Levels Of Reactive Oxygen Intermediates Research Articles

Innate inflammatory response to acute inhalation exposure of riot control agent oleoresin capsicum in female rats: An interplay between neutrophil mobilization and inflammatory markers

Aim: The use of oleoresin capsicum (OC) sprays, due to their irreversible health effects has now grown into a matter of heated debate. In the present study, the early phase pulmonary events involving chemotactic and inflammatory mediators after short-exposure duration to OC have been presented.Materials and methods: Female Wistar rats used in the evaluation of respiratory parameters at 1 h, 3 h, and 24 h post-exposure, were sacrificed for the evaluation of blood cell counts, BALF cytokine estimation, lung capillary leakage, study of oxidative stress and histopathology of the lungs.Results: Results confirmed a dose-dependent effect of OC exposure on serum clinical chemistry and hematological parameters. Subsequent upregulation of IL-l and TNF-α indicated lung’s responses to acute oxidant-induced injury and inflammation after OC exposure. Significant alterations in the pulmonary levels of reactive oxygen intermediates were seen following the inhalation of OC. Infiltration of polymorphonuclear leukocytes, mostly neutrophils, into the site of infection was evident in the cytocentrifuged samples of BALF. Histological samples of rat lung sections revealed the recruitment of inflammatory cells in the airways and around blood vessels in the subepithelium of conducting airways.Conclusion: Results of the present study demonstrated that, exposure to OC spray may mitigate inflammatory response and development of acute lung injury in rats. However, it can be concluded that although OC spray causes pulmonary hazards in the aforementioned concentrations, it can be used as a non-lethal riot control agent in minimal concentration. Understanding the in-depth mechanism of action in the molecular and receptor level will help in developing effective antagonist against OC.

The immunomodulatory effect of Zingiber cassumunar ethanolic extract on phagocytic activity, nitrit oxide and reaxtive oxygen intermediate secretions of macrophage in mice

Immunomodulators could protect the body from a variety of infectious agents and boost immunity. Zingiber cassumunar rhizome or bangle potentially showed as an immunomodulator through increasing of macrophage activity in vitro. The objective of the study was to determine the effect of Z. cassumunar rhizome ethanolic extract on phagocytic activity, nitrite oxide (NO) and reactive oxygen intermediate (ROI) secretions in macrophages in vivo. A total of 200 g of Z. cassumunar rhizome was powdered, macerated in 96% ethanol and evaporated to get concentrated extract. Mice were divided into 5 groups as follow: the normal group was given by water only, the negative control group was given by a 0.94% CMC-Na suspension, the treatment groups were given by 250, 500 and 1000 mg/kgBW, respectively, of Z. cassumunar ethanolic extract. The extract was administered orally for 7 days. On the 8th day the mice were injected intraperitoneally 0.7 mg/kg BW of lipopolysaccharide. Four hours later macrophage was isolated. Furthermore, the determination of the phagocytic activity, NO and ROI secretions levels of macrophage were performed. The treatments of 250, 500 and 1000 mg/kg BW of Z. cassumunar ethanolic extract significantly increase the ROI and NO secretions levels (p<0.05), but did not increase the phagocytic activity (p>0.05) of macrophage. Z. cassumunar ethanolic extract have immunomodulatory effect in vivo.

Seed Priming Alters the Production and Detoxification of Reactive Oxygen Intermediates in Rice Seedlings Grown under Sub-optimal Temperature and Nutrient Supply.

The production and detoxification of reactive oxygen intermediates (ROIs) play an important role in the plant response to nutrient and environmental stresses. The present study demonstrated the behavior of growth, ROIs-production and their detoxification in primed and non-primed rice seedlings under chilling stress (18°C) and nitrogen-(N), phosphorus-(P), or potassium-(K) deprivation. The results revealed that chilling stress as well as deprivation of any mineral nutrient severely hampered the seedling growth of rice, however, seed priming treatments (particularly selenium- or salicylic acid-priming), were effective in enhancing the rice growth under stress conditions. The N-deprivation caused the maximum reduction in shoot growth, while the root growth was only decreased by P- or K-deprivation. Although, N-deprivation enhanced the root length of rice, the root fresh weight was unaffected. Rate of lipid peroxidation as well as the production of ROIs, was generally increased under stress conditions; the K-deprived seedlings recorded significantly lower production of ROIs than N- or P-deprived seedlings. The responses of enzymatic and non-enzymatic antioxidants in rice seedlings to chilling stress were variable with nutrient management regime. All the seed priming were found to trigger or at least maintain the antioxidant defense system of rice seedlings. Notably, the levels of ROIs were significantly reduced by seed priming treatments, which were concomitant with the activities of ROIs-producing enzymes (monoamine oxidase and xanthine oxidase), under all studied conditions. Based on these findings, we put forward the hypothesis that along with role of ROIs-scavenging enzymes, the greater tolerance of primed rice seedlings can also be due to the reduced activity of ROIs-producing enzymes.

Abstract 2356: Pro-inflammatory macrophages promote prostate cancer initiation and progression via NOX2-mediated oxidative stress

Abstract In recent years, the causal relationship between inflammation and cancer has gained wider acceptance; however, many of the molecular and cellular mechanisms mediating this relationship remain unresolved. A key regulator of the link between inflammation and cancer are tumor-associated macrophages (TAMs). Derived primarily from circulating peripheral blood monocytes, TAMs form the major leukocytic infiltrate found within the stroma of many tumor types. Their interaction with neoplastic cells shape their differentiation and functional orientation into two phenotypically distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages. Macrophages within the tumor microenvironment are thought to acquire an M2 phenotype characterized by production of pro-angiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. In contrast, M1 macrophages are regarded as anti-microbial and anti-tumorigenic due to their efficient production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during pro-inflammatory immune responses is an important aspect of host defense, excessive ROS production has also been implicated in the pathogenesis of various degenerative diseases, including cancer. However, despite the well-established role of M1 macrophages in generating high levels of reactive oxygen intermediates, they are still largely viewed as anti-tumorigenic. Interestingly, the membrane-associated enzymes, NADPH oxidases (NOXs), are major sources of ROS in macrophages. Thus, the goals of the current study is to aid in understanding the complex interaction between tumor cells and TAMs, and to investigate a potentially novel function of M1 macrophages in promoting prostate tumorigenesis. We hypothesize that prostate cancer (PCa) cells promote a pro-tumor microenvironment, characterized by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated NOX-derived ROS production. Accordingly, our lab observed that PCa cell conditioned media efficiently recruited monocytes with increased recruitment ability directly proportional to increasing PCa cell line aggressiveness. Secondly, when compared with PCa cells cultured alone, PCa cells co-cultured with M1 macrophages exhibited significantly higher oxidative stress levels. Furthermore, upon silencing of NOX2 in M1 macrophages, we noted a decrease in fluorescent ROS staining when compared to control siRNA treated macrophages, suggesting the involvement of NOX2 in M1 macrophage-mediated ROS production. Lastly, we observed that M1-mediated ROS generation through NOX enzymes promotes prostate cancer cell invasiveness. Taken together, our results suggest a potentially novel pro-tumorigenic function of M1 macrophages in PCa, with specific regard to NOX2. Citation Format: Kia J. Jones, Latoya K. Bryant, Cimona V. Hinton. Pro-inflammatory macrophages promote prostate cancer initiation and progression via NOX2-mediated oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2356. doi:10.1158/1538-7445.AM2015-2356

The effect of leptin on the respiratory burst of human neutrophils cultured in synovial fluid.

ObjectivesLeptin is a hormone responsible for nutritional status and immune competence coordination. In rheumatoid arthritis (RA) increased leptin levels were observed in both serum and synovial fluid. Its influence on development of the disease still remains unclear. So far, research on leptin's influence on the emission of reactive oxygen intermediates (ROI) measured with chemiluminescence (CL) has provided unclear and contradictory results. In this study, we evaluated the influence of leptin on oxidative activity of neutrophils isolated from blood of healthy volunteers and cultured in different amounts of synovial fluid (SF) from patients with RA.Material and methodsNeutrophils’ oxidative metabolism was measured by two types of CL. The first one, luminol-dependent CL (CL-lum), allows one to determine phagocytic activity and the level of ROI generated in a myeloperoxidase-dependent manner. The second method used was lucigenin-dependent CL (CL-luc), which monitors ROI production dependent on the NADPH oxidase enzyme complex located in the cell membranes of neutrophils and enables one to determine the scope of extracellular ROI emission.ResultsNeutrophils stimulated by opsonized zymosan show a decrease in the level of CL-lum, proportional to the increasing concentration of both SF and serum collected from healthy donors. The observed effect of decreased CL-lum may, therefore, be dependent on the physical conditions (viscosity of fluids used). None of these experiments showed any effect of leptin on the level of CL-lum.ConclusionsThe present study showed that leptin does not affect the level of any of the CL types in inactive neutrophils incubated in normal serum, and it does not affect the level of oxidative activity in resting neutrophils incubated with SF. However, leptin influences extracellular ROI emission (measured by CL-luc). Leptin reduces extracellular emission of ROI, and this effect is dependent on concentration and duration of exposure to leptin.

Exogenous rhTRX reduces lipid accumulation under LPS-induced inflammation

Redox-regulating molecule, recombinant human thioredoxin (rhTRX) which shows anti-inflammatory, and anti-oxidative effects against lipopolysaccharide (LPS)-stimulated inflammation and regulate protein expression levels. LPS-induced reactive oxygen intermediates (ROI) and NO production were inhibited by exogenous rhTRX. We identified up/downregulated intracellular proteins under the LPS-treated condition in exogenous rhTRX-treated A375 cells compared with non-LPS-treated cells via 2-DE proteomic analysis. Also, we quantitatively measured cytokines of in vivo mouse inflammation models using cytometry bead array. Exogenous rhTRX inhibited LPS-stimulated production of ROI and NO levels. TIP47 and ATP synthase may influence the inflammation-related lipid accumulation by affecting lipid metabolism. The modulation of skin redox environments during inflammation is most likely to prevent alterations in lipid metabolism through upregulation of TIP47 and ATP synthase and downregulation of inflammatory cytokines. Our results demonstrate that exogenous rhTRX has anti-inflammatory properties and intracellular regulatory activity in vivo and in vitro. Monitoring of LPS-stimulated pro-inflammatory conditions treated with rhTRX in A375 cells could be useful for diagnosis and follow-up of inflammation reduction related with candidate proteins. These results have a therapeutic role in skin inflammation therapy.

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

The reversible formation of cysteine sulfenic acid promotes B‐cell activation and proliferation

B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 min. Increased ROIs preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration-dependent block in anti-IgM-induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to TRAIL-Induced Apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.

Antenatal maternal corticosteroid administration and markers of oxidative stress and inflammation in umbilical cord blood from very low birth weight preterm newborn infants

To investigate the association between antenatal maternal corticosteroid administration and blood levels of reactive oxygen intermediates (ROI), reduced glutathione (GR) and interleukin-6 (IL-6) in preterm, very low birth weight infants. This was a cohort study in which cord blood samples were used for the following tests: baseline and stimulated granulocyte ROI were measured by flow cytometry; GR was assayed by spectrophotometry; and IL-6 by enzyme-linked immunosorbent assay. Two different comparative analyses of antenatal corticosteroid (betamethasone) were conducted: the first compared administration against no administration and the second compared mothers who received the complete cycle with those given only a partial antenatal corticosteroid cycle. Maternal and neonatal variables were analyzed in order to compare groups. Categorical variables were compared using the chi-square or Fischer tests, and blood marker test results were compared using the Mann-Whitney test. The different corticoid therapy groups were similar in terms of all of the maternal and neonatal variables with the exception of vaginal delivery, which was significantly associated with not receiving antenatal corticosteroid. The results for ROI, GR and IL-6 did not differ when the comparison was based on simple presence or absence of administration of the steroid. However, when the complete cycle was compared against incomplete administration, median ROI and IL-6 were lower among those given the complete cycle. Administration of the complete cycle of betamethasone to the mother had a suppressive effect on baseline ROI and IL-6 production in very low birth weight preterm newborn infants.

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H 2O 2), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H 2O 2 inhibit insulin secretion. Menadione, which produces H 2O 2 via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H 2O 2 production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H 2O 2 formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H 2O 2 and menadione on insulin secretion.

The role of Reactive Oxygen Intermediates in B cell activation and differentiation (45.12)

Abstract Generating a protective humoral immune response requires proper B cell activation. Ligation of the BCR increases the level of reactive oxygen intermediates (ROI). While they have been implicated in oxidative stress, recent studies suggest ROI act as secondary messengers facilitating normal cellular function. Our focus is on the mechanisms by which ROI control B cell function. Following BCR ligation in vitro, the levels of the first oxidation product of cysteine, sulfenic acid, increased in the proteome. Using 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent further oxidation or reduction, we determined reversible cysteine sulfenic acid formation is critical for sustained tyrosine phosphorylation, calcium flux, and proliferation of B cells. To address the role of ROI in vivo, lymphocytic choriomeningitis virus (LCMV) infected mice were treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0-8 postinfection. On day 38, LCMV specific IgG antibody secreting cells (ASC) were 5-fold lower in the bone marrow. In contrast, the number of LCMV specific memory B cells was unaffected. The ASC decrease was not detected when mice were only treated from days 0-4 or 4-8 postinfection or during secondary viral infection. These data demonstrate ROI and reversible cysteine sulfenic acid formation are critical to B cell function and differentiation.

Regulation of Surface Free Thiol Levels During CD8+ T Cell Activation, Proliferation and Differentiation (132.7)

Abstract Upon both B and T cell activation the level of reactive oxygen intermediates (ROI) increases. Prior studies in the literature demonstrated that activated lymphocytes have increased total levels of surface free thiols, potentially as a compensatory mechanism to prevent surface oxidation. In this study, we determined how surface free thiol levels were regulated during the activation, proliferation, and differentiation of naïve CD8+ T cells both in vitro and in vivo. In vitro, we found that the levels of surface free thiols increased within 24 hours regardless of the activation stimulus and were maintained for at least three divisions. To understand how levels were modulated in vivo, we examined antigen-specific CD8+ T cells using lymphocytic choriomeningitis virus (LCMV) infection of mice and MHC Class I tetramers. On day 5 postinfection, the levels of surface free thiols were 8-fold higher on effector cells compared to those found on naïve CD8+ T cells. By day 8 postinfection this had decreased to 4-fold. After the infection was cleared, memory CD8+ T cells had 1.5-fold higher levels of surface free thiols than naïve cells. This contrasted with the results during chronic LCMV-Clone 13 infection. On day 8 postinfection surface free thiol levels were 11-fold higher and remained elevated until day 35 postinfection. These results suggest that surface free thiol levels are elevated most highly in rapidly proliferating cells and do not correlate with lymphocyte function.

Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice

2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR).Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10−5 to 10−7 M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM).Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-β cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-β (500μg per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs).In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CYand its metabolites can be a promising accessory tool when upregulation of the immune response is desired.

Identification of mitochondrial genome concatemers in AIDS-associated lymphomas and lymphoid cell lines

Since most oncogenic viruses persist as extrachromosomal covalently closed circular DNA (cccDNA) in tumor cells, we developed an assay to visualize and identify cccDNA in primary lymphomas. We identified concatemers of the mitochondrial genome in all samples analyzed, but not in normal lymphocytes. One AIDS-associated lymphoma (EL) was further studied in detail as its mitochondrial genome consisted of tandem head-to-tail duplications. Insertion of C-residues was noted near the origin of replication of EL mtDNA. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen intermediates. Screening of several AIDS-associated lymphomas and established lymphoid cell lines also revealed the presence of mitochondrial genome concatemers consisting of interlinked monomer molecules. Taken together, our results suggest that formation of mtDNA concatemers is associated with oncogenic transformation in lymphoid cells.

Mutants of GABA Transaminase (POP2) Suppress the Severe Phenotype of succinic semialdehyde dehydrogenase (ssadh) Mutants in Arabidopsis

BackgroundThe γ-aminubutyrate (GABA) shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-T; POP2) and succinic semialdehyde dehydrogenase (SSADH). We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported.Principal FindingsTo elucidate the role of succinic semialdehyde (SSA), γ-hydroxybutyrate (GHB) and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants.SignificanceWe conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

The requirement of reactive oxygen intermediates for lymphocytic choriomeningitis virus binding and growth

Multiple viruses induce reactive oxygen intermediate (ROI) generation during infection that plays an important role in growth. We have examined the importance of ROI during lymphocytic choriomeningitis virus (LCMV) infection of immortalized BHK-21 cells and murine peritoneal macrophages. Within 15 min of virus addition, intracellular ROI levels increased. To examine the contribution of ROI to LCMV infection, cells were pretreated with antioxidant prior to virus addition. Antioxidant treatment inhibited low and high MOI growth of virus. The requirement for ROI was greatest during the initial phase of infection, as antioxidant treatment after 6 h post infection had a weaker inhibitory effect. Furthermore, antioxidant treatment of cells inhibited virus binding, while treatment of virus stocks with N-ethyl malemide, which blocks free thiols, eliminated infectious virus. This illustrates that ROI are critical to the regulation of virus binding and growth and has important implications for understanding the infectivity of related viruses.

Peroxiredoxin II Regulates CD8+ T Cell Expansion and Survival During Acute and Chronic Viral Infections

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. We examined the role of the antioxidant enzyme, Peroxiredoxin II (PrxII), during T cell activation and throughout the immune response to acute and chronic viral infections. Following TCR stimulation, PrxII levels increased 50‐fold by 30 hours. Examination of differentiated T cells demonstrated that effector cells possessed lower PrxII levels than naïve or memory CD8+ T cells. Utilizing mice deficient in PrxII, we determined that ROI levels measured in T cells, either directly ex vivo or in response to stimulation, were higher than wild‐type mice. Deletion of PrxII also increased in vitro T cell proliferation, but decreased cell survival during division. Acute infection of PrxII−/− mice with LCMV Armstrong revealed an increased expansion of effector CD8+ T cells on day 8, but yielded no significant difference in memory CD8+ T cells by day 97. Additionally, no differences were observed in virus‐specific CD4+ T cells or antibody secreting cells at any time during infection. In contrast, chronic infection with LCMV Clone 13 resulted in death by day 13 post infection. Together, these results demonstrate that redox regulation of T cell activation through PrxII is critical during acute and chronic viral infections.Supported by ACS‐ #RSG‐04‐066‐01‐MBC to J.G.

Differential production of reactive oxygen intermediates by TCR stimulation in SLE T cells (129.10)

Abstract Systemic lupus erythematosus (SLE) is a protean autoimmune disease characterized by a wide array of intrinsic T cell defects. Reactive oxygen intermediates (ROI) modulate T cell activation, cytokine production, and proliferation at multiple levels. The purpose of this study was to measure T cell proliferation and ROI production in SLE versus healthy control T cells. CD4+ and CD8+ T cells were isolated by negative selection magnetic micro-beads. We characterized the memory phenotype and activation markers on the cells from both groups by flow cytometry. CD62Lhigh and CCR7high were the most abundant subsets in CD8+ T cells from healthy subjects. The frequency of CCR7highCD8+ T cells in SLE patients was significantly lowered compared to healthy subjects (p=0.0001). For proliferation and ROI studies, cells were stimulated with anti-CD3 and anti-CD28 antibodies. CFSE staining following activation revealed that SLE CD8+ T cells had decreased division in response to TCR-mediated stimulation as compared to healthy subjects. Both subsets of SLE T cells showed increased ROI in response to activation. Interestingly, among the SLE subjects, the maximum increase in H2O2 production was recorded in the CD8+ T cells of one patient with severe SLE disease. In conclusion, we demonstrate that T cells from SLE patients produce increased levels of ROI upon stimulation implicating ROI in SLE pathogenesis. Supported by NIH RO1-AR39501

PKC-α and TAK-1 are intermediates in the activation ofc-Jun NH2-terminal kinase by hypoxia-reoxygenation

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.