Reactive Oxygen Species Quencher Research Articles

NOX2 interacts with podocyte TRPC6 channels and contributes to their activation by diacylglycerol: essential role of podocin in formation of this complex

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetyl-sn-glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-β-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.

Methyl anthranilate as generator and quencher of reactive oxygen species: A photochemical study

Abstract Methyl anthranilate (MA) is a naturally occurring aromatizer and flavorant agent frequently employed in food industry and in personal care products. Due to the property of MA of being a UV-A radiation-absorber it is incorporated to topical sunscreen formulations. Within the framework of the wide variety of practical uses and applications, the photodegradation of MA becomes a topic of interest from health to an economic point of view. In this context, kinetics and mechanistic aspects of its photo-oxidation has been studied, in order to know the degradation pathways of MA under natural-like photoirradiation. The assays were performed in the absence and in the presence of riboflavin (Rf, vitamin B2) acting the later as a native photosensitizer. The results showed that MA is capable to generate singlet molecular oxygen (O2(1Δg)) upon direct UV-A irradiation, with a quantum yield value of 0.13 ± 0.02. The anthranilate also quenches the oxidative species with an overall rate constant value kt = 2.1 ± 0.2 × 107 M−1 s−1 while the reactive interaction between MA and O2(1Δg) showed a rate constant value in the range of 5.9–6.8 × 106 M−1 s−1. The kr/kt ratio, lower than 0.35 indicates that only one third of the O2(1Δg)-quenching events effectively lead to oxidation of the scavenger, being this an acceptable property for a sunscreen component. Regarding the Rf-photosensitized visible-light irradiation of MA–Rf mixtures, the anthranilate quenches the electronically excited singlet and triplet states of the vitamin. As a result the oxidative species O2 − and H2O2, are generated, being the later effectively deactivated by MA. At first, the described results would show MA as a candidate for UV-A and visible-light mediated degradation. Nevertheless, at the same time MA acts as a moderate physical scavenger of photogenerated oxidative species in a sort of self-protector against photoinduced degradation and protector of oxidizable surrounding compounds.

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole‐resistant Candida albicans strains

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm⁻²). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log₁₀ CFU reduction (0.5 McFarland) was 0.62 μmol l⁻¹ for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l⁻¹. Increasing the fluence to 37 J cm⁻² allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log₁₀ reduction (4 McFarland). H₂O₂ was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas ¹O₂ was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.

A comparative photochemical study on the behavior of 3,3′-dihydroxyflavone and its complex with La(III) as generators and quenchers of reactive oxygen species

A 1:1 complex between 3,3′-dihydroxyflavone (DHF) and La(III) (DHF–La(III)) is formed in methanolic solution with the relatively high apparent stability constant value of 2.3×106 and a calculated standard entropy change of 88.2Jmol−1K−1, both at 25°C.The photophysical properties of the complex and the free flavonoid are discussed in comparison to the well known related compound 3-hydroxyflavone. The ligand photogenerates O2(1Δg) by energy transfer from its excited triplet state (3DHF*) to dissolved ground state oxygen, with a quantum yield of 0.13. 3DHF* is quenched by La(III) with a rate constant close to the diffusion-controlled value.The respective abilities of the free flavonoid and DHF–La(III) as quenchers of the riboflavin-photogenerated reactive oxygen species singlet molecular oxygen (O2(1Δg)) and superoxide radical anion (O2-) have been investigated. Both individual compounds were photoirradiated with visible light in the presence of the flavin as the only light-absorbing compound. A detailed kinetics and mechanistic study employing polarographic monitoring of oxygen uptake and time resolved detection of O2(1Δg) phosphorescence indicates that DHF and the complex react with O2(1Δg) and O2- by a non simple mechanism. The former deactivates O2(1Δg) in a predominant physical fashion, a fact that constitutes a desirable property for antioxidants. It was found that metal chelation greatly enhances the ability of DHF as an overall O2(1Δg) quencher.

Abstract 163: Common food additive carrageenan activates wnt signaling pathway in mouse colon and human colonic epithelial cells

Abstract The Wnt-β-catenin signaling pathway plays a pivotal role in development and disease. It is crucial for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division. Recent studies suggest that self-renewal of stem/progenitor cells by the canonical Wnt cascade is subverted in cancer cells to allow malignant proliferation in intestine. Carrageenans are highly sulfated polysaccharides, which are widely used as food additives to improve the texture of processed foods, and are consumed at levels of about 250 mg/day in the average diet. Reports of many investigators have demonstrated that carrageenan induces inflammation, colonic ulcerations, polyps, and colorectal tumors. We have reported distinct pathways by which carrageenan induces the activation of NF-κB and secretion of IL-8, mediated through TLR4-BCL10 or reactive oxygen species (ROS) in colonic epithelial cells. Our previous work also indicated that carrageenan exposure caused activation of the Wnt-β-catenin pathway in colonic epithelial cells. This investigation was undertaken to elucidate the Wnt signaling mechanisms activated by carrageenan exposure, using NCM460 (INCELL) human colonic epithelial cells exposed to carrageenan (1 μg/ml) x 24 h and colonic tissue from ten-week old C57BL/6J mice exposed to carrageenan (10 - 100 μg/ml) in their water for four weeks. Nuclear β-catenin in NCM460 cells and mouse colonic tissue was determined by Western blot and ELISA and increased to two times the baseline level following carrageenan. This increase in nuclear β-catenin in NCM460 cells was inhibited by the ROS quencher Tempol, but not by TLR4 or BCL10 siRNA. TCF/Lef-1 reporter assay demonstrated increased nuclear binding post carrageenan treatment in the mouse colon (to 4x baseline) and in the NCM460 cells (to 6.3x baseline). Also, mRNA expression of cyclin D1 and wnt9a was increased more than two-fold, and BMP4 expression was decreased. In the NCM460 cells, these responses to carrageenan were inhibited by Tempol, but not BCL10 silencing. Also, carrageenan-induced increases in ROS and lipid peroxidation were inhibited by Tempol. Since Wnt signaling is affected by free dishevelled 2 (dsh2), and dsh2 binding to nucleoredoxin (NRX) is greater when NRX is in its reduced state, we tested whether carrageenan exposure could impact upon the Wnt signaling pathway by modifying the redox status of NRX. The ratio of free NRX to NRX bound to dsh2 increased to six times baseline following carrageenan, and was reversed by Tempol. These profound effects of carrageenan on the Wnt signaling pathway in colonic cells can help to explain how diet can contribute to development of colonic neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 163. doi:1538-7445.AM2012-163

Uncoupling Protein 2 Negatively Regulates Mitochondrial Reactive Oxygen Species Generation and Induces Phosphatase-Mediated Anti-Inflammatory Response in Experimental Visceral Leishmaniasis

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.

ROS Quenching Potential of the Epidermal Cornified Cell Envelope

The cornified cell envelope (CE) is a specialized structure assembled beneath the plasma membrane of keratinocytes in the outermost layers of the epidermis. It is essential for the physical and permeability properties of the barrier function of the skin. Our skin is continuously exposed to atmospheric oxygen and threatened by reactive oxygen species (ROS). Here, we identify the CE as a first line of antioxidant defense and show that the small proline-rich (SPRR) family of CE precursor proteins have a major role in ROS detoxification. Cysteine residues within these proteins are responsible for ROS quenching, resulting in inter- and intramolecular S-S bond formation, both in isolated proteins and purified CEs. The related keratinocyte proline-rich protein is also oxidized on several cysteine residues within the CE. Differences in antioxidant potential between various SPRR family members are likely determined by structural differences rather than by the amount of cysteine residues per protein. Loricrin, a major component of the CE with a higher cysteine content than SPRRs, is a weak ROS quencher and oxidized on a single cysteine residue within the CE. It is inferred that SPRR proteins provide the outermost layer of our skin with a highly adaptive and protective antioxidant shield.

Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii

In many phototrophic microorganisms and plants, chloroplasts change their positions relative to the incident light to achieve optimal photosynthesis. In the case of motile green algae, cells change their swimming direction by switching between positive and negative phototaxis, i.e., swimming toward or away from the light source, depending on environmental and internal conditions. However, little is known about the molecular signals that determine the phototactic direction. Using the green alga Chlamydomonas reinhardtii, we found that cellular reduction-oxidation (redox) poise plays a key role: Cells always exhibited positive phototaxis after treatment with reactive oxygen species (ROS) and always displayed negative phototaxis after treatment with ROS quenchers. The redox-dependent switching of the sign of phototaxis may contribute in turn to the maintenance of cellular redox homeostasis.

Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by CD70 signaling is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway (66.23)

Abstract CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70+ tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from mitochondria and cleavage of caspases. CD70-induced apoptosis was inhibited by ER stress inhibitor salubrinal, ROS quencher NAC, and Ca2+ chelator BAPTA. We supposed that ROS generation might be the first event because NAC blocked increases of ROS and Ca2+, but BAPTA did not block ROS. We also found that CD70 stimulation activated JNK and p38. JNK inhibitor and p38 inhibitor blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation blocked phosphorylation of JNK and p38 and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBVt- B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

Adding ROS Quenchers to Cold K+Cardioplegia Reduces Superoxide Emission During 2-Hour Global Cold Cardiac Ischemia

We reported that the combination of reactive oxygen species (ROS) quenchers Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), catalase, and glutathione (MCG) given before 2 hours cold ischemia better protected cardiac mitochondria against cold ischemia and warm reperfusion (IR)-induced damage than MnTBAP alone. Here, we hypothesize that high K(+) cardioplegia (CP) plus MCG would provide added protection of mitochondrial bioenergetics and cardiac function against IR injury. Using fluorescence spectrophotometry, we monitored redox balance, ie reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide (NADH/FAD), superoxide (O(2) (•-)), and mitochondrial Ca(2+) (m[Ca(2+)]) in the left ventricular free wall. Guinea pig isolated hearts were perfused with either Krebs Ringer's (KR) solution, CP, or CP + MCG, before and during 27°C perfusion followed immediately by 2 hours of global ischemia at 27°C. Drugs were washed out with KR at the onset of 2 hours 37°C reperfusion. After 120 minutes warm reperfusion, myocardial infarction was lowest in the CP + MCG group and highest in the KR group. Developed left ventricular pressure recovery was similar in CP and CP + MCG and was better than in the KR group. O(2) (•-), m[Ca(2+)], and NADH/FAD were significantly different between the treatment and KR groups. O(2) (•-) was lower in CP + MCG than in the CP group. This study suggests that CP and ROS quenchers act in parallel to improve mitochondrial function and to provide protection against IR injury at 27°C.

The role of the pyridoxine (vitamin B6) biosynthesis enzyme PDX1 in ultraviolet-B radiation responses in plants.

Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B(6)) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type.

A dual role of selenium in the growth control of seedlings of Stylosanthes humilis

The growth of seedlings of Townsville sytlo (Stylosanthes humilis H.B.K.) is inhibited by aluminium (Al) ions, their elongation being re-covered with sodium selenate at 1.0 µM. Methyl viologen and hydrogen peroxide, reactive oxy-gen species (ROS)-generating compounds, also inhibited seedling elongation and again growth was relieved by selenate. Selenate, thus, seemed to be operating as a ROS quencher, since N-acetylcysteine (NAC), an antioxidant com-pound, also stimulated largely the growth of Al-inhibited seedlings. At a higher concentra-tion (0.1 mM), however, selenate inhibited seed-ling growth and elongation was recovered by NAC. Ethylene production by selenate plus NAC-treated seedlings was very higher and thus the gaseous hormone was not responsible for the seedling growth inhibition caused by sele-nate. Hence, it seems that at high levels sele-nate operates as a ROS-generating compound whose effects were counteracted by NAC. It can be deduced that, at low concentration, sele-nates behave as a ROS quencher and at high level as a ROS-promoting species.

Endoplasmic Reticulum Stress-Mediated Apoptosis of EBV-Transformed B Cells by Cross-Linking of CD70 Is Dependent upon Generation of Reactive Oxygen Species and Activation of p38 MAPK and JNK Pathway

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 2. Reaction Mechanism Investigated by Product Analysis and Deuterium Isotope Effect

Transmetalated (Pd) bacteriochlorophyll derivatives are currently being clinically tested as sensitizers for photodynamic therapy. Protocols using short delay times between injection and irradiation generate interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the mechanism of photo-oxidation in two lipoprotein fractions, low- and high-density lipoproteins, derived from human blood plasma that preferentially accumulate these pigments (Dandler et al. [2009] Photochem. Photobiol., 85, in press). Using quenchers of reactive oxygen species, and chemical reporters, in particular peroxides generated from cholesterol as an inherent component of the lipoproteins, a Type II mechanism generating singlet oxygen has been demonstrated for Pd- and Zn-bacteriopheophorbides. In homogeneous systems, accelerated bleaching in D(2)O, compared with H(2)O, supports this mechanism. An unusual deuterium isotope effect was observed, by contrast, in heterogeneous amphiphilic-water systems. In the early phase, and under high oxygen concentrations, again a positive D-isotope effect is observed which later, in a second phase, is reversed to a negative D-isotope effect. The latter cannot be explained by heterogeneous pigment populations in the amphiphilic system; we, therefore, conclude a mechanistic switch, and discuss a possible mechanism.

Fungicidal activity of miconazole against Candida spp. biofilms

Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined. The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3. Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis. ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.

Overexpression of rice isoflavone reductase-like gene (OsIRL) confers tolerance to reactive oxygen species

Isoflavone reductase is an enzyme involved in isoflavonoid biosynthesis in plants. However, rice isoflavone reductase-like gene (OsIRL, accession no. AY071920) has not been unraveled so far. Here, we have characterized its behavior in response to oxidizing agents. Using Northern and Western blot analyses, the OsIRL gene and protein were shown to be down-regulated in young seedling roots treated with reduced glutathione (GSH) and diphenyleneiodonium (DPI), known quenchers of reactive oxygen species (ROS). The OsIRL transcript level in rice suspension-cultured cells was also found to be induced by oxidants such as hydrogen peroxide (H(2)O(2)), ferric chloride (FeCl(3)), methyl viologen (MV) and glucose/glucose oxidase (G/GO), but down-regulated when co-treated with GSH. Furthermore, to investigate whether overexpression of OsIRL in transgenic rice plants promotes resistance to ROS, we generated transgenic rice lines overexpressing the OsIRL gene under an abscisic acid (ABA) inducible promoter. Results showed that the OsIRL transgenic rice line activated by ABA treatment was tolerant against MV and G/GO-induced stress in rice leave and suspension-cultured cells. Our results strongly suggest the involvement of OsIRL in homeostasis of ROS.

Analysis of a Horizontally Transferred Pathway Involved in Vitamin B6 Biosynthesis from the Soybean Cyst Nematode Heterodera glycines

Heterodera glycines is an obligate plant parasite capable of biochemically and developmentally altering its host's cells in order to create a specialized feeding cell. Although the exact mechanism of feeding cell morphogenesis remains a mystery, the nematode's ability to manipulate the plant is thought to be due in part to horizontal gene transfers (HGTs). A bioinformatic screen of the nematode genome has revealed homologues of the genes SNZ and SNO, which comprise a metabolic pathway for the de novo biosynthesis of pyridoxal 5'-phosphate, the active form of vitamin B(6) (VB(6)). Analysis of the 2 genes, HgSNZ and HgSNO, show that they contain nematode-like introns, generate polyadenylated mRNAs, and map to the soybean cyst nematode genetic linkage map, indicating that they are part of the nematode genome. However, gene synteny, protein homology, and phylogenetic evidence suggest prokaryotic origin. This would represent the first case of the HGT of a complete pathway into a nematode or terrestrial animal. VB(6) acts as a cofactor in over 140 different enzymes, and recent studies point toward an important role as a potent quencher of reactive oxygen species. With H. glycines' penchant for acquiring parasitism genes through HGT along with the absence of this pathway in other land-based animals suggests a specific need for VB(6) which may involve the parasite-host interaction.

Comparison of the Abilities of Ambient and Manufactured Nanoparticles To Induce Cellular Toxicity According to an Oxidative Stress Paradigm

Nanomaterial properties differ from those bulk materials of the same composition, allowing them to execute novel activities. A possible downside of these capabilities is harmful interactions with biological systems, with the potential to generate toxicity. An approach to assess the safety of nanomaterials is urgently required. We compared the cellular effects of ambient ultrafine particles with manufactured titanium dioxide (TiO2), carbon black, fullerol, and polystyrene (PS) nanoparticles (NPs). The study was conducted in a phagocytic cell line (RAW 264.7) that is representative of a lung target for NPs. Physicochemical characterization of the NPs showed a dramatic change in their state of aggregation, dispersibility, and charge during transfer from a buffered aqueous solution to cell culture medium. Particles differed with respect to cellular uptake, subcellular localization, and ability to catalyze the production of reactive oxygen species (ROS) under biotic and abiotic conditions. Spontaneous ROS production was compared by using an ROS quencher (furfuryl alcohol) as well as an NADPH peroxidase bioelectrode platform. Among the particles tested, ambient ultrafine particles (UFPs) and cationic PS nanospheres were capable of inducing cellular ROS production, GSH depletion, and toxic oxidative stress. This toxicity involves mitochondrial injury through increased calcium uptake and structural organellar damage. Although active under abiotic conditions, TiO2 and fullerol did not induce toxic oxidative stress. While increased TNF-alpha production could be seen to accompany UFP-induced oxidant injury, cationic PS nanospheres induced mitochondrial damage and cell death without inflammation. In summary, we demonstrate that ROS generation and oxidative stress are a valid test paradigm to compare NP toxicity. Although not all materials have electronic configurations or surface properties to allow spontaneous ROS generation, particle interactions with cellular components are capable of generating oxidative stress.

UVA1 radiation (340–400 nm) interferes with the perforin-granule system of CD8 hi+ cytotoxic T lymphocytes in vitro

UVA1-irradiation was introduced as an innovative and effective phototherapy of atopic dermatitis (AD) and other skin diseases. In AD, a defect of a central apoptosis inducing effector system involved in immunoregulation and immune defense, i.e., the system of perforin-granules in cytotoxic T lymphocytes (CTL), was recently reported: perforin-reduction and perforin-hyperreleasability. We now investigated UVA1-effects on the perforin-granule system in vitro. Peripheral blood CTLs were exposed in vitro to 10–100 J/cm 2 UVA1 (340–400 nm), and to 30–150 mJ/cm 2 UVB (280–315 nm) as a control. A time-dependent perforin-granule release was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin. This release was inhibited dose-dependently by UVA1, but not by UVB. An UVA1-dose dependent pattern of sensitive (80%) and insensitive (20%) individuals was found. The kinetics of perforin release in AD-CTLs, i.e. hyperreleasability, was normalized by 50 J/cm 2 UVA1 in vitro. Sodium azide as a quencher of reactive oxygen species prevented the UVA1-mediated inhibition of perforin-granule release. Our data demonstrate for the first time a dose- and wavelength-dependent UVA1-effect in vitro on a major effector system of cytotoxic lymphocytes, the system of perforin-granules. This might contribute to the further understanding of immunomodulatory UVA1-effects in vivo.