Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response

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Oxidative Stress and Heat Shock Protein Induction in Human Cells

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis.

Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.     

Oxidative Stress and Heat Shock Protein Induction in Human Cells

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis.

Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.     

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.     

砷暴露诱导人肝细胞氧化应激过程中NADPH氧化酶作用机制

目的：探讨砷暴露诱导细胞氧化应激的分子机制。方法：采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽（reducedglutathione,GSH）预处理,检测细胞超氧阴离子（02。）和细胞整体ROS的水平。WestemBlot方法检测细胞氧化／抗氧化重要酶微粒体谷胱甘肽硫转移酶（microsomalglutathioneS-transferase-l,Mgst．1）、半胱氨酸双加氧酶l（cysteinedioxygenasel,Cd01）和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂（diphenyleneiodoniumchloride,DPI）进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果：砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷（亚砷酸钠,A矿）诱导氧化应激作用显著强于五价砷（砷酸钠,As5＋）。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论：NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。     

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.     

人线粒体tRNALeu(UUR)基因氧化损伤与修复研究

人mtDNA比核DNA更易受到自由基的氧化损伤,这些损伤可以被线粒体内的DNA修复机制所修复,损伤与修复是决定突变是否产生的两个重要因素.为了确定氧化损伤与损伤后修复对mtDNA突变的具体影响,采用四氧嘧啶处理LO2细胞,这种试剂进入细胞后,经氧化还原反应生成的自由基与线粒体自身代谢产生的自由基类似,然后观察自由基对细胞mtDNA的氧化损伤与损伤后DNA修复的动力学变化.由于线粒体的正常功能为修复机制所必需,采用MTT细胞活力实验检测不同浓度四氧嘧啶处理下线粒体酶活力,发现9 mmol/L四氧嘧啶培养细胞1h后,线粒体琥珀酸脱氢酶功能在撤去药物后0,2,8和24 h时间点均无明显变化.提取各组细胞的mtDNA,用EndoⅢ和Fgp两种酶切除受氧化损伤的核苷酸,然后用碱性琼脂糖凝胶电泳分离大小不等的mtDNA,进行DNA印迹实验,地高辛-抗体-碱性磷酸酶系统显色,检测完整与断裂的mtDNA量,利用Poisson公式(s=-lnP0/P,P0为未断裂链光密度值,P为所有链光密度值总和)计算一个mtDNA分子的平均损伤频率,结果显示,9 mmol/L四氧嘧啶处理细胞1 h,链平均损伤频率由对照的0.11个/分子增加至5.60个/分子,明显增加了mtDNA上核苷酸的氧化损伤,除去药物后8 h,绝大部分损伤可被修复,损伤频率减至0.40个/分子,除去药物后24h核苷酸的氧化损伤恢复至正常水平.采用接头介导PCR(LM-PCR)检测MTTL1基因区域内单个核苷酸的损伤与修复动力学.这种方法可以检测各组mtDNA上MTTL1基因75 bp区域内单个核苷酸损伤的部位及频率.结果显示,人MTTL1基因存在20个易受氧化损伤的核苷酸热点,经与相应区域内文献报道的16个突变热点比较,有12个热点部位重合,而修复未显示热点部位或区域.结果提示,自由基对核苷酸的选择性氧化损伤是决定mtDNA点突变发生及发生部位的主要原因.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Oxidative Stress Markers Induced by Hyperosmolarity in Primary Human Corneal Epithelial Cells

Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350–450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4–hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.     

Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced,p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

Background

The aging gene p66^Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66^Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66^Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66^Shc activation.

Methods and Results

Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66^Shc at Ser-36, resulting in an enhanced production of superoxide anion (O₂ ^-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66^Shc phosphorylation, suggesting that basal NO production inhibits O₂ ^- production. oxLDL-induced, p66^Shc-mediated O₂- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66^Shc activation and reduced O_2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66^Shc activation, and reduced O₂- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.

Conclusions

The present study reports that in primary cultured HAEC treated with oxLDL, p66^Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66^Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.     

A Possible Role for Calcium in Oxidative Plant Stress

Disruption of calcium homoeostasis may be crucially important in damaging animal cells under oxidising conditions. In hepatocytes, oxidative stress causes an increase in cytosolic calcium, a process which is thought to be largely responsible for subsequent cell death. The oxidation of glutathione precedes a flux of calcium into the cytoplasm from internal and external sources. The possibility that similar events occur in plant cells has been tested using strips of epidermis taken from leaves of Commelina communis. The closure of stomata in response to many chemical or physical stimuli is thought to involve a transient rise in cytosolic calcium. Paraquat (10^-4 M) and hydrogen peroxide (10^-3 M) caused a marked reduction in stomatal aperture when included in the incubation medium of illuminated epidermal strips. The calcium channel blocker verapamil (10^-5 M) protected stomata from hydrogen peroxide-induced closure while the ion chelator EGTA (2 × 10^-3 M) protected stomata in both oxidising treatments. These results are discussed in so far as they support the hypothesis that a rise in cytosolic calcium may play a role in the pathology of oxidative stress in plant cells through a mechanism analogous to the liver cell system.     

Experimental Evidence for the Physiological Role of Bacterial Luciferase in the Protection of Cells Against Oxidative Stress

The origin and function of bioluminescence was considered a problematic question of the Charles Darwin theory. Early evolution of bacterial luminescence and its current physiological importance seem to be especially mysterious. Recently, it was proposed that stimulation of DNA repair may be a physiological role for production of light by bacterial cells. On the other hand, it was also proposed that primary role of luminescent systems could be detoxification of the deleterious oxygen derivatives. Although some previous results might suggest that this hypothesis can be correct, until now experimental evidence for such a mechanism operating in bacterial cells and having physiological importance was generally lacking. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, and ferrous ions) at certain concentrations in the culture medium, growth of Vibrio harveyi mutants luxA and luxB, but not of the mutant luxD, is severely impaired relative to wild-type bacteria. This deleterious effect of oxidants on the mutants luxA and luxB could be significantly reduced by addition of the antioxidants A-TEMPO or 40H-TEMPO. We conclude that bacterial luciferase may indeed play a physiological role in the protection of cells against oxidative stress.     

Apelin Attenuates Oxidative Stress in Human Adipocytes

It has been recently recognized that the increased oxidative stress (ROS overproduction) in obese condition is a key contributor to the pathogenesis of obesity-associated metabolic diseases. Apelin is an adipocytokine secreted by adipocytes, and known for its anti-obesity and anti-diabetic properties. In obesity, both oxidative stress and plasma level of apelin are increased. However, the regulatory roles of apelin on oxidative stress in adipocytes remain unknown. In the present study, we provide evidence that apelin, through its interaction with apelin receptor (APJ), suppresses production and release of reactive oxygen species (ROS) in adipocytes. This is further supported by the observations that apelin promotes the expression of anti-oxidant enzymes via MAPK kinase/ERK and AMPK pathways, and suppresses the expression of pro-oxidant enzyme via AMPK pathway. We further demonstrate that apelin is able to relieve oxidative stress-induced dysregulations of the expression of anti- and pro-oxidant enzymes, mitochondrial biogenesis and function, as well as release of pro- and anti-inflammatory adipocytokines. This study, for the first time, reveals the antioxidant properties of apelin in adipocytes, and suggests its potential as a novel therapeutic target for metabolic diseases.     

Luminol-Enhanced Chemiluminescent Response of Human Melanocytes and Melanoma Cells to Hydrogen Peroxide Stress

The response of human melanocytes and melanoma cells to hydrogen peroxide stress was measured. Cells were exposed to glucose/glucose oxidase or free H₂O₂ and reactive oxygen species measured by luminol-enhanced chemiluminescence. The response was distinctly different between the two types and the addition of superoxide dismutase to melanoma cells paradoxically enhanced the chemiluminescent signal. These findings coupled with other known differences between the way these two types of cells handle oxidative stress at a molecular level suggests that a therapeutic window may be avail-able for exploitation.