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Oxygen radical mediated modification of a proteins and neurodegenerative disorders in aged patients
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AMYLOID p PEPTIDES DO NOT FORM RADICAL ADDUCTS.
OXYGEN RADICAL MEDIATED MODIFICATION OF A PROTEINS AND NEURODEGENERATIVE DISORDERS IN AGED PATIENTS
Sereev I. Dikalov*, Michael Viteckg, Kirk P. Maples#, Ronald P. Mason* *NIEHS, NIH P.O. Box 12233, RTP, NC 27709. #Duke University Medical Center, Durham NC 277 IO #Centaur Pharmaceuticals, Sunnyvale, CA 94086. Amyloid p (AP) peptides play an important role in the pathogenesis of Alzheimer’s disease. Free radical generation by amyloid p peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from A!3 (l-40) and A!3 (25-35) peptides react with the spin trap N-tert-butyla-phenylnitrone (PBN) to form a PBN/‘AP peptide radical adduct with a specific triplet ESR signal indicate that the peptide itself was the source of free radicals. We have reinvestigated this spin-trapping study of free radical formation by AD peptides. We now report that three AP peptides, A@ (l-40), AP (25-35) and AP (40-l), do not yield radical adduct ESR signals with PBN from the Oklahoma Medical Research Foundation (OMRF) even though the A!3 (l-40) and AD (25-35) peptides were toxic to cells in culture. In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without AP peptides produced a three-line ESR spdctrum. This triplet spectrum is identical to that reported as a PBNrAP peptide radical adduct. These PBN-derived ESR spectra are identical to the highly specific ESR spectra of di-terf-butyl n&oxide and tert-butyl hydronitroxide. In this report we show that these nitroxides are formed in the PBN solution as a result of transition metal-catalyzed autooxidation of their respective hydroxylamine impurities in the Sigma PBN. The data obtained lead to the conclusion that A!3 peptides do not form ESR radical adducts, leading us to reinterpret previous reports of spontaneous formation of free radicals by AD peptides.
REDOX SENSITIVE P38 KINASE IS ACTIVATED IN THE ALZHEIMER BRATN K. Henslev*, G.Y. Bing. R. Nael. N.Y. Zhene. K.A. Robinson. X. Nmven. E. Patel. W.R. Markesbery and R.A. Flovd. Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73 104. The p38 protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. The p38 system is activated by inflammatory cytokines or oxidants such as Hz02 and is antagonized by antioxidants. Consequences of p38 activation include expression of inducible nitric oxide synthase (INOS) and de tzovo cytokine biosynthesis. Using an antibody directed against the active (dually-phosphorylated) form of ~38, we have documented p38 hyperphosphorylation in the Alzheimer’s diseased (AD) brain, in spatial association with neuritic plaques and neurofibrillary tangle (NFT)-bearing neurons. Extending upon this finding, we report that treatment of retinoic acid-differentiated human SKN-MC neuroblastoma cells with the cytokine ILla causes p38 and tau phosphorylation similar to that seen in the AD brain, as well as inducing transcription of apoptotic genes. Moreover, tau hyperphosphorylation (a prelude to NFT formation) and apoptotic correlates were both suppressed by a specific inhibitor of ~38 catalysis. These findings support a neuroinflammatory mechanism of AD and suggest a specific pathway which may be largely responsible for neuronal damage in the Alzheimer’s brain. Supported by grant from NIH NS35747, POl-AG-05 119.
OXYGEN
N. Leonova, P. Konovalov, I. Solitemova, S. Kovmgina, M.Morosova, 0. Kudrjashova Department of Biochemistry, Bekhterev Psychoneurological Research Institute, St.Petersburg Russia E. Dubinina,
We have examined the oxidative modification of proteins bovine serum albumin, human serum albumin, superoxide dismutase, thrombin, inhibitor of the trypsin and proteins plasma from aged patients with dementia. We determined the content of protein carbonyl groups (PCG), and measured flurescence of dityrosine and hyptophan. Oxidized proteins were analysed by using HPLC system. The expressive aggregation of the thrombin has been discovered according, to our experiments with the HPLC system. The dependence between the level of formed PCG, the decrease of fluorescence tryptophan and increased of
fluorescence dityrosine, concentration of active oxygen species and time incubation have been exposed. The level of metal-ioncatalyzed oxidation (MCO) proteins in aged patients with and without dementia is higher in comparison with healthy persons. The aged patients with dementia have the most low values of MC0 proteins in comparison with aged healthy people. Patients with severe dementia show a lower amount of oxidized proteins. Decreased level of oxidized proteins correlated with degree of dementia.
4-HYDROXYBENZOATE HYDROXYLATION IN RAT STRIATUM OR CORTEX DURING FOCAL CEREBRAL ISCHEMIA, Jane Montoomery, Chantal BBmeur and Line Ste-Marie, Notre Dame Campus, CHUM Research Center, Montrkal, QuBbec. Using a three vessel occlusion (3VO) rat model of focal cerebral ischemia, combined with bilateral microdialysis in the cortex or striatum, we have investigated the hydroxylation of 4-hydroxybenzoate (an analogue of salicylate). Basal production of the hydroxylation product, 3,4-dihydroxybenzoate (34DHB), was higher in the striatum (0.1 pmol/min) than the cortex (0.05 pmol/min). Induction of 3V0 ischemia caused a transient 40% increase in the deeply ischemic cortex and no change in the mildly ischemic striatum. Release of the left carotid artery resulted in an increase of cortical 34DHB (0.1 pmol/min) in the left hemisphere, while 34DHB in the ischemic right cortex returned to basal levels. In both hemispheres, 3h after ischemia induction, striatal 34DHB rose to 0.25 pmol/min and cortical 34DHB to 0.07 pmol/min. The transient increases observed in both the contralateral and ischemic cortex are indicative of increased hydroxyl radical production during 3V0. Compared to the cortex, the higher levels of striatal 34DHB at all time points are consistent with the greater vulnerability of the striatum to various stresses including ischemia. (Funded by the MRC of Canada and the Heart and Stroke Foundations of Canada and QuBbec)
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I315 I
AMYLOID p PEPTIDES DO NOT FORM RADICAL ADDUCTS.
OXYGEN RADICAL MEDIATED MODIFICATION OF A PROTEINS AND NEURODEGENERATIVE DISORDERS IN AGED PATIENTS
Sereev I. Dikalov*, Michael Viteckg, Kirk P. Maples#, Ronald P. Mason* *NIEHS, NIH P.O. Box 12233, RTP, NC 27709. #Duke University Medical Center, Durham NC 277 IO #Centaur Pharmaceuticals, Sunnyvale, CA 94086. Amyloid p (AP) peptides play an important role in the pathogenesis of Alzheimer’s disease. Free radical generation by amyloid p peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from A!3 (l-40) and A!3 (25-35) peptides react with the spin trap N-tert-butyla-phenylnitrone (PBN) to form a PBN/‘AP peptide radical adduct with a specific triplet ESR signal indicate that the peptide itself was the source of free radicals. We have reinvestigated this spin-trapping study of free radical formation by AD peptides. We now report that three AP peptides, A@ (l-40), AP (25-35) and AP (40-l), do not yield radical adduct ESR signals with PBN from the Oklahoma Medical Research Foundation (OMRF) even though the A!3 (l-40) and AD (25-35) peptides were toxic to cells in culture. In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without AP peptides produced a three-line ESR spdctrum. This triplet spectrum is identical to that reported as a PBNrAP peptide radical adduct. These PBN-derived ESR spectra are identical to the highly specific ESR spectra of di-terf-butyl n&oxide and tert-butyl hydronitroxide. In this report we show that these nitroxides are formed in the PBN solution as a result of transition metal-catalyzed autooxidation of their respective hydroxylamine impurities in the Sigma PBN. The data obtained lead to the conclusion that A!3 peptides do not form ESR radical adducts, leading us to reinterpret previous reports of spontaneous formation of free radicals by AD peptides.
REDOX SENSITIVE P38 KINASE IS ACTIVATED IN THE ALZHEIMER BRATN K. Henslev*, G.Y. Bing. R. Nael. N.Y. Zhene. K.A. Robinson. X. Nmven. E. Patel. W.R. Markesbery and R.A. Flovd. Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73 104. The p38 protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. The p38 system is activated by inflammatory cytokines or oxidants such as Hz02 and is antagonized by antioxidants. Consequences of p38 activation include expression of inducible nitric oxide synthase (INOS) and de tzovo cytokine biosynthesis. Using an antibody directed against the active (dually-phosphorylated) form of ~38, we have documented p38 hyperphosphorylation in the Alzheimer’s diseased (AD) brain, in spatial association with neuritic plaques and neurofibrillary tangle (NFT)-bearing neurons. Extending upon this finding, we report that treatment of retinoic acid-differentiated human SKN-MC neuroblastoma cells with the cytokine ILla causes p38 and tau phosphorylation similar to that seen in the AD brain, as well as inducing transcription of apoptotic genes. Moreover, tau hyperphosphorylation (a prelude to NFT formation) and apoptotic correlates were both suppressed by a specific inhibitor of ~38 catalysis. These findings support a neuroinflammatory mechanism of AD and suggest a specific pathway which may be largely responsible for neuronal damage in the Alzheimer’s brain. Supported by grant from NIH NS35747, POl-AG-05 119.
OXYGEN
N. Leonova, P. Konovalov, I. Solitemova, S. Kovmgina, M.Morosova, 0. Kudrjashova Department of Biochemistry, Bekhterev Psychoneurological Research Institute, St.Petersburg Russia E. Dubinina,
We have examined the oxidative modification of proteins bovine serum albumin, human serum albumin, superoxide dismutase, thrombin, inhibitor of the trypsin and proteins plasma from aged patients with dementia. We determined the content of protein carbonyl groups (PCG), and measured flurescence of dityrosine and hyptophan. Oxidized proteins were analysed by using HPLC system. The expressive aggregation of the thrombin has been discovered according, to our experiments with the HPLC system. The dependence between the level of formed PCG, the decrease of fluorescence tryptophan and increased of
fluorescence dityrosine, concentration of active oxygen species and time incubation have been exposed. The level of metal-ioncatalyzed oxidation (MCO) proteins in aged patients with and without dementia is higher in comparison with healthy persons. The aged patients with dementia have the most low values of MC0 proteins in comparison with aged healthy people. Patients with severe dementia show a lower amount of oxidized proteins. Decreased level of oxidized proteins correlated with degree of dementia.
4-HYDROXYBENZOATE HYDROXYLATION IN RAT STRIATUM OR CORTEX DURING FOCAL CEREBRAL ISCHEMIA, Jane Montoomery, Chantal BBmeur and Line Ste-Marie, Notre Dame Campus, CHUM Research Center, Montrkal, QuBbec. Using a three vessel occlusion (3VO) rat model of focal cerebral ischemia, combined with bilateral microdialysis in the cortex or striatum, we have investigated the hydroxylation of 4-hydroxybenzoate (an analogue of salicylate). Basal production of the hydroxylation product, 3,4-dihydroxybenzoate (34DHB), was higher in the striatum (0.1 pmol/min) than the cortex (0.05 pmol/min). Induction of 3V0 ischemia caused a transient 40% increase in the deeply ischemic cortex and no change in the mildly ischemic striatum. Release of the left carotid artery resulted in an increase of cortical 34DHB (0.1 pmol/min) in the left hemisphere, while 34DHB in the ischemic right cortex returned to basal levels. In both hemispheres, 3h after ischemia induction, striatal 34DHB rose to 0.25 pmol/min and cortical 34DHB to 0.07 pmol/min. The transient increases observed in both the contralateral and ischemic cortex are indicative of increased hydroxyl radical production during 3V0. Compared to the cortex, the higher levels of striatal 34DHB at all time points are consistent with the greater vulnerability of the striatum to various stresses including ischemia. (Funded by the MRC of Canada and the Heart and Stroke Foundations of Canada and QuBbec)
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