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The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2'-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.

Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions.DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay.None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation.Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm this inference from the data.

In the event of a radionuclear attack or nuclear accident, the skin would be the first barrier exposed to radiation, though skin injury can progress over days to years following exposure. Chronic oxidative stress has been implicated as being a potential contributor to the progression of delayed radiation-induced injury to skin and other organs. To examine the causative role of oxidative stress in delayed radiation-induced skin injury, including impaired wound healing, we tested a synthetic superoxide dismutase (SOD)/catalase mimetic, EUK-207, in a rat model of combined skin irradiation and wound injury. Administered systemically, beginning 48 hours after irradiation, EUK-207 mitigated radiation dermatitis, suppressed indicators of tissue oxidative stress, and enhanced wound healing. Evaluation of gene expression in irradiated skin at 30 days after exposure revealed a significant upregulation of several key genes involved in detoxication of reactive oxygen and nitrogen species. This gene expression pattern was primarily reversed by EUK-207 therapy. These results demonstrate that oxidative stress has a critical role in the progression of radiation-induced skin injury, and that the injury can be mitigated by appropriate antioxidant compounds administered 48 hours after exposure.

Superoxide dismutase/catalase mimetics, such as salen Mn complexes and certain metalloporphyrins, catalytically neutralize reactive oxygen and nitrogen species, which have been implicated in the pathogenesis of many serious diseases. Both classes of mimetic are protective in animal models of oxidative stress. However, only AEOL11207 and EUK-418, two uncharged Mn porphyrins, have been shown to be orally bioavailable. In this study, EUK-418 and several new analogs (the EUK-400 series) were synthesized and shown to exhibit superoxide dismutase, catalase, and peroxidase activities in vitro. Some also protected PC12 cells against staurosporine-induced cell death. All EUK-400 compounds were stable in simulated gastric fluid, and most were substantially more lipophilic than the salen Mn complexes EUK-189 and EUK-207, which lack oral activity. Pharmacokinetics studies demonstrate the presence of all EUK-400 series compounds in the plasma of rats after oral administration. These EUK-400 series compounds are potential oral therapeutic agents for cellular damage caused by oxidative stress. Electronic supplementary material The online version of this article (doi:10.1007/s00775-009-0550-4) contains supplementary material, which is available to authorized users.

Animal models, and human postmortem studies, of prion disease have demonstrated the presence of heightened oxidative stress in the brain, with additional findings supporting the likelihood that the normal isoform of prion protein directly contributes to neuronal antioxidant defences. Although such data are consistent with the postulate that oxidative stress plays a salient pathogenic role in prion disease, it remains possible that oxidative damage represents a secondary or relatively less important phenomenon in neurons already rendered dysfunctional from other primary insults. To provide further insights into the relative pathogenic importance of oxidative stress, we employed a potent manganese-superoxide dismutase/catalase mimetic, EUK-189, as a therapeutic in our mouse model of human prion disease. A significant but relatively modest prolongation of survival in EUK-189-treated mice was observed, which correlated with reductions in oxidative, especially nitrative, damage to proteins when compared to untreated disease controls. Lesion profiling also revealed reductions in spongiform change in specific brain regions of terminally sick EUK-189-treated mice. Our results are consistent with heightened oxidative stress playing a pathogenic role in prion disease but underscore the need for more biologically potent and, most likely, broader spectrum antioxidant treatments if more successful amelioration is to be achieved.

Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neurogenesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse peroxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H(2)O(2), as is known for CuZnSOD, may be potentially deleterious. Our results also show that the ability of catalase mimetics, like true catalases, to catalyse peroxidase reactions can complicate the interpretation of data obtained with certain fluorescent ROS-indicator dyes.

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.

Alzheimer disease is characterized by cerebral Abeta deposition, which we have recently discovered occurs also in the lens as cataracts in Alzheimer disease patients. Here we report the presence of significantly increased cataracts in the lenses of an Abeta-transgenic mouse model for Alzheimer disease and their amelioration upon treatment with EUK-189, a synthetic SOD/catalase mimetic. These data support an oxidative etiology for AD-associated lens cataracts and their potential to be treated preventatively with antioxidants.

SOD2 is an antioxidant protein that protects cells against mitochondrial superoxide. Hematopoietic stem cells (HSCs) lacking SOD2 are capable of rescuing lethally irradiated hosts, but reconstituted animals display a persistent hemolytic anemia characterized by increased oxidative damage to red cells, with morphologic similarity to human “sideroblastic” anemia. We report further characterization of this novel SOD2-deficiency anemia. Electron micrographs of SOD2-deficient reticulocytes reveal striking mitochondrial proliferation and mitochondrial membrane thickening. Peripheral blood smears show abundant iron-stainable granules in mature red cells (siderocytes). Fluorescence-activated cell sorting (FACS) analysis of cells labeled with oxidation-sensitive dyes demonstrates enhanced production of superoxide and hydrogen peroxide by SOD2-deficient cells. Oxidative damage to proteins is increased in SOD2-deficient cells, with much of the damage affecting membrane/insoluble proteins. Red cell proteome analysis demonstrates that several proteins involved in folding/chaperone function, redox regulation, adenosine triphosphate (ATP) synthesis, and red cell metabolism show altered expression in SOD2-deficient cells. This data, combined with information on how protein expression levels change upon antioxidant therapy, will aid in identification of proteins that are sensitive to oxidative damage in this model, and by extension, may have a role in the regulation of red cell lifespan in other hemolytic disorders.

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.

Oxidative stress has been implicated in cognitive impairment in both old experimental animals and aged humans. This implication has led to the notion that antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. Here we report a dramatic loss of learning and memory function from 8 to 11 months of age in mice, associated with marked increases in several markers of brain oxidative stress. Chronic systemic administration of two synthetic catalytic scavengers of reactive oxygen species, Eukarion experimental compounds EUK-189 and EUK-207, from 8 to 11 months almost completely reversed cognitive deficits and increase in oxidative stress taking place during this time period in brain. In particular, increase in protein oxidation was completely prevented, whereas increase in lipid peroxidation was decreased by ≈50%. In addition, we observed a significant negative correlation between contextual fear learning and levels of protein oxidation in brain. These results further support the role of reactive oxygen species in age-related learning impairment and suggest potential clinical applications for synthetic catalytic scavengers of reactive oxygen species.

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.

A series of novel manganese(III) complexes is described based on a 6,6'-bis(2-hydroxyphenyl)-2,2'-bipyridine template. These complexes show superoxide dismutase and catalase activity. The effect of the aromatic substitution pattern on the SAR is described.

Cranial irradiation with 56Fe, a form of space radiation, causes hippocampus-dependent cognitive changes. 56Fe irradiation also increases reactive oxygen species (ROS) levels, which may contribute to these changes. Therefore, we investigated the effects of the antioxidant alpha lipoic acid (ALA) on cognition following sham-irradiation and irradiation. Male mice were irradiated (brain only) with 56Fe (3 Gy) or sham-irradiated at 6–9 months of age. Half of the mice remained fed a regular chow and the other half of the mice were fed a caloric-matched diet containing ALA starting two-weeks prior to irradiation and throughout cognitive testing. Following cognitive testing, levels of 3-nitrotyrosine (3NT), a marker of oxidative protein stress, and levels of microtubule-associated protein (MAP-2), a dendritic protein important for cognition, were assessed using immunohistochemistry and confocal microscopy. ALA prevented radiation-induced impairments in spatial memory retention in the hippocampal and cortical dependent water maze probe trials following reversal learning. However, in sham-irradiated mice, ALA treatment impaired cortical-dependent novel object recognition and amygdala-dependent cued fear conditioning. There was a trend towards lower 3NT levels in irradiated mice receiving a diet containing ALA than irradiated mice receiving a regular diet. In the hippocampal dentate gyrus of mice on regular diet, irradiated mice had higher levels of MAP-2 immunoreactivity than sham-irradiated mice. Thus, ALA might have differential effects on the brain under normal physiological conditions and those involving environmental challenges such as cranial irradiation.

We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.

Considerable evidence exists that the brains of individuals with Alzheimer's disease are subject to elevated levels of oxidative stress, particularly in regions exhibiting pathological damage. A major contributor to this oxidative stress appears to be the inflammatory process. Activation of rodent microglial cells by LPS or beta-amyloid peptide results in a marked up-regulation of inducible nitric oxide synthase (iNOS) and corresponding nitric oxide (NO) production. Elevated levels of iNOS are also observed in the brains of Alzheimer patients. The reaction of NO with superoxide leads to the generation of the highly reactive and damaging peroxynitrite free radical species. Peroxynitrite appears to play a key role in the generation of an oxidative stress in the Alzheimer brain as evidenced by widespread nitrotyrosine immunoreactivity. We have employed SIN-1 as a peroxynitrite generating system in cell cultures in order to characterize the effects of this free radical on neurons. SIN-1 treatment of primary rat hippocampal neurons in culture results in neurotoxicity by a necrosis mechanism according to electron microscopic criteria. One approach to limiting peroxynitrite mediated damage is to limit superoxide production. An approach we have evaluated is treatment with salen manganese compounds, a class of catalytic antioxidant compounds which behave as superoxide dismutase (SOD)/catalase mimetics to detoxify superoxide. A number of such salen manganese compounds, including EUK-8 and EUK-134, can markedly protect primary rat cortical neurons from hydrogen peroxide mediated oxidative stress. Such salen manganese compounds can similarly afford marked neuroprotection to an oxidative stress imposed by SIN-1, potentially attributable at least in part to their inherent SOD activity. The salen manganese SOD/catalase mimetics represent a promising class of catalytic antioxidant for attenuating oxidative stress.

A reduction in stress tolerance is a hallmark of the aging process, and the lowered functional capacity observed in aged organisms is associated with an increased rate of oxidative stress and a greater susceptibility of aged tissues to oxidative injury. In this report, we show that chronic systemic administration of a superoxide dismutase (SOD)/catalase mimetic (EUK-189), delivered over a 1 month period via osmotic pump, prevents heat stress-induced liver injury by dramatically decreasing oxidative damage in aged animals. Widespread liver injury was present in old but not young vehicle-treated rats in response to a 2 day heating protocol. However, SOD/catalase mimetic treatment markedly decreased the hyperthermia-induced liver injury associated in old animals. The reversal of damage with EUK-189 was associated with an improvement in intracellular redox status and a striking reduction in hepatocellular lipid peroxidation. EUK-189 treatment also blocked the activation of activator protein-1 (AP-1), which is a redox-sensitive early response transcription factor involved in the regulation of cellular stress responses. These results demonstrate that oxidative stress plays a unique role in age-related hyperthermic injury and suggest that therapeutic strategies aimed at improving redox potential, such as chronic SOD/catalase mimetic treatment, can prevent the oxidative-mediated damage associated with environmental stress.

Ataxia-telangiectasia is caused by mutations in the ATM gene, the protein product of which is essential for effective response to double-stranded DNA breaks. Loss of ATM function explains most aspects of the disease, but not the cerebellar neurodegeneration characteristic of the disease. Mice lacking ATM provide an excellent model of the human disorder. In addition to deficient response to DNA damage, these mice exhibit oxidative stress, which we hypothesized is the cause of cerebellar dysfunction. We show that treatment with a catalytic antioxidant corrects the neurobehavioral deficit in these mice.

Excessive extracellular zinc may contribute to neuronal cell death following ischemia and seizures, although the mechanisms mediating zinc-induced cell death remain largely unknown. In this study, we examined potential cellular and molecular mechanisms associated with zinc neurotoxicity and determined the neuroprotective effects of the superoxide dismutase (SOD)/catalase mimetic, EUK-134. Cortical neuron cultures exposed to zinc for 24 h exhibited concentration-dependent increases in lactate dehydrogenase (LDH) release and number of apoptotic cell bodies. Both effects were prevented by treatment with EUK-134. Zinc exposure resulted in increased release of cytochrome c from the mitochondria into the cytosol. Treatment with EUK-134 blocked this parameter of mitochondrial dysfunction. Exposure of cultures to zinc for 4 h produced an elevation of reactive oxygen species (ROS) as determined by increased 2,7-dichlorofluorescein (DCF) fluorescence, which was followed by an increase in lipid peroxidation. EUK-134 completely attenuated ROS production and subsequent oxidative damage. Finally, zinc exposure activated NF-κB, an effect also prevented by EUK-134. These data indicate that multiple cellular and molecular mechanisms are involved in zinc neurotoxicity. As all these mechanisms appear to be sensitive to treatment with EUK-134, our data suggest that oxidative stress occurs early in the cascade of events triggered by zinc.

Publisher Summary Molecular oxygen, although essential for aerobic metabolism, can be converted to potentially deleterious substances, including superoxide ion, hydrogen peroxide, and hydroxyl radical, known collectively as reactive oxygen species (ROS). Increased ROS formation under pathological conditions is believed to cause cellular damage through chemical interactions with proteins, lipids, and DNA. Some degree of tissue protection is provided by endogenous ROS scavengers such as superoxide dismutases (SOD) and catalase, which catalytically destroy superoxide ion and hydrogen peroxide, respectively. Also, molecules synthesized endogenously or acquired through diet, such as ascorbate (vitamin C), α-tocopherol (vitamin E), and glutathione, have antioxidant properties. Salen-manganese complexes, the focus of this chapter and porphyrin-manganese complexes have been reported to have SOD activity. Synthetic salen-manganese has catalase activity as well. Salen-manganese complexes have characteristics that might facilitate their potential usefulness as therapeutic agents. First, as low molecular weight, synthetic molecules rather than proteinaceous antioxidant enzymes they have the potential advantages that relate to cost-effectiveness and pharmaceutical formulation and delivery. Second, they act catalytically, presumably enhancing their efficiency over noncatalytic low-molecular-weight ROS scavengers. Third, their ability to destroy both superoxide anion and hydrogen peroxide should enhance their protective potential in various disease states involving the production of multiple ROS species. In this chapter are described the catalytic properties of EUK-8, a prototype salen-manganese complex, the efficacy of EUK-8 in experimental models for disease and the future directions in the development of salen–manganese complexes as novel, broadly applicable potential therapeutic agents. The chapter discusses how EUK-8 and other salen–manganese complexes exhibit superoxide scavenging activity, the catalase activity of EUK-8: catalatic and peroxidatic reaction pathways, how salen–manganese complexes have advantages over other antioxidants, and how EUK-8 is protective in a model for adult respiratory distress syndrome and neurological disorders. Salen–manganese complexes have a novel mode of action and an extremely broad potential clinical utility.

Dear Editor: We write to correct some of the misleading descriptions of our research in a Comprehensive Invited Review “Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential” by Dr. Ines Batinic-Haberle and colleagues (1). We wish to correct two factual errors and then discuss a matter of opinion. On page 888, in a section entitled Neutral Porphyrins, our research describing a series of such compounds is cited (2). The review states “Yet, critical analytical data, such as elemental analysis, were again not provided.” This statement is incorrect. Analytical data are summarized for all porphyrins in the Materials and Methods section under “Compound Synthesis” (pp. 981–983), with complete elemental analysis and other detailed analytical data provided in the Supplementary Information. Another line in the same section of the review states “The SOD-like activity has been assayed by NBT assay to avoid the artifact problems with cyt c assay, which the authors incorrectly claim were previously observed with MnTBAP.” In fact, our article refers to “ … interference observed when using MnTBAP analogs,” citing Trova et al. (3), who describe a series of “MnTBAP analogues,” nine of which exhibited artifacts in the cytochrome c assay. Specifically, they state “Those porphyrin analogues derived from dipyrromethane 3b and glyoxylate esters 1a-f … generally interfered with the SOD assay (employing cytochrome c).” Our citation, therefore, is by no means an incorrect claim. While the above comments reflect objective, factual errors in the Batinic-Haberle Review, we wish to add one comment that is more reflective of a difference in opinion. It is further stated in the same paragraph of the review that the compounds we describe have “ … no structural features … [that] would predict them to be good SOD mimics.” Our article reports the superoxide dismutase (SOD) and other catalytic activities of our compounds, pointing out that the SOD activity of these Mn porphyrins is, indeed, low when compared with salen–Mn complexes, which we have also described. Unlike Dr. Batinic-Haberle, we believe that SOD activity is not as important as other properties, such as catalase and other reactive oxygen species or reactive nitrogen species scavenging properties, lipophilicity, lack of toxicity, or intracellular stability, in determining biological efficacy (discussed in our article) (2). Our position is further supported by our finding that these neutral Mn porphyrins are potently antiapoptotic in two cellular injury models (2, 4), an important point that Dr. Batinic-Haberle et al. fail to mention.

It is well established that inflammation and oxidative stress are key components of the pathology of Alzheimer's disease (AD), but how early in the pathological cascade these processes are involved or which specific molecular components are key, has not been fully elucidated. This paper describes the pharmacological approach to understand the molecular components of inflammation and oxidative stress on the activation of microglial cells and neuronal cell viability. We have shown that activation of microglia with the 42-amino-acid form of the ϐ-amyloid peptide (Aϐ42) activates the production of cyclooxygenase-2, the inducible form of nitric oxide synthase and tumour necrosis factor-α and there appears to be little interactive feedback between these three mediators. Moreover, we explore the effects of a series of salen-manganese complexes, EUK-8, -134 and -189, which are known to possess both superoxide and catalase activity. These compounds are able to protect cells from insults produced by hydrogen peroxide or peroxynitrite. Moreover, EUK-134 was also able to limit the output of prostaglandin E2 from activated microglial cells. The mechanisms underlying these effects are discussed. Together, these data support a pivotal role for oxidative stress and inflammation as key mediators of the pathological cascade in AD and provide some ideas about possible therapeutic targets.

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5‘-chloro-5‘-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.

Adenosine and 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of phosphatidylinositol in membranes prepared from aortic smooth muscle. The nucleosides did not affect the breakdown of phosphatidylinositol-4-phosphate. Under certain conditions, the membrane-bound phosphatidylinositol kinase phosphorylated exogenous phosphatidylinositol. The nucleosides inhibited the enzyme competitively with respect to magnesium-ATP and non-competitively with respect to phosphatidylinositol. Adenosine analogs modified in the ribose moiety were inhibitors with potencies comparable to that of adenosine, whereas adenine nucleotides and purine-modified adenosine analogs were much weaker inhibitors. Density gradient fractionation studies showed that phosphatidylinositol kinase is primarily associated with the sarcoplasmic reticulum. Vascular smooth muscle contraction is associated with increased phosphatidylinositol turnover. Inhibition of phosphatidylinositol kinase by intracellular adenosine may, therefore, be a factor involved in regulating vasodilation.

Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5′-amino-5′-deoxyadenosine covalently linked to agarose through the 5′-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2′,5′-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5′-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5′-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.