Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.     

Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation

A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD.     

Effects of the spin trap-alpha-phenyl-N-tert-butyl nitrone (PBN) in transient forebrain ischaemia in the rat

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity of Carcinus maenas Y-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals. In vitro incubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (-cGMP/ +cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.     

Coupled diffusional/displacive transformations: Part II. Solute trapping

An earlier theory for the nonequilibrium transformation of austenite in Fe-C alloys to partially supersaturated plates of ferrite is extended to include a recent solute trapping model by Aziz. The previous model yielded a relationship between interface velocity and supersaturation for a specified transformation temperature. However, a unique growth velocity was determined from a velocity maximization criterion that could not be justified. By including the Aziz solute trapping function as a third interface response function (in addition to functions describing the diffusion field velocity and interface mobility), it has been possible to obtain a more physically based unique solution to the growth velocity for a specified temperature. The new calculations on an Fe-C alloy indicate a smoothly increasing supersaturation during both nucleation and growth, as the transformation temperature is reduced. Both the nucleation and growth processes tend to become completely diffusionless only below the martensite start temperature. This paper is based on a presentation made in the symposium “International Conference on Bainite” presented at the 1988 World Materials Congress in Chicago, IL, on September 26 and 27, 1988, under the auspices of the ASM INTERNATIONAL Phase Transformations Committee and the TMS Ferrous Metallurgy Committee.     

Persistence of negative priming: II. Evidence for episodic trace retrieval.

Responses to recently ignored stimuli may be slower or less accurate than to new stimuli. This negative priming effect decays over time when delay is randomized within Ss, but not when delay varies between Ss. In Exp 1, response–stimulus intervals (RSI) of 500 and 4,000 msec were randomized within Ss in a target localization task. Negative priming of ignored locations diminished with longer delay. However, no significant decay was obtained when RSI and the preceding RSI were equal. Similar results were obtained when RSI and preceding RSI were deliberately confounded by blocking (Exp 2). Negative priming appears to depend on temporal discriminability of the priming episode. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Photoreduction of the fluorescent dye 2'-7'-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/*OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

Impact of shock on pain reactivity: II. Evidence for enhanced pain.

Shocked rats (Rattus norvegicus) often exhibit longer tail withdrawal latencies to radiant heat, which suggests that exposure to shock reduces pain. But at the same time, rats appear hyperreactive to shock, suggesting that pain is enhanced. Exp 1 replicated these findings and showed that when tail movement was monitored, shocked rats were less responsive to heat and hyperreactive to shock even when the same behavioral criteria were used. When latency to vocalize was measured, shocked rats appeared hyperreactive to both test stimuli (Exps 2 and 3). Prior exposure to shock also enhanced the acquisition of conditioned fear in a different context (Exp 4) and the speed with which rats learned a response to avoid a thermal stimulus (Exp 5). The results suggest that exposure to shock enhances pain. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Formation of spin trap adducts during the decomposition of peroxynitrite

To investigate Ca2+ handling in compensated hypertrophied cardiomyocytes, we measured Ca2+ transients and contraction of hypertrophied rat left ventricular myocytes induced by aortic constriction (AC). The fluorescence ratio (I405/I480) after indo-1/AM loading and circumferential length were simultaneously measured in isolated myocytes. The amplitude of Ca2+ transients (Ca-Amp) was higher in rats with AC than in sham-operated rats (Sham) (0.25 +/- 0.08 vs 0.17 +/- 0.05). There was a positive correlation between Ca-Amp and fractional shortening (FS) in both AC and Sham rats, whereas the ratio of FS/Ca-Amp was smaller in AC rats. These observations suggest that compensated hypertrophied cardiomyocytes exhibit an adaptive increase in Ca-Amp, associated with reduced myofilament responsiveness to an increase in Ca2+. Isoproterenol and forskolin increased Ca-Amp and FS, and decreased time to 50% decline of Ca2+ transients. Although myocytes from AC rats exhibited reduced responsiveness to isoproterenol, responses to forskolin did not differ between the 2 groups. The reduced beta-adrenergic response in Ca2+ handling was probably due to altered beta-adrenoceptor numbers, G-protein function and/or their coupling process.     

Determination of the rate of superoxide generation from biological systems by spin trapping: use of rapid oxygen depletion to measure the decay rate of spin adducts

A method was developed to measure the superoxide generation rate from biological systems using the spin trapping method. Kinetic treatment of the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the EPR signal intensity of the system is proportional to the superoxide generation rate. Rapid depletion of oxygen in the sample was utilized to terminate superoxide generation so that the decay rate of the DMPO superoxide adduct (DMPO-OOH) could be determined. For this decay measurement, a controlled atmosphere EPR cavity was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophils and for the hypoxanthine-xanthine oxidase system were comparable to those obtained with the cytochrome c reduction method. This method is specifically applicable to the system in which dissolved oxygen supplied from the gas phase is utilized as a source of superoxide.     

Keratin decomposition by dermatophytes. II. Presence of s-sulfocysteine and cysteic acid in soluble decomposition products

The dermatophyte Microsporum gypseum was grown on human hair in a mineral medium. Filtrates of cultures of various age were chromatographed on Sephadex gels and further characterized by chemical analysis and thin-layer chromatography. The products of keratin decomposition were separated into two fractions. The first fraction formed 10 to 20% of the bulk and contained proteins low in sulfur content. The second, very wide fraction contained oligopeptides and polypeptides with molecular weights from several hundreds up to approximately 13,000 with a peak at 1,000-2,000 daltons. This fraction contained a relatively high amount of cystine, S-sulfocysteine, and cysteic acid. S-sulfocysteine was present in the free state and also combined in peptides where its amount exceeded that of cystine. This supports the author's hypothesis on keratin decomposition by dermatophytes. According to this hypothesis, the fungus excretes sulfite which cleaves the disulfide bonds of keratin to cysteine and S-sulfocysteine. The substrate, denatured by sulfitolysis, is then more easily digestible by fungal proteases. Besides S-sulfocysteine, comparable amounts of cysteic acid were found in all analyses. This compound most probably originated by air oxidation of sulfur amino acids in the alkaline cultivation fluid.     

Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps

alpha-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O2(-.)) and hydroxyl radical ((.)OH) trapping activity as determined biochemically and using electron spin resonance (ESR) spectroscopy. All cyclic nitrones tested were much more potent as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the analogs of MDL 101,002 revealed a direct correlation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, inasmuch as the calculated octanol/water partition coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nitrones are inherently more effective radical traps than PBN in a membrane system. The most active compound was a dichloro analog in the seven-membered ring series (MDL 104,342), which had an IC50 of 26 mum, which was 550-fold better than that of PBN. The cyclic nitrones were shown to trap (.)OH with MDL 101,002 being 20 25 times more active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of (.)OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the (.)OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O2(-.) radical to yield a six-line spectrum with coupling constants very distinct from that of the (.)OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of (.)OH and O2(-.) suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury.     

Solid solution decomposition mechanisms in cast and microcrystalline Al-Sc alloys: II. Model for the decomposition of a solid solution during the formation of coherent second-phase particles

A model is proposed to calculate the kinetic parameters of the precipitation of coherent second-phase particles from a supersaturated solid solution. This model is based on the assumption of barrier-free nucleation of coherent particles. In this case, the decomposition rate of a solid solution and the particle precipitation kinetic parameters are mainly determined by the characteristics of nucleus diffusion growth. The decomposition kinetics of a solid solution during the precipitation of coherent particles is described by the Johnson-Mehl-Avrami-Kolmogorov equation W = 1 ? exp[-(t/??) ⁿ ], where W is the degree of decomposition and ?? = ??₀exp(Q/k _B T) is the decomposition time. The values of parameters n and Q characterizing the decomposition kinetics are determined. Expressions are derived to describe the dependences of the volume fraction and size of precipitating coherent particles on the supersaturation of a solid solution, the annealing temperature and time, and the thermodynamic and diffusion parameters of a material.     

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.     

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.     

The acid decomposition of silicate minerals part II. Hydrometallurgical applications

A review of processes for the recovery of metals involving the acid decomposition of silicate ores and slags is presented. Emphasis is placed on how consideration of the modes of dissolution and reactivities of silicates in acid solutions can aid process development. The polymerisation and precipitation behaviour of silica under acidic conditions is briefly described.     

Amyloid beta-peptide and its fragments induce acetylcholine release in in vitro basal forebrain tissue slices of rat brain, but do not affect the choline uptake

OBJECTIVE: Although stressor uncontrollability has been shown to suppress immune responses in animals and for human subjects, the results have been inconsistent. We reanalyzed results of our previous study regarding stress-related immune deviation in man, to establish whether perceived uncontrollability of an acute stressor acts as a co-determinant in the observed changes in immunological parameters. METHOD: Three types of cognitive reactions to an acute interpersonal stressor were assessed: "motivation," "uncontrollability," and "guiltiness." Stress-induced changes in the number of several types of immune cells in peripheral blood and proliferative responses of lymphocytes to antigens and mitogens were assessed. RESULTS: In comparison with control subjects and with subjects perceiving high control over the experimental stress situation, the subject perceiving low control showed a stressor-induced decrease in the number of T helper cells. Reversely, subjects perceiving high control showed an increase in the number of B cells as opposed to the other two groups. The effects of perceived uncontrollability could not be accounted for by mood changes, but they were related to previously experienced life stress. CONCLUSIONS: Perceived uncontrollability of an acute stressor can have immuno-modulating effects over and above those of the stressor per se.     

Anodic dissolution of copper sulphides in sulphuric acid solution: II. The anodic decomposition of CuS

The anodic decomposition of CuS in sulphuric acid solutions was investigated by means of cyclic voltammetry. The pH of the electrolyte and the scan rate were varied. Our results are interpreted using the experience of semiconductor electrochemistry and of passivation of pure metals. At potentials E < E_np (E_np = potential of pitting nucleation), the current/voltage curves were interpreted by the formation of metastable nonstoichiometric copper oxide or hydroxide. After the dissolution of these layers at the potential E_np, total decomposition of CuS starts. These results were confirmed by rest potential measurements and by scanning electron microscopy.