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J M Carney

Publications and source records attributed to J M Carney.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies.

Nitrone-based spin trapping compounds have been shown to protect experimental animals from pathology associated with ischaemia/reperfusion injury, endotoxaemia, natural and accelerated aging, certain xenobiotics, and physical trauma. Moreover, these compounds have an intriguing nootropic action. Nitrones affect pathophysiological correlates in both the central nervous system and peripheral organ systems. These compounds have been shown to affect cellular oxidation state and oxidatively sensitive enzyme systems, but the precise mode of nitrone action has not been elucidated. Recent discoveries regarding the ability of nitrones to suppress gene transcriptional events associated with pathophysiological states, particularly the elaboration of NF kappa B-regulated cytokines and inducible nitric oxide synthase, argue that nitrones may act at a proximal level to oxidatively sensitive signal amplification systems.

Animals↗
PubMed1997

Effect of 2-cyclohexene-1-one-induced glutathione diminution on ischemia/reperfusion-induced alterations in the physical state of brain synaptosomal membrane proteins and lipids.

Glutathione is able to protect membrane proteins from oxidative stress. In ischemia/reperfusion injury, free radicals cause synaptosomal membrane protein and lipid oxidation that is prevented by the free radical scavenger N-tert-butyl-alpha-phenylnitrone (Hall N. C. et al. (1995) Neuroscience 64, 81-89; 69, 591-600). We wondered if diminution of glutathione would lead to further membrane alterations. Accordingly, the effects of glutathione depletion, by intraperitoneal administration of 2-cyclohexene-1-one, on the physical state of cortical synaptosomal membrane proteins and lipids, with and without global ischemia/reperfusion, were studied in vivo and in vitro in adult and aged gerbils utilizing electron paramagnetic resonance spectrometry. 2-Cyclohexene-1-one (100 mg/kg, i.p.) was administered 30 min prior to 10-min ischemia followed by 1 or 14 h reperfusion. This glutathione reduction agent was also administered to gerbils under the same temporal schedule in the absence of ischemia and compared to untreated controls. Synaptosomal membranes were labeled with a protein-specific spin label, 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl, or a lipid-specific spin probe, 5-doxylstearic acid. There were no significant changes in the physical state of the lipid portion of synaptosomal membranes when comparing ischemia reperfusion and 2-cyclohexene-1-one-treated ischemia reperfusion in either the adult or aged gerbils. However, glutathione depletion without ischemia/reperfusion caused significant changes in the physical state of the protein portion of cortical synaptosomal membranes in both the adult and aged models. Glutathione depletion, without ischemia/reperfusion, in the adult model showed a maximum change at 3 h that returned to control values by 14 h. In contrast, the aged model showed significant changes at 1 h reperfusion, which did not return to control values by 14 h reperfusion. Glutathione depletion combined with ischemia/reperfusion caused initial protein change in both adult and aged models at 1 h reperfusion, which did not return toward control values by 14 h reperfusion. The results of this study suggest that glutathione depletion increases the severity of membrane protein damage associated with ischemia/reperfusion injury.

Animals↗
PubMed1997

Aging and caloric restriction affect mitochondrial respiration and lipid membrane status: an electron paramagnetic resonance investigation.

Previous studies have indicated that reactive oxygen species (ROS) are likely involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). ROS, generated by succinate-stimulated mitochondria, have been reported to be spin trapped and detected by electron paramagnetic resonance (EPR). Our aim in the current study was to study the impact of aging on the effect of increased metabolic stimuli on mitochondrial respiration in terms of oxy-radical generation and possible lipid peroxidative changes in brain neocortical membranes. A mixed population of brain synaptosomes and mitochondria from brown norway male rats of differing ages being fed either ad lib (AL) or a caloric-restricted diet (DR) was prepared and labeled with 5-nitroxyl stearate (5-NS), a membrane lipid-specific spin label. The changes in anisotropic motion of the intercalated 5-NS spin probe also allows one to evaluate the status of the membrane fluidity in the lipid microenvironment via the order parameter. Upon succinate stimulation of mitochondria, the ROS generated resulted in a decrease in the EPR signal amplitude of the 5-NS reporter molecule indicative of the flux of oxy-radicals produced and possible peroxidation-induced changes in the synaptosomal lipid membrane. The line width remained constant, indicating that the overall intensity was reduced. The results showed a significant overall age effect in the ability to generate oxygen-derived radicals following metabolic stimulation (p < .0001). Stimulation of state 4 mitochondrial respiration with 20 mM succinate resulted in greater oxy-radical production in 25-month-old animals as compared to younger animals, suggesting increased mitochondrial leakage with age. Free radical stress induced by metabolic stimulation also causes a concomitant increase in membrane fluidity (p < .0001). There was also a significant age effect (p < .0007) on the order parameter of the mixed population of membranes. Although caloric restriction attenuated the membrane rigidization caused by aging, it was found to play a role in limiting the oxy-radical production following metabolic stimulation of mitochondria. The overall effect of age on membrane spin-label intensities EPR signal upon succinate stimulation suggests that progressive mitochondrial dysfunction may be a key factor in the aging process and in development of age-associated diseases.

Aging↗
PubMed1997

Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer's disease.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated beta-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

Alzheimer Disease↗
PubMed1997

Oxidative modification of glutamine synthetase by amyloid beta peptide.

beta-Amyloid peptide (A beta), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of A beta form free radical species in aqueous solution. It was proposed that A beta-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic A beta can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic A beta(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by A beta, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of A beta is not iron or peroxide-dependent. Our present observations consistently show that toxic A beta does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that A beta(25-35) induces carbonyl formation in BSA. Our results demonstrate that beta-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.

Amyloid beta-Peptides↗
PubMed1997

Effect of alpha-phenyl-tert-butylnitrone on endotoxin toxemia in horses.

Lipopolysaccharide (LPS), or endotoxin, is a component of the cell wall of gram-negative bacteria and is toxic to humans and animals. The GI tract of horses contains large numbers of endotoxins which may cause disease if gut wall integrity is compromised. The objective of this study was to develop a unique therapeutic approach to the treatment of endotoxemia with a sulfonyl analog of the alpha-phenyl-N-tert-butyl-nitrone (PBN) spin-trap molecule which may prevent the LPS-induced cytokine cascade. Following challenge with 55 mg/kg LPS, the survivability of ICR Swiss mice was significantly improved after treatment with 100 and 175 mg/kg PBN, although survivability of mice treated with 175 mg/kg PBN was significantly less than those treated with 100 mg/kg PBN. Challenged mice treated with 300 and 1000 mg/kg PBN survived for a significantly shorter period of time (vs control). Horses treated with a sublethal dose (1 microgram/kg) of endotoxin experienced 2 periods of distress at 1 and 6 h after challenge. Disulfonyl-PBN significantly reduced the increase in heart and respiratory rates 6 h after challenge. Analogs of PBN appeared to be more beneficial following near-lethal challenge with LPS. Dramatic benefits to horses may only be observed in life-threatening situations.

Animals↗
PubMed1996

Alpha 1-antichymotrypsin interaction with A beta (1-42) does not inhibit fibril formation but attenuates the peptide toxicity.

alpha 1-Antichymotrypsin (ACT) is intimately associated with senile plaques in the Alzheimer's diseased (AD) brain. It was reported that ACT can promote the polimerization of A beta (1-42) into amyloid filaments. It was suggested that neurotoxic amyloid deposits arise when beta-peptide is induced to form fibrils by ACT or other amyloid-promoting factors (pathological chaperones) expressed in AD brain. However, it was reported recently that ACT can inhibit fibrillization of A beta (1-40) and disaggregate pre-formed beta-amyloid fibrils of this synthetic A beta peptide. Our previous study [Aksenova et al., Neurosci. Lett., 411 (1996) 43-48] confirmed that ACT is able to inhibit A beta (1-40) aggregation into fibrils, but it was shown that at the same time ACT does not change the peptide cytotoxicity. In this report we have observed that interaction of ACT with A beta (1-42), unlike that for ACT-A beta (1-40) interaction, does not prevent the formation of insoluble A beta (1-42) aggregates, but completely blocks the peptide's toxicity in rat hippocampal cell cultures. These results are discussed in terms of the potential double role of peptide-protein interactions on A beta aggregation and neurotoxicity.

Amyloid beta-Peptides↗
PubMed1996

alpha-1-antichymotrypsin interaction with A beta (1-40) inhibits fibril formation but does not affect the peptide toxicity.

Recent studies have shown that senile plaque-associated or glial-derived proteins can prevent fibril formation of beta-amyloid peptide (A beta), while increasing the neurotoxicity of the latter (in the case of glutamine synthetase, apolipoprotein J or thrombin). alpha-1-Antichymotrypsin (ACT) is a glial-derived protein associated with senile plaques in the Alzheimer's brain. In this report we show that ACT, a minor protein component of beta-amyloid deposits, is able to inhibit A beta (1-40) aggregation into fibrils, but unable to modulate the toxicity of A beta (1-40) in primary rat hippocampal cell cultures. These results are discussed in terms of the potential role of glial-derived proteins on A beta aggregation and neurotoxicity.

Amyloid beta-Peptides↗
PubMed1996

A beta (25-35) peptide displays H2O2-like reactivity towards aqueous Fe2+, nitroxide spin probes, and synaptosomal membrane proteins.

Amyloid beta peptides (A beta s) are found in abnormally high accumulations in brains of persons with Alzheimer's disease, and are believed to contribute to cognitive decline in this disorder. Synthetic A beta and its peptide fragment 25-35 [A beta (25-35)] are toxic to cells in culture; however, the exact mechanism of amyloid peptide toxicity is not known. An emerging hypothesis contends that A beta toxicity results from peptide-mediated free radical reactions and generation of reactive oxygen species. Recently, we reported that reactivity of A beta toward the oxidation-sensitive enzyme glutamine synthetase is related to the peptide's reactivity toward the spin trap phenyl-tert-butyl nitrone (PBN). Neuronal damage may be due, in part, to oxidative processes initiated by amyloid-derived free radicals species. This work presents evidence from electron paramagnetic resonance (EPR) spin labeling techniques and spectrophotometric assays that a portion of synthetic A beta (25-35) demonstrates hydrogen peroxide-like reactivity toward Fe2+, nitroxide spin probes, and neocortical synaptasomal membrane proteins. These results are discussed with reference to free radical membrane damage and neurotoxicity in Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Glutamine synthetase-induced enhancement of beta-amyloid peptide A beta (1-40) neurotoxicity accompanied by abrogation of fibril formation and A beta fragmentation.

beta-Amyloid peptide (A beta) is the main constituent in both senile plaques and diffuse deposits in Alzheimer's diseased brains. It was previously shown that synthetic A beta s were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of A beta (1-40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of A beta toxicity to hippocampal cell cultures. In the present study the enhancement of A beta toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of A beta (1-40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of A beta (1-40). The fragmentation of A beta was inhibited by immunoprecipitation of GS.

Amyloid beta-Peptides↗
PubMed1996

Amyloid beta peptide (25-35) inhibits Na+-dependent glutamate uptake in rat hippocampal astrocyte cultures.

Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid beta peptide (Abeta), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Abeta including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Abeta increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Abeta. The aim of the current study was to determine if Abetas inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Abeta can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Abeta (25-35) significantly inhibited L-glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular L-glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Abeta toxicity involving astrocytes.

ATP-Binding Cassette Transporters↗
PubMed1995

beta-Amyloid peptide-derived, oxygen-dependent free radicals inhibit glutamate uptake in cultured astrocytes: implications for Alzheimer's disease.

beta-Amyloid (A beta), the central constituent of senile plaques in Alzheimer's disease (AD) brains, was shown by us recently to generate free radicals in an oxygen dependent mechanism. A beta-derived free radicals were detected directly using electron paramagnetic resonance (EPR) spin trapping techniques employing the spin trap phenyl-alpha-tert-butylnitrone (PBN). We have extended these studies to investigate the nature of the oxyradicals derived from A beta peptides, and we show that these free radicals are able to inhibit glutamate uptake in cultured astrocytes. An implication of inhibited astrocyte glutamate uptake in brain is increased extracellular levels of glutamate, which is excitotoxic to neurons. These results support the hypothesis that A beta neurotoxicity in AD may be due in part to A beta-derived, oxygen-dependent free radical inhibition of glutamate uptake.

Alzheimer Disease↗
PubMed1995

Membrane protein alterations in rodent erythrocytes and synaptosomes due to aging and hyperoxia.

We have applied the technique of electron paramagnetic resonance (EPR) protein-specific spin labeling to the study of membrane protein alterations occurring during age and exposure to isobaric hyperoxia. Cortical synaptosomes and erythrocyte membranes (ghosts) were isolated from young rodents (Fisher 344 rats or mongolian gerbils, 3-4 months of age) and aged rodents (age 22-27 months for rats, greater than 15 months for gerbils). Membrane proteins were spin labeled with the thiol-specific spin label MAL-6 (2,2,6,6,-tetramehtyl-4-maleimido-piperdin-1-oxyl). The relevant EPR spectral parameter of MAL-6 labeled membranes, the W/S ratio, decreased significantly with age of animal in both synaptosomes and ghosts (P < 0.001). As a paradigm for accelerated oxidative stress, young and aged gerbils were exposed to an atmosphere of 90-100% O2 for 0-48 h. In both young and aged gerbils, the W/S ratio decreased significantly with hyperoxic stress (P < 0.003). The W/S ratio of synaptosomes isolated from aged gerbils decreased continually from 0-48 h hyperoxia, whereas the W/S ratio of synaptosomes from young animals demonstrated a pronounced rebound effect from 24-48 h. The results are discussed with reference to membrane protein oxidation in aging.

Aging↗
PubMed1995

Amyloid beta-peptide spin trapping. I: Peptide enzyme toxicity is related to free radical spin trap reactivity.

Synthetic beta-amyloid peptides (A betas) demonstrate lot-to-lot variation in toxicity that has not been adequately explained. Studies from our laboratory have shown that A beta toxicity may result from the ability of the peptide to promote oxidation reactions. Both A beta(1-40) and A beta(25-35) inactivate the oxidation-sensitive enzyme glutamine synthetase (GS) and generate electron paramagnetic resonance (EPR)-detectable products upon reaction with the spin trap phenyl-tert-butylnitrone (PBN). We now report that samples of synthetic A beta(1-40) and A beta(25-35) with attenuated toxicity with respect to peptide-induced GS inactivation, produce qualitatively different EPR spectra when the peptides are incubated with PBN. The results suggest an interpretation of conflicting observations regarding the toxicity of synthetic A betas, and that investigators must be careful to assess the reactivity state of A beta being studied.

Amino Acids↗
PubMed1995

Amyloid beta-peptide spin trapping. II: Evidence for decomposition of the PBN spin adduct.

Synthetic beta-amyloid peptides (A betas) react with the spin trap phenyl-tert-butyl nitrone (PBN) to form products detectable by electron paramagnetic resonance (EPR) spectroscopy. At least two EPR-detectable products can be distinguished from the A beta/PBN reaction, and peptide toxicity towards glutamine synthetase enzyme correlates with the type of PBN reaction product observed. We have reacted synthetic A beta(25-35) peptide with [12C]- or [13C]PBN to demonstrate that the two products represent alternate pathways of spin adduct decomposition. Results indicate that the C=N bond of PBN is cleaved by A beta in what we hypothesize is a radical addition-fragmentation reaction.

Amyloid beta-Peptides↗
PubMed1995

Structural alterations in synaptosomal membrane-associated proteins and lipids by transient middle cerebral artery occlusion in the cat.

We have previously reported that ischemia reperfusion injury results from free radical generation following transient global ischemia, and that this radical induced damage is evident in the synaptosomal membrane of the gerbil. [Hall et al, (1995) Neuroscience 64: 81-89]. In the present study we have extended these observations to transient focal ischemia in the cat. We prepared synaptosomal membranes from frontal, parietal-temporal, and occipital regions of the cat cerebral cortex with reperfusion times of 1 and 3 hours following 1 hour right middle cerebral artery occlusion. The membranes were selectively labeled with protein and lipid specific paramagnetic spin labels and analyzed using electron paramagnetic resonance spectrometry. There were significant motional changes of both the protein and lipid specific spin labels in the parietal-temporal and occipital regions with 1 hour reperfusion; but, both parameters returned to control values by 3 hours reperfusion. No significant changes were observed in the normally perfused frontal pole at either reperfusion time. These results support the argument that free radicals play a critical role in cell damage at early reperfusion times following ischemia.

Animals↗