Scavengers Of Reactive Oxygen Intermediates Research Articles

Synthesis, crystallographic and spectroscopic characterization and magnetic properties of dimer and monomer ternary copper(II) complexes with sulfonamide derivatives and 1,10-phenanthroline. Nuclease activity by the oxidative mechanism

Three dinuclear and one mononuclear copper(II)-1,10-phenanthroline ternary complexes, [Cu(L1)(phen)(OH)] 2 ( 1), [Cu(L2)(phen)(OH)] 2·3H 2O ( 2), [Cu(L3)(phen)(OH)] 2 ( 3) and [Cu(L4) 2(phen)(H 2O)] ( 4), with thiadiazole sulfonamide derivative ligands: HL1 ( N-(5-ethyl-1,3,4-thiadiazol-2-yl)naphthalene-1-sulfonamide), HL2 ( N-(5-ethylthio)-1,3,4-thiadiazol-2-yl)-4-methylbenzenesulfonamide), HL3 ( N-(5-ethyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide) and HL4 ( N-(5-ethyl-1,3,4-thiadiazol-2-yl)-4-methylbenzenesulfonamide) have been synthesized and characterized. In the four complexes each copper atom is five-coordinated. The structure of complexes 1, 2 and 3 consists of a dimeric unit with a C2 symmetry axis, where both coppers are bridged by two hydroxo anions. Magnetic measurements show that the dimer complexes are ferromagnetic according to the Cu–O–Cu angles. Cleavage experiments using pUC18 plasmid DNA in the presence of H 2O 2/ascorbic acid as an activating agent show that the title complexes are potent artificial chemical nucleases, the order of efficiency being 3 > 2 ∼ 1 > 4. Control cleavage experiments indicated that the dimer complexes are stronger artificial nucleases than the [Cu(phen) 2] 2+ complex under the same experimental conditions, while the monomer 4 has a lower nuclease activity than the [Cu(phen) 2] 2+ complex. The inhibition of the cleavage process in the presence of reactive oxygen intermediate scavengers suggests that the hydroxyl radical and the superoxide anion are reactive species for the breakage of the DNA strands.

Fluoranthene fumigation and exogenous scavenging of reactive oxygen intermediates (ROI) in evergreen Japanese red pine seedlings ( Pinus densiflora Sieb. et. Zucc.)

Generation of reactive oxygen intermediates (ROI) such as O 2 - , H 2O 2, and OH is known to be a major mechanism of damage in biological systems. This study investigated and compared effectiveness of scavenging ROI generated in fluoranthene (FLU) pre-fumigated Japanese red pine seedlings. Three kinds of eco-physiological assessments were used to express the impact of the different fumigants used inside the green house. Gas exchange measurements showed negative changes induced by 10 μM FLU on Japanese pine seedlings during a 10 d exposure period whilst no negative change was found during a 5 d exposure period. Moreover, during a 14 d FLU exposure incorporating ROI scavengers, results revealed that chlorophyll fluorescence, needle chemical contents and needle dry mass per unit area of the seedlings were affected. The negative effects of FLU on the conifer were dependent on both the dose and period of FLU fumigation. Peroxidase (PERO), superoxide dismutase (SOD) and mannitol (MANN) were all effective scavengers of ROI. MANN scavenged OH, the most lethal of the ROI. For practicable use, MANN is more economical, and may be the best ROI scavenger among the three considered. It can be concluded that efficient scavenging of ROI in biological systems is important to mitigate the negative effects of FLU on Japanese red pine trees.

The Aspergillus fumigatus Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model

Macrophages and neutrophils kill the airborne fungal pathogen Aspergillus fumigatus. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of A. fumigatus challenged by H(2)O(2). Since many of the identified proteins and genes are apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified and designated Afyap1. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Afyap1 gene led to drastically increased sensitivity of the deletion mutant against H(2)O(2) and menadione, but not against diamide and NO radicals. Proteome analysis of the DeltaAfyap1 mutant strain challenged with 2 mM H(2)O(2) indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Afyap1 deletion mutant did not show attenuated virulence in a murine model of Aspergillus infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of A. fumigatus in an immunocompromised host. This conclusion was further supported by the finding that killing of A. fumigatus wild-type and DeltaAfyap1 mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette–Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact‐dependent/nitric oxide‐independent mechanism

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.

TNF-alpha suppresses dendritic cell death and the production of reactive oxygen intermediates induced by plasma withdrawal.

Mature dendritic cells (DCs) were generated by culturing human peripheral blood monocytes for 7 days and, then, treating them with a cytokine cocktail for 2 days. The viability of the mature DCs (Day 9) obtained was approximately 60-70%, and this gradually declined when they were recultured in X-VIVO 15 media containing 2% human plasma (40% viability after 3 days of reculture). DC death accelerated on withdrawing plasma from the culture (20% viability after 3 days). However, the addition of tumor necrosis factor-alpha (TNF-alpha) to the medium completely restored DC viability in the absence of plasma. Such a protective effect was not afforded by other cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1alpha (IL-1alpha), IL-4, IL-6 and prostaglandin E2 which are used for the maturation of DCs. These results indicate that TNF-alpha is specifically required to maintain the viability of mature DCs. The withdrawal of plasma rapidly (within 15 min) elevated cellular levels of reactive oxygen intermediates (ROIs), which have been proposed to regulate the ability of DCs to control inflammatory reactions. The possibility that ROIs act as mediators of DC death was eliminated by the observation that scavengers of ROIs, such as catalase, N-acetylcysteine, glutathione, failed to prolong DC life span in the absence of plasma. Interestingly, TNF-alpha was found to almost completely abolish the production of ROIs induced by plasma withdrawal. To summarize, our results suggest that TNF-alpha controls not only the inflammatory functions of DCs but also their survival.

Effect of Sodium Thiosulfate on the Depletion of Photosynthetic Apparatus in Cyanobacterium Synechocystis sp. PCC 6803 Cells Grown in the Presence of Glucose

Fluorescence spectroscopy at 77 K showed that the application of glucose lead to the depletion of phycobilisomes (PBS) and photosystems (PS) 2 and 1, and that PS2 was more sensitive to glucose than PS1. The application of sodium thiosulfate, an effective scavenger of reactive oxygen intermediates, counteracted the effects of glucose. Sodium thiosulfate effectively protected photosynthetic apparatus, PS2, PS1, and PBS against glucose-induced depletion. Sodium thiosulfate showed strong capability to inhibit the disappearance of chlorophyll induced by glucose. At a relatively low concentration of glucose, the application of sodium thiosulfate can even be helpful for the assembly of photosynthetic apparatus. Hence the reactive oxygen species might be involved in the depletion of the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6803 cells grown in the presence of glucose.

7] Conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients

Publisher Summary This chapter discusses the conversion to mucoidy in pseudomonas aeruginosa, as a potent human opportunistic pathogen causing chronic respiratory infections in cystic fibrosis (CF). The persistent infection of the CF lung is facilitated by a biofilm mode of growth of mucoid, exopolysaccharide alginate-overproducing mutants of P. aeruginosa selected during the process of colonization of the CF respiratory tract. The molecular mechanism of conversion to mucoidy involves two pathways. The more common pathway depends on the activation of the alternative sigma factor Alg U via mutations in the gene termed mucA. In addition to bacterial genetic factors, the host environment contributes to the selection of P. aeruginosa mutants and formation of bio films because of mutations in the human gene encoding the CF transmembrane conductance regulator (CFTR). The alginate biofilm may play a role in resistance to phagocytosis, scavenging of reactive oxygen intermediates, and a number of other biofilm-related phenomena. Alginate biofilms counteract processes of P. aeruginosa elimination via uptake and killing by host macrophages and neutrophils, and potentially by respiratory epithelial cells. This chapter describes the genetic methods, molecular approaches, cell culture techniques, and animal models used to examine P. aeruginosa conversion to mucoid phenotype and biofilm mode of growth and their role in the pathogenesis of CF.

Amifostine: a tonic or toxin to myeloid progenitors

It should come as no surprise, that the aminothiol prodrug, Amifostine (EthyolTM), whose actions as a hematopoietic promoter were recently realized, should now be considered to exert opposing biologic effects in leukemic cells [1]. A product of the United States Defense Department research emanating from the cold war, the relative selective cytoprotection afforded by amifostine to normal cells compared to tumor cells seemed at first glance somewhat difficult to accept. Indeed, relative differences in alkaline phosphatase distribution and pH within the tumor milieu appeared plausible [2–4], but insufficient to explain the agent’s ability to potentiate antineoplastic cytotoxicity in selected malignancies while conferring no protection to leukemic cells and protecting normal tissues [5–8]. The answer to this apparent dichotomy lies in amifostine’s chemical derivation and structural resemblance to endogenous polyamines, abundant but highly regulated organic cations involved in the homeostatic balance of cell death and survival. The dephosphorylated form of amifostine, WR-1065, and its symmetrical disulfide oxidation product, WR33278, display striking homology to spermidine and spermine, respectively [9,10]. In the oxidized extracellular environment, WR-33278 is believed to predominate, gaining entry into cells as a substrate for the polyamine facilitative transporter [11,12]. The activity of these transporters are strictly regulated by the feedback inhibitor, antizyme, which serves as a gate-keeper to limit intracellular polyamine content [13,14]. Within the cell, WR-33278 can be reduced to WR-1065. These novel aminothiols can then modulate cell behavior in two ways. They can compete with the naturally occurring polyamines for binding to DNA [15] and as substrates for amine oxidases [16]. Like endogenous polyamines, they induce the activity of spermine/spermidine acetyl transferase (SSAT), the first enzyme in polyamine catabolism, which is transcriptionally activated by the erythroid-specific transcription factor GATA-1 as an early event in erythroid differentiation [16,17]. In addition, these molecules can activate unique patterns of gene expression via polyamine-independent mechanisms, such as the redox sensitive tumor suppressor gene, p53 [18]. Like the polyamines, the cationic charge of the aminothiols facilitates their avid DNA binding, where they exert their many biologic activities including activation of early response genes, thymidine kinase, NFk-B, and other genes involved in the maintenance of genomic integrity [19–21]. Polyamines and polyamine analogues, like classical thiol antioxidants, afford protection against nuclear oxidation by serving as scavengers of reactive oxygen intermediates [22–24]. How then can we expect divergent effects on cell survival in normal, myelodysplastic and leukemic progenitors? Proliferating tumor cells maintain a high cellular polyamine content via ornithine decarboxylase-controlled biosynthesis; thereby blocking cell import by the induction of antizyme. Significant changes in polyamine homeostasis in tumor cells can be catastrophic, resulting in growth arrest and/or programmed cell death. Although active import of WR33278 is inhibited [25], passive inward diffusion of WR-1065 accelerates polyamine degradation by induction of SSAT, resulting in lowered apoptotic threshold. Sensing exceedingly high intracellular polyamine content, antizyme binds to and destabilizes ornithine decarboxylase, triggering its rapid proteosome degradation [26,27]. In normal cells where polyamine generation is not upregulated, the disulfide is actively imported into the cell, affording added defense against oxidative stress and resistance to apoptosis induction. The latter is indeed the case for normal hematopoietic progenitors,

Activation of murine peritoneal macrophages by Streptococcus pneumoniae type II capsular polysaccharide: involvement of CD14-dependent pathway.

In this study we examined the ability of capsular polysaccharide type 2 (PS) from Streptococcus pnemoniae to induce secretory and cellular responses in peritoneal macrophages. Tumour cytotoxicity induced by preincubation with PS was demonstrated to be concentration-dependent. PS-induced tumouricidal activity was partially abrogated by anti-tumour necrosis factor (TNF)-alpha and inhibitor of nitric oxide, whereas anti-interferon (IFN)-alpha/beta antibody and the scavengers of reactive oxygen intermediates had no effect. In addition, supernatants from macrophages treated with PS contained TNF-alpha, and their iNOS-enzymatic activity was significantly increased. Thus, the tumouricidal activity induced by PS appeared to be mediated by the production of TNF-alpha and nitrite. Treatment of macrophages with PS increased the expression of CD14, the receptor for lipolysaccharide (LPS)/LPS-binding protein. Moreover, blocking antibody to CD14 abrogated partially TNF-alpha and nitrite induction by PS, suggesting that the PS-induced CD14 upregulation was correlated with secretion of TNF-alpha and nitrite. Taken together, these results demonstrate that PS may induce macrophage-secretory and cellular activities, in part, possibly via CD14-dependent pathway.

N-acetylcysteine decreases severity of acute pancreatitis in mice.

Oxidative stress plays a major role in the early stage of acute pancreatitis. This study assessed the effects of N-acetylcysteine (NAC), a reduced glutathione (GSH) provider and a direct scavenger of reactive oxygen intermediates, in the course of acute pancreatitis in mice. Acute pancreatitis (AP) was induced by intraperitoneal (i.p.) injections of cerulein. Mice received NAC (1,000 mg/kg, i.p.) every 3 h, starting either 1 h before the first cerulein injection (prophylactic group) or 1 h after the first cerulein injection (therapeutic group), or i.p. saline injections for controls. Severity of AP was evaluated by histology, serum hydrolase levels, and serum and intrapancreatic levels of MCP-1 and interleukin 6 (IL-6). Pancreatic conjugated dienes and intrapancreatic and intrahepatic GSH levels were measured to assess the local and systemic oxidative processes. Acute pancreatitis was also induced with a CDE diet in controls and mice receiving either both NAC ad libidum in drinking water and 1,000 mg/kg i.p. injection once daily. The severity of pulmonary lesions was assessed by arterial blood gases (pO2) and intrapulmonary myeloperoxidase (MPO content) measurements as well as the survival of mice. The severity of cerulein-induced AP was significantly decreased in the prophylactic group compared with the therapeutic and control groups. Prophylactic administration of NAC also decreased the intrapancreatic levels of conjugated dienes compared with controls. The intrapancreatic and systemic release of MCP- 1 and IL-6 was also decreased in the prophylactic group 3 and 6 hours after AP induction. In addition, NAC pretreatment also reduced hepatic IL-6 production at 3 and 6 hours after starting cerulein challenge. In CDE-induced AP, the severity of lung injury (hypoxemia, MPO content) was decreased, and survival was improved by NAC. NAC administered in a prophylactic protocol limits the severity of experimental acute pancreatitis in mice, as well as its systemic complications and related mortality.

Copper, zinc–superoxide dismutase protects from ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes: the protection is associated with the increased levels of antioxidant enzymes

It has been reported that cellular oxidative stress induces apoptosis. Ultraviolet radiation that generates reactive oxygen intermediates (ROIs) also induces apoptosis. Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Mammalian cells express two isozymes of SOD, copper, zinc–SOD (Cu, Zn–SOD) and manganese–SOD (Mn–SOD). Using SV40-transformed human keratinocytes (SVHK cells), we investigated the role of SODs in the ultraviolet B (UVB) irradiation-induced apoptosis. UVB irradiation decreased transiently Cu, Zn– and Mn–SOD activities and their protein levels, with subsequent recovery to the basal levels by 24 h. The UVB-induced decrease in SOD activity was dose-dependent and the maximal effect was obtained at 75 mJ/cm 2. The decrease in Cu, Zn–SOD was more marked than that in Mn–SOD. The cell death assay, annexin-V/propidium iodide flow cytometry, and DNA fragmentation analysis revealed that UVB irradiation induces apoptosis in SVHK cells. The UVB-induced apoptosis was suppressed by the treatment of antioxidants, catalase, glutathione, and α-tochopherol. The stable transfection of Cu, Zn–SOD expression vectors into SVHK cells was accompanied by the increased activities of antioxidant enzymes, catalase, and glutathione reductase, as well as glutathione and the cells were shown to be more resistant to UVB-induced apoptosis. In contrast, the transfection of Mn–SOD affected neither activities of antioxidant enzymes nor the UVB-induced apoptosis. The transfection of Cu, Zn–SOD antisense oligomers but not sense oligomers into SVHK or Cu, Zn–SOD cDNA-transfected SVHK (C2) cells significantly decreased the antioxidant enzyme activities and increased the UVB-induced apoptosis. On the other hand, the transfection of Mn–SOD antisense oligomers did not affect the UVB-induced apoptosis. These results suggest that the transfection of Cu, Zn–SOD expression vector, which is accompanied by the increased level of antioxidant enzymes, suppresses the UVB-induced apoptosis of SVHK cells.

Requirement of nuclear factor kappaB for the constitutive expression of nitric oxide synthase-2 and cyclooxygenase-2 in rat trophoblasts.

Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.

Different Mechanisms of c-Jun NH2-terminal Kinase-1 (JNK1) Activation by Ultraviolet-B Radiation and by Oxidative Stressors

Irradiation of mammalian cells with ultraviolet-B radiation (UV-B) triggers the activation of a group of stress-activated protein kinases known as c-Jun NH(2)-terminal kinases (JNKs). UV-B activates JNKs via UV-B-induced ribotoxic stress. Because oxidative stress also activates JNKs, we have addressed the question of whether the ribotoxic and the oxidative stress responses are mechanistically similar. The pro-oxidants sodium arsenite, cadmium chloride, and hydrogen peroxide activated JNK1 with slow kinetics, whereas UV-B potentiated the activity of JNK1 rapidly. N-acetyl cysteine (a scavenger of reactive oxygen intermediates) abolished the ability of all oxidative stressors tested to activate JNK1, but failed to affect the activation of JNK1 by UV-B or by another ribotoxic stressor, the antibiotic anisomycin. In contrast, emetine, an inhibitor of the ribotoxic stress response, was unable to inhibit the activation of JNK1 by oxidative stressors. Although UV-A and long wavelength UV-B are the spectral components of the ultraviolet solar radiation that cause significant oxidative damage to macromolecules, the use of a filter to eliminate the radiation output from wavelengths below 310 nm abolished the activation of JNK1 by UV. Our results are consistent with the notion that UV-B and oxidative stressors trigger the activation of JNK1 through different signal transduction pathways.

Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells

Nitric oxide (NO) has been implicated in a number of important brain functions, such as long-term potentiation (LTP) and long-term depression (LTD), and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO production is increased by an inducible enzyme (iNOS), which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide (LPS). This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 and/or cyclooxygenase-2 (e.g., acetyl salicylic acid, SC 58125, L 745337), but is decreased by approx. 50% with PDTC, a scavenger of reactive oxygen intermediates (ROI) that inhibits nuclear factor κB (NF-κB) activation. Furthermore, inhibitors of protein kinase C (PKC) strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component (besides NF-κB) in the signaling pathway regulating iNOS expression in microglia.

Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells.

The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells.

Interferon- γ-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF- α

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF- α on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF- α in the presence of interferon- γ (IFN- γ) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN- γ, no increase in Mn-SOD activity was detected. The induction of IFN- γ-dependent Mn-SOD activity by anti-Fas antibody or TNF- α was concentration-dependent; the maximal effect was observed at 1–10 μg/ml and 5–10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF- α in the presence of IFN- γ resulted in an additive increase in Mn-SOD activity. Although the addition of 12- o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN- γ-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF- α. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN- γ and that TPA augments the process through the activation of protein kinase C.

Exposure of macrophages to an enzymatically inactive macrophage mannose receptor ligand augments killing of Candida albicans.

Macrophages (Mphi) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mphi exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mphi-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mphi were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P < or = 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mphi via ROI, and that iMPO which is released at a site of inflammation induces Mphi-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Engineering stress tolerance in transgenic plants

Research in our laboratory has focused on the analysis of the functions of a variety of enzymes that are involved in the scavenging of reactive oxygen intermediates (ROI) such as superoxide radicals (·O2−) and hydrogen peroxide (H2O2). Recent work has been on transgenic plants that over-express glutathione S-transferases (GST) that also have glutathione peroxidase activity. Transgenic tobacco plants that contain gene constructs that encode two different tobacco GST’s had elevated levels of both GST and GPX activity. Analysis of mature vegetative transgenic tobacco plants that over-express GST/GPX failed to show any increase in paraquat tolerance or protection from photooxidative stress. However, seeds of these GST/GPX-expressing tobacco lines are capable of more rapid germination and seedling growth at low temperatures and at elevated salt concentrations. Reduced levels of lipid peroxidation were noted in GST/GPX-expressing seedling compared to control seedlings under both stressful and non-stressful conditions. In addition, GST/GPX-expressing seedlings significantly accumulated more oxidized glutathione (GSSG) than control seedlings during stress. These characteristics clearly indicate that over-expression of GST/GPX in transgenic seedlings can have substantial effects on their stress tolerance. Furthermore, it appears that this effect is due primarily to the elevated levels of GPX activity.

Effect of arsenite, arsenate, and the herbicide monosodium methyl arsonate (MSMA) on hepatic metallothionein expression and lipid peroxidation in channel catfish

Arsenic exerts its toxicity by the generation of reactive oxygen intermediates which cause lipid peroxidation and cellular damage. Metallothioneins (MTs) have been shown to be induced by oxidative stress and act as scavengers of reactive oxygen intermediates. Thus, hepatic MT was examined in channel catfish treated with the herbicide monosodium methyl arsonate (MSMA) and compared to equal doses of trivalent and pentavalent arsenic. Fish were exposed to 0.01, 0.1, and 1.0 mg/L of each compound for 1 week by static renewal. Hepatic MT was measured by the cadmium/hemoglobin ( Cd Hb ) saturation assay, ELISA using antibodies raised against the first 10 amino acids of piscine MT, and Northern blot analysis using a cDNA encoding winter flounder hepatic MT. Cd Hb and ELISA measurements of low molecular weight fractions from the hepatic cytosolic component of fish exposed to MSMA revealed a dose dependent increase in MT. MTs and MT mRNA of fish receiving the 1.0 mg/L dose were significantly induced vs control. Responses to arsenate exposure were more variable, but showed a trend toward a dose-dependent induction of MT and MT mRNA. MT mRNA and protein also showed a dose-dependent increase with arsenite exposure with no significant differences with untreated animals. Hepatic lipid peroxidation (as determined by TBARS) and glutathione was unaltered by any of the arsenical treatments. Thus, the lack of correlation between oxidative stress and MT expression suggest MT may not be a reliable indicator of oxidative stress. In addition, the induction of hepatic MT by various forms of As does not appear to be mediated through an oxidative stress mechanism in the liver.