活性氧诱发人类11号染色体基因突变

对体外产生的和内源性刺激产生的活性氧 (ROS)诱发人类 11号染色体 (Hchr 11)基因突变规律及其突变谱进行研究 .体外羟自由基 (·OH)用过氧化氢 (H2 O2 )与Fe2 + 反应产生 ,并用化学发光(CL)进行相对定量分析 ;内源性ROS用佛波醇酯 (PMA)刺激人外周血白细胞产生 ,并用CL和特异性抗氧化物检测和鉴定 ;用包含单条Hchr 11的人 中国仓鼠卵巢细胞 (AL)为靶 ,经CD59表面抗原抗体筛选突变细胞克隆 ,研究ROS诱发的Hchr 11基因突变 ;突变克隆细胞DNA用Hchr 11上 5种标志基因引物进行多重PCR分析 ,结合琼脂糖凝胶电泳绘制基因突变谱 .结果表明 ,体外ROS可诱发Hchr 11基因突变 ,且·OH诱发基因突变的能力明显强于H2 O2 ,两者的突变谱也存在明显差异 ;PMA可刺激人外周血白细胞产生大量的多种ROS ,并诱发Hchr 11基因突变 ,突变谱综合了H2 O2 和·OH的所有特征 ;一些抗氧化物对内源性产生的ROS诱发Hchr 11基因突变有明显抑制作用 .提示体外和内源性ROS可诱发Hchr 11基因突变 ,不同的活性氧分子诱发的基因突变可能具有特异性     

叶绿体中活性氧的产生和清除机制

正常情况下植物细胞内活性氧（reactive oxygen species ROS）的产生和清除是平衡的,但是,一旦植物遭受环境胁迫,ROS的积累超过抗氧化剂防护系统清除能力,就会产生氧胁迫损伤细胞。由于叶绿体作为光合作用的场所与其他细胞器相比更易遭受氧化胁迫的伤害。因此,叶绿体进化了更强的防御机制调控电子传递链的氧化还原平衡及叶绿体基质中的氧化还原状态。活性氧具有双重效应．高浓度的活性氧对植物细胞有很强的毒害作用,低浓度时可充当信号分子参与植物的某些防卫反应过程,本文就叶绿体中活性氧的产生（三线态叶绿素、PSI和PSI I电子传递链）、网络清除（抗氧化剂,SOD,As—Glu循环系统,硫氧还蛋白）机制以及功能作用进行了综述。     

信号配体诱导的活性氧生成

活性氧(reactiveoxygenspecies,ROS)是生物体内一类活性含氧化合物的总称,主要包括超氧阴离子、羟自由基和过氧化氢等。细胞内有多种部位能生成ROS,主要包括线粒体、内质网、NADPH氧化酶复合体、脂氧合酶系、环氧合酶系等。静息条件下,细胞内ROS的水平被控制在很低的范围。而在细胞受到各种生理或病理因素作用时,当多种细胞外信号分子作用于其膜受体,ROS生成可以受到受体活化的诱导而“有目的”地快速增加,从而作为细胞内信号分子参与细胞增殖,分化和凋亡等各种细胞行为。     

Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions

The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O₂] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O₂]. In the current study, we used a wide variety of substrates and inhibitors to probe the O₂ sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent K_m for O₂ of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 μm O₂ for the complex I flavin site, complex I electron backflow, complex III Q_O site, and electron transfer flavoprotein quinone oxidoreductase of β-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O₂]. Based on these data, we hypothesize that at physiological [O₂], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O₂]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O₂]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.The production of reactive oxygen species (ROS)² by mitochondria has been implicated in numerous disease states, including but not limited to sepsis, solid state tumor survival, and diabetes (1). In addition, mitochondrial ROS (mtROS) play key roles in cell signaling (reviewed in Refs. 2 and 3). There exist within mitochondria several sites for the generation of ROS, with the most widely studied being complexes I and III of the electron transport chain (ETC). However, there is currently some debate regarding the relative contribution of these complexes to overall ROS production (4–9) and the factors that may alter this distribution. One such factor considered herein is [O₂]. Estimates of physiological [O₂] within tissues (i.e. interstitial [O₂]) range from 37 down to 6 μm at 5–40 μm away from a blood vessel (10). More recently, EPR oximetry has estimated tissue [O₂] to be in the 12–60 μm range (11). In addition, elegant studies with hepatocytes have shown that O₂ gradients exist within cells, such that an extracellular [O₂] of 6–10 μm yields an [O₂] of ∼5 μm close to the plasma membrane, dropping to 1–2 μm close to mitochondria deep within the cell (12). In cardiomyocytes, at an extracellular [O₂] of 29 μm, intracellular [O₂] varied in the range 6–25 μm (13). Clearly, different tissues consume O₂ at different rates, so these gradients can vary considerably between tissue and cell types.By definition, the generation of reactive oxygen species by any mechanism, is an O₂-dependent process. However, measurements in intact cells have indicated that mtROS generation increases at lower O₂ levels (1–5% O₂) (14). Proponents of an increase in mtROS in response to hypoxia suggest that under such conditions, reduction of the ETC results in increased leakage of electrons to O₂ at the Q_O site of complex III (14). Such a model posits that increased hypoxic ROS is a mitochondria-autonomous signaling mechanism (i.e. it is an inherent property of the mitochondrial ETC). Therefore, mtROS generation should increase in hypoxia regardless of the experimental system being studied, including isolated mitochondria. In contrast to this hypothesis, we and others have demonstrated that ROS generation by mitochondria is a positive function of [O₂] across a wide range of values (0.1–1000 μm O₂) (15–18), suggesting that signaling mechanisms external to mitochondria may be required to facilitate the increased hypoxic mtROS production observed in cells.One limitation of our previous work (15) was that only a single respiratory condition was studied, namely succinate as respiratory substrate (feeding electrons into complex II) plus rotenone to inhibit backflow of electrons through complex I (5, 7). The possibility exists that under different metabolic conditions, which may lead to differential redox states between the cytochromes in the ETC (19, 20), ROS generation may exhibit a different response to [O₂]. Thus, in the current study, we examined the response of mtROS generation to [O₂] under 11 different conditions, using a variety of respiratory substrates and inhibitors (for a thorough review of electron entry points to the ETC under various substrate/inhibitor combinations, see Ref. 21). Fig. 1 shows a schematic of the mitochondrial ETC, highlighting sites of electron entry resulting from various substrates, binding sites of inhibitors, and major sites of ROS generation. Fig. 2 shows the specific details of each experimental condition, indicating the predicted sites of ROS generation resulting from the use of each substrate/inhibitor combination. The legend to Fig. 2 provides an explanation of each condition.Open in a separate windowFIGURE 1.Mitochondrial pathways of electron flow resulting from the substrates and inhibitors used in this study. Substrates used were glutamate/malate (which generates NADH via the tricarboxylic acid cycle, feeding into complex I), succinate (which feeds electrons directly into complex II), and palmitoyl-carnitine (which feeds electrons into the ETC via acyl-CoA dehydrogenase as well as through the β-oxidation pathway). (For a more thorough explanation, refer to Ref. 21.) Inhibitors used were rotenone (which inhibits at the downstream Q binding site of complex I (9)), malonate (a competitive inhibitor of complex II (25, 26)), and antimycin A (a complex III inhibitor that prevents electron flow to the Q_I site of complex III, thus stabilizing QH^˙ at the Q_O site (6, 28)).Open in a separate windowFIGURE 2.Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO_{2 max}; nmol of O₂/min/mg of protein) measured under each condition. A, glutamate/malate/malonate. Electrons enter through complex I, whereas electron entry at complex II is inhibited by malonate. ROS generation occurs at the FMN site of complex I as well as the Q_O site of complex III. B, glutamate/malate/malonate/rotenone. Electrons enter through complex I. Electron passage through complex I is inhibited by rotenone binding at the downstream Q site, resulting in maximal ROS production at the FMN site of complex I. ROS production at the Q_O site of complex III is prevented due to no electrons reaching the complex from either complexes I or II, both of which are inhibited. C, glutamate/malate/malonate/antimycin A. Electrons enter through complex I only, since complex II is blocked. Flow of electrons is inhibited by the complex III inhibitor antimycin A, resulting in ROS production at the Q_O site of complex III, as well as the FMN site of complex I. D, succinate. Electrons enter at complex II. ROS is generated by the flow of electrons though the Q_O site of complex III as well as the backflow of electrons through complex I. E, succinate/rotenone. Electrons enter at complex II, and ROS is generated at the Q_O site of complex III, because rotenone is present to inhibit backflow of electrons through complex I. F, succinate/antimycin A. Electrons enter through complex II. ROS is generated at both complex I via backflow and complex III Q_O, with an increased rate at the latter due to inhibition by antimycin A. G, succinate/rotenone/antimycin A. Electrons enter through complex II. Backflow of electrons through complex I is inhibited by rotenone, whereas ROS generation at complex III Q_O is augmented due to the presence of antimycin A. H, glutamate/malate/succinate. Electrons enter at both complexes I and II. ROS is generated from the complex I FMN site and the complex III Q_O site. J, glutamate/malate/succinate/antimycin A. Electrons enter at complexes I and II. ROS generation occurs at the complex I FMN and is augmented at the complex III Q_O site by antimycin A. K, palmitoyl-carnitine. Electrons enter at the ETFQOR. ROS is generated at the ETFQOR as well as complex I via backflow and at the complex III Q_O site. L, palmitoyl-carnitine/rotenone. Electron entry is at the ETFQOR. ROS is generated at the ETFQOR as well as at the complex III Q_O site, whereas ROS due to complex I backflow is blocked by rotenone. Glu, glutamate; Mal, malate; Suc, succinate; PC, palmitoyl-carnitine; Rot, rotenone; AntiA, antimycin A; Malon, malonate.The results of these studies indicated that although ROS generation under all experimental conditions exhibited the same overall response to [O₂] (i.e. hyperbolic, with decreased ROS at low [O₂]), the apparent K_m for O₂ varied widely between metabolic states.     

The Influence of Reactive Oxygen Species on the Adhesion of Pancreatic Carcinoma Cells to the Peritoneum

Postoperative peritoneal carcinomatosis is a significant clinical problem after “curative” resection of pancreatic carcinoma. Preoperative surgical trauma activates a cascade of peritoneal defense mechanisms responsible for postoperative intra-abdominal tumor recurrence. Reactive oxygen species (ROS) play a pivotal role in this postoperative inflammatory reaction. This study explores the influence of ROS on adhesion of human pancreatic carcinoma cells to human mesothelial cells. Furthermore this study explores the influence of ROS on the presentation of adhesion molecules on Panc-1 and mesothelial cells. ROS were produced using the enzymatic reaction of xanthine with xanthine oxidase (X/XO). A reproducible in vitro assay to study adhesion of human Panc-1 carcinoma tumor cells to a mesothelial cell monolayer of primary human mesothelial cells was used. Mesothelial monolayers were incubated with ROS produced prior to adhesion of the tumor cells. Incubation of the mesothelial cells with X/XO resulted in a significant increase (69.5%) in adhesion of Panc-1 in all patients. SOD/catalase, anti-oxidants, could reduce this increase by 56.7%. ROS significantly influenced the expression of the adhesion molecules ICAM-1, VCAM-1 and CD44h on mesothelial cells, but did not influence adhesion molecule expression on Panc-1. The ROS released during the post-operative inflammatory reaction may play an important role in the adhesion of pancreatic tumor cells to the mesothelium-possibly by influencing adhesion molecule expression on mesothelial cells. Therefore ROS can partly be responsible for the enhanced post-operative intra-abdominal tumor recurrence.Key words: reactive oxygen species, mesothelium, Panc-1     

The Influence of Reactive Oxygen Species on the Adhesion of Pancreatic Carcinoma Cells to the Peritoneum

Postoperative peritoneal carcinomatosis is a significant clinical problem after “curative” resection of pancreatic carcinoma. Peroperative surgical trauma activates a cascade of peritoneal defense mechanisms responsible for postoperative intra-abdominal tumor recurrence. Reactive oxygen species (ROS) play a pivotal role in this postoperative inflammatory reaction. This study explores the influence of ROS on adhesion of human pancreatic carcinoma cells to human mesothelial cells. Furthermore this study explores the influence of ROS on the presentation of adhesion molecules on Panc-1 and mesothelial cells. ROS were produced using the enzymatic reaction of xanthine with xanthine oxidase (X/XO). A reproducible in-vitro assay to study adhesion of human Panc-1 carcinoma tumor cells to a mesothelial cell monolayer of primary human mesothelial cells was used. Mesothelial monolayers were incubated with ROS produced prior to adhesion of the tumor cells. Incubation of the mesothelial cells with X/XO resulted in a significant increase (69.5%) in adhesion of Panc-1 in all patients. SOD/catalase, anti-oxidants, could reduce this increase by 56.7%. ROS significantly influenced the expression of the adhesion molecules ICAM-1, VCAM-1 and CD44h on mesothelial cells, but did not influence adhesion molecule expression on Panc-1. The ROS released during the post-operative inflammatory reaction may play an important role in the adhesion of pancreatic tumor cells to the mesothelium. Possibly by influencing adhesion molecule expression on mesothelial cells. Therefore ROS can partly be responsible for the enhanced post-operative intra-abdominal tumor recurrence.     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

活性氧调控植物细胞自噬的研究进展

真核生物通过双层膜结构包裹细胞内受损的蛋白、细胞器或外源物质, 经溶酶体(或液泡)将内含物降解并进行循环利用, 这种高度保守的生物学过程称为自噬。活性氧是细胞有氧代谢的副产物, 作为一种信号分子广泛参与不同生物学过程的调控。研究表明, 真核生物中自噬与活性氧之间存在密切联系。该文结合近年的研究进展, 对植物细胞中活性氧的种类及作用和自噬的分子机制等进行概述, 旨在探讨活性氧对自噬的调控作用。     

Decreased Reactive Oxygen Species Production in Cells with Mitochondrial Haplogroups Associated with Longevity

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function. Zhang and colleagues found a strikingly higher frequency of a C150T transition in the D-loop of mtDNA from centenarians and twins of an Italian population, and also demonstrated that this base substitution causes a remodeling of the mtDNA 151 replication origin in human leukocytes and fibroblasts [1]. The C150T transition is a polymorphism associated with several haplogroups. To determine whether haplogroups that carry the C150T transition display any phenotype that may be advantageous for longevity, we analyzed cybrids carrying or not the C150T transition. These cybrids were obtained by fusing cytoplasts derived from human fibroblasts with human mtDNA-less cells (ρ⁰ cells). We chose for cybrid construction and analysis haplogroup-matched pairs of fibroblast strains containing or not the C150T transition. In particular, we used, as one pair of mtDNA donors, a fibroblast strain of the U3a haplogroup, carrying the C150T transition and a strain of the U-K2 haplogroup, without the C150T transition, and as another pair, fibroblasts of the J2b haplogroup, carrying the C150T transition and of the J1c haplogroup, without the C150T transition. We have found no association of respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate with the presence of the C150T transition. However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids examined.     

Generation of Active Oxygen Species in Blood Platelets—Spin Trapping Analysis

Generation of active oxygen species by bovine blood platelets was examined by the electron spin resonance (ESR) spin trapping technique with 5,5-dimethyl-l-pyroline-l-oxide (DMPO). The hydroxyl spin-trapped adduct 5,5-dimethyl-2-hydroxy-l-pyrolidinyloxy (DMPO-OH) was formed in the presence of platelets, indicating the generation of hydroxyl radicals (· OH) by the platelets. Generation of · OH was observed even with platelets in the resting state, but was markedly enhanced when the platelets were activated with stimulants. Stronger stimulants such as the calcium ionophore ionomycin, induced greater radical gener-ation than the weaker stimulant ADP. When the platelets were stimulated by thrombin, generation of · OH was greatest after l.5 min, and depended on the dose of the stimulant. It was inhibited by inhibitors of platelet activation such as forskolin and phenolic antioxidants.     

Involvement of Reactive Oxygen Species in Capsaicinoid-induced Apoptosis in Transformed Cells

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.     

Reactive Oxygen Species-Mediated DNA Damage and Apoptosis in Human Skin Epidermal Cells After Exposure to Nickel Nanoparticles

Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Photodynamic Inactivation of Candida albicans with Imidazoacridinones: Influence of Irradiance,Photosensitizer Uptake and Reactive Oxygen Species Generation

The increasing applicability of antifungal treatments, the limited range of available drug classes and the emergence of drug resistance in Candida spp. suggest the need for new treatment options. To explore the applicability of C. albicans photoinactivation, we examined nine structurally different imidazoacridinone derivatives as photosensitizing agents. The most effective derivatives showed a >10⁴-fold reduction of viable cell numbers. The fungicidal action of the three most active compounds was compared at different radiant powers(3.5 to 63 mW/cm²), and this analysis indicated that 7 mW/cm² was the most efficient. The intracellular accumulation of these compounds in fungal cells correlated with the fungicidal activity of all 9 derivatives. The lack of effect of verapamil, an inhibitor targeting Candida ABC efflux pumps, suggests that these imidazoacridinones are not substrates for ABC transporters. Thus, unlike azoles, a major class of antifungals used against Candida, ABC transporter-mediated resistance is unlikely. Electron paramagnetic resonance (EPR)-spin trapping data suggested that the fungicidal light-induced action of these derivatives might depend on the production of superoxide anion. The highest generation rate of superoxide anion was observed for 1330H, 1610H, and 1611. Singlet oxygen production was also detected upon the irradiation of imidazoacridinone derivatives with UV laser light, with a low to moderate yield, depending on the type of compound. Thus, imidazoacridinone derivatives examined in the present study might act via mixed type I/type II photodynamic mechanism. The presented data indicate lack of direct correlation between the structures of studied imidazoacridinones, cell killing ability, and ROS production. However, we showed for the first time that for imidazoacridinones not only intracellular accumulation is necessary prerequisite of lethal photosensitization of C. albicans, but also localization within particular cellular structures. Our findings present IA derivatives as efficient antifungal photosensitizers with a potential to be used in local treatment of Candida infection.     

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.     

植物活性氧的产生及其作用和危害

活性氧(ROS)是一类由O2转化而来的自由基或具有高反应活性的离子或分子。植物消耗的O2约有1%在叶绿体、线粒体、过氧化物酶体等多种亚细胞单位中被转化成了ROS。ROS有益或有害取决于它在植物体内的浓度。低浓度的ROS作为第二信使能在植物细胞信号转导途径中介导多种应答反应,高浓度的ROS则引起生物大分子的氧化损伤甚至细胞死亡。植物体内ROS产生和清除之间的平衡十分重要,并由一套有效的酶促和非酶促抗氧化系统来监控。该文主要系统介绍了植物ROS的种类、产生部位、在信号转导中的作用及其对植物细胞造成的主要伤害等方面的研究进展,为利用基因工程手段来提高植物对环境胁迫的抗性提供信息和思路。