effect on the molecular geometry of the lipid, but only if the lipids had a net negative charge (the protein has a net positive charge in the pH range investigated). Examination of the ESR spectra indicated that this protein altered the geometry of the lipid structure without causing major changes in the mobility of the individual lipid molecules.     

Studying the effect of Dimebon and NT-1505 on microviscosity of mice brain synaptosomal membranes in vivo by EPR spin labeling method

In this work membrane fluidity alterations in synaptosomes, isolated from mice brain tissue, at chronic injection of neuroprotectors Dimebon and NT-1505 in vivo were studied. Membrane microviscosity was measured by electron paramagnetic resonance spin labeling of 2,2,6,6-tetramethyl-4-capryloyl-oxylpiperidine-1-oxyl (lipid probe) and 5,6-benzo-2,2,6,6-tetramethyl-1,2,3,4-tetrahydro-γ-carboline-3-oxyl (near protein probe). It was shown that the neuroprotectors Dimebon and NT-1505 affect a membrane structure. Despite the difference in membrane structures, fluidity of the lipid bilayer in time returned to control values.     

The effect of imazalil in the control of decay in yellow yam caused by Penicillium sclerotigenum

A strain of Penicillium sclerotigenum isolated from decaying yellow yams (Dioscorea cayenensis) was found to have developed resistance to benzimidazole fungicides and the use of imazalil was investigated as an alternative agent for controlling it. Two formulations were tested and proved to be equally effective in controlling decay at a concentration of 50 mg imazalil litre^-1 if the yams were dipped in it for 5 s; concentrations down to 10 mg litre^-1 were effective if the immersion time was increased to 5 min or more. These treatments gave good control of decay when applied up to 24 h after inoculation but were less effective when application was 48 h after inoculation, although at 500 mg litre^-1 there was some indication that levels of decay were decreased when compared with untreated tubers. Fungal penetration was unaffected or increased by increasing delays in the time of the fungicide application depending on the concentration applied. In trial shipments of yams from Jamaica to Britain imazalil alone was less effective in controlling overall decay because of the presence of a Rhizopus species. This could be controlled by the addition of dichloran to the dip. Imazalil residues were highest in the peel; levels in the peeled tuber varied and tended to decrease during storage but were generally in the range of 0·5 - 0·8 mg kg^-1 which is higher than the maximum levels accepted for other produce. A simple cooking test showed that residues in treated yam slices declined by about one third after boiling the slices in water for 45 min.     

Inhibition of superoxide dismutase by nitroprusside and electron spin resonance observations on the formation of a superoxide-mediated nitroprusside nitroxyl free radical

Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal prosthetic group and regardless of the source from which the enzymes were isolated. Thus the copper-zinc enzyme from bovine erythrocyte or Neurospora crassa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E. coli and a blue-green alga. The inhibition was dose dependent with a Ki = 2.5 X 10(-5) for nitroprusside. Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with O2- for superoxide dismutase. These inhibitions by nitroprusside, which were elicited not only in purified enzymes but also in crude soluble extracts of biological samples, were rapidly reversible. Nitroprusside was found to react with O2- to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028. The spin adduct appears to be a nitroxide radical and was stable for several minutes.     

Elevation of antioxidant potency in mice brain by low-dose X-ray irradiation and its effect on Fe-NTA-induced brain damage

The increase in lipid peroxide levels in mice brain following Fe3+ administration was about 50% of that when 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was administered. This may be due to excessive oxidation by Fe3+, and was supported by the decrease in activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), Na+,K(+)-ATPase activity and membrane fluidity after Fe3+ administration. Relatively low-dose X-ray irradiation (0.5 Gy) inhibited lipid peroxidation associated with Fe3+ administration and restored the decreased activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity to the levels in the non-Fe(3+)-administered group. In the purine metabolism system, uric acid decreased after Fe3+ administration, which may be due to transient impairment of the system for production of uric acid from xanthine by excessive oxidation by Fe3+. However, 0.5 Gy irradiation inhibited this decrease in uric acid, increasing its level to that in the non Fe(3+)-administrated group. This may be due to factors such as rapid recovery of the activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity after 0.5 Gy irradiation. In addition, since no changes were observed in xanthine and uric acid, increased inosine and hypoxanthine may have advanced to a salvage pathway leading to not xanthine but inosine 5'-monophosphate (IMP).     

Identification of candidate biomarkers of brain damage in a mouse model of closed head injury: a metabolomic pilot study

We aim to identify candidate brain biomarkers for, and to elucidate the pathophysiology of closed traumatic brain injury (TBI). Nuclear magnetic resonance (NMR) based metabolomic analysis was performed on the whole brain of mice undergoing TBI using a validated technique. There were 10 TBI mice compared to 8 sham operated controls. A total of 45 metabolites were evaluated. There was a statistically significant alteration in concentrations of 29 metabolites in TBI brains as compared to controls (FDR <0.05). Profound disturbances of several metabolic pathways (FDR <1E-07), including pathways associated with purine, alanine, aspartate and glutamine and glutathione metabolism were observed. Also, a significant elevation in glutamate (the main excitatory neurotransmitter) and depression of GABA (the main inhibitory neurotransmitter) was observed. Four metabolites, ADP, AMP, NAD+, and IMP were the most important indicators of TBI, relative to normal controls. All were elevated in the TBI mice. A combination of these 4 biomarkers produced a perfect predictor of TBI status, AUC (95 % CI) = 1.0 (1.0, 1.0). We also detected significant disturbances in mitochondrial function, energy metabolism, neurotransmitter metabolism and other important biochemical pathways in TBI mouse brains. Further studies to assess the utility of metabolomics to detect and classify the severity of and assess the prognosis of TBI is warranted.     

Flash photolysis-electron spin resonance study of the effect of o-phenanthroline and temperature on the decay time of the ESR signal B1 in reaction-center preparations and chromatophores of mutant and wild strains of Rhodopseudomonas spheroides and Rhodospirillum rubrum

We have studied the effect of o-phenanthroline and temperature on the decay rate of Signal B1 in reaction-center preparations and in chromatophores from Rhodopseudomonas spheroides and Rhodospirillum rubrum. We have shown that o-phenanthroline binds specifically to the reaction center protein (the binding center is probably at the iron) and when so bound inhibits the transfer of electrons from primary to secondary acceptors. We have also shown that the direct return decay time (A^? → P865⁺) increases with increasing temperature above approx. 150 K. This phenomenon has been interpreted within a quantum mechanical tunnelling model in which the distance of closest approach between P865⁺ and A^? increases about 2 Å between approx. 150 and 300 K.     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

Direct observations of the redox states of frozen cherry buds by a unique in vivo ESR

Low temperatures cause cellular damage in flower buds of the sweet cherry (Prunus avium L. cv. Satohnishiki). In this study, the redox states within the cherry buds suffering freezing damage were non-destructively observed by a unique in vivo electron spin resonance (ESR) technique with a spin probe such as carbamoyl-PROXYL. The ESR signals of carbamoyl-PROXYL-treated bud were continuously recorded under freezing and thawing condition, which was decreased to approximately -4 degrees C and maintained for 1.5h, and then returned to room temperature. Most of the buds began to freeze at -2.5 to -3.9 degrees C. The peak areas of the ESR signals significantly increased during the period of temperature rise. These results show that the reduced carbamoyl-PROXYL within the frozen bud was re-oxidized and became ESR-detectable while the bud was thawing. Our in vivo ESR technique has confirmed the oxidative transition of the redox states within the buds during thawing.     

Reorganization of lipid domain structure in membranes by a transmembrane peptide: an ESR spin label study on the effect of the Escherichia coli outer membrane protein A signal peptide on the fluid lipid domain connectivity in binary mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine.

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their point of connectivity (Sankaram, M.B., D. Marsh, and T.E. Thompson. 1992. Biophys. J. 63:340-349). In the absence of the peptide, the number of fluid lipid domains does not vary with the fraction of lipid that is in the fluid phase, and phase conversion is accomplished solely by changes in the domain size. The phase boundaries of the lipid mixture remain largely unchanged by the presence of the peptide at mole fractions up to 0.02, but both the size and number of the fluid domains is changed, and the point at which they become connected is shifted to lower fractions of the fluid phase. In addition, the number of domains in the presence of the peptide no longer remains constant but increases from a domain density at low fractions of the fluid phase that is much lower than that in the absence of peptide to one that is comparable to the natural state in the absence of peptide at the point of domain connectivity. A simple model is presented for the process of domain fission, where the latter is determined by a balance between the effects of peptide concentration in the fluid domains, the line tension at the domain boundaries, and the distributional entropy of the domains.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Protective effect of unsymmetrical dichalcogenide, a novel antioxidant agent, in vitro and an in vivo model of brain oxidative damage

Unsymmetrical dichalcogenides, a class of organoselenium compounds, were screened for antioxidant activity in rat brain homogenates in vitro. Unsymmetrical dichalcogenides (1-3) were tested against lipid peroxidation induced by sodium nitroprusside (SNP) or malonate, and reactive species (RS) production induced by sodium azide in rat brain homogenates. Compounds 1 (without a substituent at the phenyl group), 2 (chloro substituent at the phenyl group bounded to the sulfur atom) and 3 (chloro substituent at the phenyl group bounded to the selenium atom) protected against lipid peroxidation induced by SNP. The IC50 values followed the order 3<2<1. Lipid peroxidation induced by malonate was also reduced by dichalcogenides 1, 2 and 3. The IC50 values were 3相似文献   

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.