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assessed from 1 H MRS using point-resolved spectroscopy (PRESS) [TE=24ms; TR=2500ms; variable pulse power and optimized relaxation delay (VAPOR) water suppression; 27-m La nd 8-mL voxels in rats and mice, respectively] at 7T. Alterations in metabolites (Totally Automatic Robust Quantitation in NMR, TARQUIN) in tumors were characterized by increases in lipids (Lip1.3: 8.8–54.5mM for C6 and GL261) and decreases in NAA (1.3–2.0mM for RG2, GL261 and C6) and tCr (0.8–4.0mM for F98, RG2, GL261 and C6) in some models. F98, RG2, GL261 and C6 models all showed significantly decreased (p

The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.

The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle-bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

Choline deficiency (CD) is involved in hepatocellular carcinoma and CD-induced apoptosis may be implicated in cellular malignant transformation. In this report, we studied the effects of choline deficiency on generation of reactive oxygen species (ROS) using the fluorescent probe dichlorodihydrofluorescein diacetate and the possible role of ROS on CD-induced apoptosis in cultured CWSV-1 cells, an immortalized rat hepatocyte. This cell line is reported to become tumorigenic by step-wise culturing in lower levels of choline. Our data demonstrate that CD induces a time- and dose-dependent increase in ROS in CWSV-1 cells. The increase in ROS production may be related to dysfunction of the mitochondrial respiratory chain. Our data also demonstrated that ROS generation occurred before CD-induced apoptosis, suggesting ROS may play a key role in signaling CD-induced apoptosis in CWSV-1 cells.

Vitamin E (alpha-tocopherol or alphaT) has long been recognized as a classic free radical scavenging antioxidant whose deficiency impairs mammalian fertility. In actuality, alpha-tocopherol is one member of a class of phytochemicals that are distinguished by varying methylation of a chroman head group. Early studies conducted between 1922 and 1950 indicated that alpha-tocopherol was specific among the tocopherols in allowing fertility of laboratory animals. The unique vitamin action of alphaT, combined with its prevalence in the human body and the similar efficiency of tocopherols as chain-breaking antioxidants, led biologists to almost completely discount the "minor" tocopherols as topics for basic and clinical research. Recent discoveries have forced a serious reconsideration of this conventional wisdom. New and unexpected biological activities have been reported for the desmethyl tocopherols, such as gamma-tocopherol, and for specific tocopherol metabolites, most notably the carboxyethyl-hydroxychroman (CEHC) products. The activities of these other tocopherols do not map directly to their chemical antioxidant behavior but rather reflect anti-inflammatory, antineoplastic, and natriuretic functions possibly mediated through specific binding interactions. Moreover, a nascent body of epidemiological data suggests that gamma-tocopherol is a better negative risk factor for certain types of cancer and myocardial infarction than is a alpha-tocopherol. The potential public health implications are immense, given the extreme popularity of alphaT supplementation which can unintentionally deplete the body of gamma-tocopherol. These findings may or may not signal a major paradigm shift in free radical biology and medicine. The data argue for thorough experimental and epidemiological reappraisal of desmethyl tocopherols, especially within the contexts of cardiovascular disease and cancer biology.

Purpose Inorganic catalytic nanoceria or cerium oxide nanoparticles (CeNPs) are bona fide antioxidants that possess regenerative radical scavenging activities in vitro. Previously, we demonstrated that CeNPs had neuroprotective and anti-angiogenic properties in rodent retinal degeneration and neovascularization models. However, the cellular mechanisms and duration of the catalytic activity of CeNPs in preventing photoreceptor cell loss are still unknown. In this study, we sought to answer these questions using the P23H-1 rat, an autosomal dominant retinitis pigmentosa (adRP) model. Methods A single dose of either saline or CeNPs was delivered intravitreally into the eyes of P23H-1 rats at 2–3 weeks of age. Retinal functions were examined at 3 to 7 weeks post injection. We quantified retinal proteins by Western blot analyses and counted the number of apoptotic (TUNEL+) profiles in the outer nuclear layer (ONL) of retinal sections. We measured free 8-isoprostanes to quantify lipid peroxidation in retinal tissues. Results We observed increased rod and cone cell functions up to three weeks post injection. Apoptotic cells were reduced by 46%, 56%, 21%, and 24% at 3, 7, 14, 21 days, respectively, after CeNPs injection compared to saline. Additionally, reduction of lipid peroxidation in the retinas of CeNPs-treated vs saline-treated animals was detected 14 days post injection. Conclusions We validated that CeNPs were effective in delaying loss of photoreceptor cell function in an adRP rat model. This represents the fourth rodent retinal disease model that shows delay in disease progression after a single application of CeNPs. We further demonstrated that CeNPs slowed the rate of photoreceptor cell death. We deduced that the catalytic activity of CeNPs in vivo in this rat model to be undiminished for at least 7 days and then declined over the next 14 days after CeNPs administration.

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O 2 to hydrogen peroxide (H 2 O 2 ) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H 2 O 2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O 2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H 2 O 2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H 2 O 2 flux was accelerated by rotenone. α-Phenyl-N-tert-butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex 1-stimulated H 2 O 2 flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H 2 O 2 synthesis, possessing an electron leak site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H 2 O 2 production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.

Phosphorylation of the serine/threonine kinase Akt has previously been shown to be increased by treatment of cells with H2O2; the target of H2O2 has not been clearly identified. Here we show that treatment of rat primary astrocytes with H2O2 resulted in increased Akt phosphorylation that was blocked by wortmannin. The thiol-reducing agent N-acetylcysteine had only a slight inhibitory effect. Treatment with rotenone or antimycin A also resulted in increased wortmannin-sensitive Akt phosphorylation, probably by increasing intracellular H2O2 generation by blocking mitochondrial electron transport. Addition of phosphatidylinositol 3,4-bisphosphate to cells also resulted in an increase in Akt phosphorylation. This increase was additive to that induced by H2O2 and was also blocked by wortmannin. These results suggest that activation of Akt by H2O2 occurs upstream of phosphatidylinositol 3-kinase (PI 3-K) activity in astrocytes. The data indicate that major oxidative effects do not occur at the level of the PI 3-K-antagonizing phosphatase PTEN.

Methylene blue (MB) is being used as a sensitizer for the photodynamic inactivation of viral contaminants, including the human immunodeficiency virus, in blood and blood components used in medical treatment. We recently showed that oxygen-dependent photodynamic inactivation of the RNA bacteriophage Q beta with MB plus light (MB + L) is associated with the formation of 8-oxo-7,8-dihydroguanine, protein carbonyls, RNA-protein crosslinkages and minor amounts of RNA strand breaks. We report herein, with the use of infectious RNA assays, that the lethal lesions in Q beta phage following MB + L exposure can be accounted for, and thereby most likely reside in, the RNA component of the phage but that the protein component of the virion contributes to the inactivation. The formation of RNA-protein crosslinkages as the primary inactivating type of lesion is put forth as the most probable model of the inactivation mechanism due to the sensitivity with which RNA-protein crosslinks are formed in response to MB + L exposure and the expectation of the powerful inactivating power of this type of lesion.

Nitrone-based free radical traps (NFTs) have been shown to be protective in several neurodegenerative models. Our research has strongly implicated that: A) several neurodegenerative conditions exhibit increased levels of pro-inflammatory cytokines which consequently result in increased levels of oxidative stress and B) that NFTs act in part by suppressing oxidative stress through suppression of the action of the cytokine cascade. Acquired Immune Deficiency Syndrome (AIDS) dementia complex (ADC) is one of several conditions where the data collected helped to develop these concepts. Novel observations include demonstration that IL-1beta acts on cultured brain glia cells to invoke protein nitration and oxidative stress and that low levels of PBN (alpha-phenyl tert-butyl nitrone) inhibit this effect. We interpret these data as indicating that PBN prevents IL-1beta mediated peroxynitrite formation. Additionally, we have found that the AIDS viral envelope protein gp120 upregulates mRNA for the cytokines TNF alpha and TNF beta in rat neonatal brain, and that PBN prevents this. Western blots of protein extracts showed upregulation of inducible nitric oxide synthase (iNOS) in gp120 treated neonatal rat brains, and that PBN prevented induction of this enzyme as well. These observations underscore the general concept that PBN inhibits the induction of genes which produce neurotoxic products, one of which is peroxynitrite formed by the reaction of nitric oxide with superoxide, and may act also by inhibiting the induction of cytokines which mediate pro-inflammatory conditions in the brain.

Reversible protein phosphorylation regulates a wide array of cellular functions. Cells respond to cytokines and various stressors via phosphorylation and thus activation of one or more of the mitogen-activated protein kinase (MAPK) pathways. Involvement of these signal transduction pathways has been implicated in numerous pathologies, including inflammation. Using a primary glia cell culture, we show here that the antioxidant N-acetylcysteine (NAC) and the nitrone-based free radical trap, alpha-phenyl-N-tert-butyl nitrone (PBN), reduce total basal protein phosphorylation in a concentration-dependent manner as assessed by phosphotyrosine analysis and by [gamma-32P]ATP transfer radioassay. In addition we show that NAC inhibits H2O2-induced phosphatase inactivation in glia cell lysate. The PBN- and NAC-induced reduction in protein phosphorylation is accompanied by an increase in phosphatase activity, suggesting that PBN and NAC reduce protein phosphorylation by globally augmenting oxidant-sensitive phosphatase activities. These results partly explain why certain antioxidants also possess anti-inflammatory actions.

Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.

Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses. Published version

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.

The central nervous system (CNS) presents both challenges and opportunities to researchers of redox biochemistry. The CNS is sensitive to oxidative damage during aging or disease; excellent transgenic models of specific neurodegenerative diseases have been created that reproduce oxidative stress components of the corresponding human disorder. Mouse models of familial amyotrophic lateral sclerosis (ALS) based on overexpressed mutant human Cu, Zn-superoxide dismutase (SOD1) are cases in point. These animals experience predictably staged, age-dependent motor neuron degeneration with profound cellular and biochemical damage to nerve fibers and spinal cord tissue. Severe protein and lipid oxidation occurs in these animals, apparently as an indirect consequence of protein aggregation or cytopathic protein-protein interactions, as opposed to aberrant redox catalysis by the mutant enzyme. Recent studies of G93A-SOD1 mice and rats suggest that oxidative damage is part of an unmitigated neuroinflammatory reaction, possibly arising in combination from mitochondrial dysfunction plus pathophysiologic activation of both astrocytes and microglia. Lesions to redox signal-transduction pathways in mutant SOD1+ glial cells may stimulate broad-spectrum upregulation of proinflammatory genes, including arachidonic acid-metabolizing enzymes [e.g., cyclooxygenase-II (COX-II) and 5-lipoxygenase (5LOX)]; nitric oxide synthase (NOS) isoforms; cytokines (particularly tumor necrosis factor alpha, TNF-alpha); chemokines; and immunoglobulin Fc receptors (FcgammaRs). The integration of these processes creates a paracrine milieu inconsistent with healthy neural function. This review summarizes what has been learned to date from studies of mutant SOD1 transgenic animals and demonstrates that the G93A-SOD1 mouse in particular is a robust laboratory for the study of neuroinflammation and redox biochemistry.

Detailed study of glial inflammation has been hindered by lack of cell culture systems that spontaneously demonstrate the "neuroinflammatory phenotype". Mice expressing a glycine → alanine substitution in cytosolic Cu, Zn-superoxide dismutase (G93A-SOD1) associated with familial amyotrophic lateral sclerosis (ALS) demonstrate age-dependent neuroinflammation associated with broad-spectrum cytokine, eicosanoid and oxidant production. In order to more precisely study the cellular mechanisms underlying glial activation in the G93A-SOD1 mouse, primary astrocytes were cultured from 7 day mouse neonates. At this age, G93A-SOD1 mice demonstrated no in vivo hallmarks of neuroinflammation. Nonetheless astrocytes cultured from G93A-SOD1 (but not wild-type human SOD1-expressing) transgenic mouse pups demonstrated a significant elevation in either the basal or the tumor necrosis alpha (TNFα)-stimulated levels of proinflammatory eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4); inducible nitric oxide synthase (iNOS) and •NO (indexed by nitrite release into the culture medium); and protein carbonyl products. Specific cytokine- and TNFα death-receptor-associated components were similarly upregulated in cultured G93A-SOD1 cells as assessed by multiprobe ribonuclease protection assays (RPAs) for their mRNA transcripts. Thus, endogenous glial expression of G93A-SOD1 produces a metastable condition in which glia are more prone to enter an activated neuroinflammatory state associated with broad-spectrum increased production of paracrine-acting substances. These findings support a role for active glial involvement in ALS and may provide a useful cell culture tool for the study of glial inflammation.

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc-superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up-regulation of tumor necrosis factor alpha (TNFalpha) and its primary receptor TNF-RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFalpha antagonists. Walker EOC-20 microglia cells were stimulated with recombinant TNFalpha, with or without inhibitors, and the cell response was indexed by NO2- output. Three hundred and fifty-five rationally selected compounds were included in this bioassay. The arachidonic acid 5-lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non-cytotoxic antagonists tested (IC50 8 +/- 3 microm). Investigation of the G93A-SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A-SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease-associated gliosis and cleaved microtubule-associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFalpha antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25-40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE-Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE-keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Recent data indicate that certain pro-inflammatory cytokines are transcriptionally upregulated in the spinal cords of G93A-SOD1 mice, a model of amyotrophic lateral sclerosis (ALS). We previously showed that the receptor for tumor necrosis factor alpha (TNF-R1) was notably elevated at late presymptomatic as well as symptomatic phases of disease (J. Neurochem. 82 (2002) 365). We now extend these findings by showing that message for TNFalpha, as well as mRNA for interferon gamma (IFNgamma) and transforming growth factor beta1/2 (TGFbeta1, TGFbeta2), is simultaneously increased. Furthermore, TNFalpha protein is significantly increased in G93A-SOD1 mouse spinal cords, as are protein levels for interleukin-6 (IL6), IFNgamma, and the chemokines RANTES (CCL5) and KC. The interaction of TNFalpha, IL6, and IFNgamma proteins was modeled in vitro using Walker EOC-20 murine microglia with nitrite (NO(2)(-)) efflux as a quantitative index of cell response. TNFalpha alone caused robust NO(2)(-) flux, while IL6 had a lesser effect and neither IFNgamma nor IL1beta was active when applied singly. The TNFalpha stimulus was potently magnified in the presence of IL6 or IFNgamma. When applied in combination at very low concentrations, IFNgamma co-synergized with IL6 to produce a multiplicative increase in NO(2)(-) after stimulation with TNFalpha. Taken together, these data suggest that modest increases in multiple synergistic cytokines could produce a disproportionately severe activation of microglia within the degenerating spinal cord. Our data support a model wherein TNFalpha acts as a principal driver for neuroinflammation, while several co-stimulating cytokines and chemokines act to potentiate the TNFalpha effects.

Removal of choline from the diet results in accumulation of triglycerides in the liver, and chronic dietary deficiency produces a non-genotoxic model of hepatocellular carcinoma. An early event in choline deficiency is the appearance of oxidized lipid, DNA and protein, suggesting that increased oxidative stress may facilitate neoplasia in the choline deficient liver. In this study, we find that mitochondria isolated from rats fed a choline-deficient, L-amino acid defined diet (CDAA) demonstrate impaired respiratory function, particularly in regard to complex I-linked (NADH-dependent) respiration. This impairment in mitochondrial electron transport occurs coincidentally with alterations in phosphatidylcholine metabolism as indicated by an increased ratio of long-chain to short-chain mitochondrial phosphatidylcholine. Moreover, hydrogen peroxide (H(2)O(2)) generation is significantly increased in mitochondria isolated from CDAA rats compared with mitochondrial from normal rats, and the NADH-specific yield of H(2)O(2) is increased by at least 2.5-fold. These findings suggest an explanation for the rapid onset of oxidative stress and energy compromise in the choline deficiency model of hepatocellular carcinoma and indicate that dietary choline withdrawal may be a useful paradigm for the study of mitochondrial pathophysiology in carcinogenesis.

The p38 mitogen-activated protein kinase (p38-MAPK) is a central enzyme in one of the major protein kinase cascades that regulate proapoptotic and proinflammatory signal transduction. p38-MAPK is activated by receptor/ligand recognition events or by exposure to extracellular stressors, including oxidative stress. Activation of p38-MAPK is affected by dual phosphorylation on a specific inhibitory domain. Dual phosphorylation causes a structural change in the p38-MAPK enzyme which allows binding of ATP and target substrate. Agents which block ATP docking to phosphoactivated p38-MAPK are being investigated for treatment of inflammatory diseases and neurodegenerative pathologies. An alternative strategy for p38-MAPK antagonism would be the inhibition of p38-MAPK phosphoactivation. We now report potent inhibition of p38-MAPK phosphorylation by a synthetic benzamide (CPI-1189) which displays protective action against tumor necrosis factor- α (TNF α )-induced neurodegeneration. In primary astrocytes treated with interleukin 1 β (IL1 β ), CPI-1189 inhibits p38-MAPK phosphorylation at concentrations of 10 nM or less. While the precise molecular target of CPI-1189 remains unknown, these findings suggest a novel mechanism for the neuroprotective properties of the compound. These findings also indicate that antagonism of the p38-MAPK may be achieved through pharmacological inhibition of p38-MAPK phosphorylation, a strategy that is conceptually distinct from direct inhibition of ATP binding to the active enzyme.

As organs age, the likelihood of severe dysfunction increases steadily. The brain is particularly sensitive to age-related, chronic and acute oxidative pathologies. An emerging paradigm holds that diverse neurodegenerative conditions share a common etiological factor, namely, enhanced brain tissue oxidation owing to exacerbated production of reactive oxygen species (ROS) or to compromise of antioxidant defense and repair mechanisms. Brain is particularly susceptible to oxidative stress owing to its high content of unsaturated lipids, high metabolic rate, relative dearth of antioxidant enzymes, and inability to regenerate lost neurons. Pathogenic ROS generation may result from metabolic enzyme dysregulation, impaired mitochondrial respiration, excitotoxic stimulation, and secondarily as a function of intracellular calcium stress (summarized in Fig. 1 and elaborated below). Natural variation in antioxidant systems may explain why humans differ so greatly with respect to pathways and rates of neurodegeneration. If this is the case, antioxidant supplementation of the aging brain may forestall certain aspects of age-related neurodegeneration. Accordingly, much research has focused on antioxidant management of aging brain and on antioxidant interdiction of postischemic brain damage. Recent findings indicate that specific antioxidants do more than scavenge ROS, but may indirectly affect cellular signal transduction, genetic response, and inflammatory events in such a way as to modulate beneficially brain response to oxidative challenge.

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.

We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.

Ferritin heavy chain (FH), an embryonically-expressed protein in the erythroid lineage, localizes to the nucleus and represses the human adult β-globin promoter in transient expression assays (Broyles et al., PNAS98: 9145, 2001). Recently, we have performed chromatin immunoprecipitation (ChIP) assays with cross-linked chromatin of K562 cells in which the β-globin gene is repressed, using anti-FH polyclonal antisera. These results strongly indicate that FH occupies the repression site (a CAGTGC motif) in vivo. Binding to this -150 site has been previously demonstrated to be required for β-promoter repression in co-transfections. EMSA assays (competitive gel shifts) have revealed that the mouse βMajor-globin promoter has an analogous CAGTGN motif at -160 bp from the cap site that competes specifically with the human CAGTGC site for FH binding. The mouse βMinor-globin promoter lacks the -150/-160 CAGTGN motif and, therefore, the FH binding site. Thus, a human FH transgenic mouse, in which the FH gene is driven by a truncated β-promoter lacking the CAGTGN motif, should express human FH in definitive erythroid cells where the FH would be predicted to repress βMajor-globin but not βMinor-globin. Such a mouse would be predicted to survive but be born with a mild β-thalassemia due to the decreased βMajor/βMinor ratio in its definitive erythroid cells. Preliminary results from the litters of F1 generation FH-tg mice indicate that such is indeed the case, i.e., that human FH functions as a βMajor-globin repressor in vivo.

The ninaC gene is one of eight nina (neither inactivation nor afterpotential) genes identified from mutations that drastically reduce the amount of rhodopsin in the compound eye of Drosophila melanogaster. The gene has been cytogenetically localized to the 27E-28B region of the second chromosome. NaDodSO4/PAGE analysis of eye proteins of flies carrying one, two, or three copies of the ninaC region shows that two eye-specific proteins of molecular weight 170,000 and 130,000 display a strong dependence on the dosage of the ninaC gene, although the dependence is evident only when the dosage is decreased and not when it is increased. All mutations in the ninaC gene studied to date have pronounced effects on these two polypeptides. These results suggest that the ninaC locus encodes these two polypeptides. Ultrastructural studies show that the polypeptides encoded by ninaC are very likely to be important components of the cytoskeletal structure of rhabdomeral microvilli.

Mutants displaying generalized behavioral defects and one mutant having an enzyme deficiency were examined for electroretinogram (ERG) defects. Mutations in nine genes were examined that cause ERG defects. Two, parats4 and slrpD, cause reversibly temperature dependent loss of the off-transients in the ERG. stnC and Tyr-2 cause loss of the on and off-transients. The transient defect in Tyr-2 mapped close to a site shown to affect tyrosinase activity in this strain. Mutations bas, rex and sesD delay recovery from the prolonged depolarization afterpotential. The visual defects of mutations elavjl and nbAEE171 are not complemented by lethal mutations, which, presumably, affect other tissues.