Responses of the photosynthetic machinery of Spirulina maxima to induced reactive oxygen species

The photosynthetic machinery of Spirulina maxima was studied when subjected to induced reactive oxygen species (ROS) to examine the organism's responses to stress. Significant decreases in both photosynthetic efficiency and growth rate were observed. Exposure to 0.01 mmol H(2)O(2)/(g cell), which induced the lowest specific intracellular ROS level (siROS) led to a 15% decrease in specific growth rate; an increase in siROS by 70-fold led to a 25% decrease in specific growth rate. Similarly, siROS induced by 0.01 mmol H(2)O(2)/(g cell) led to 15% inhibition in photosynthetic efficiency, while an increase in siROS by 40- or 70-fold led to about 60% inhibition in photosynthetic efficiency. To further understand the effects of induced ROS on photosynthetic machinery, we performed a detailed pigmentation analysis as well as analyzed Phycobilisomes (PBS), Photosystem II (PSII), and Photosystem I (PSI), the three important components of cyanobacterial photosynthetic apparatus. We found carotenoids (beta-carotene and lutein) to be most sensitive to siROS. Also, specific levels of phycocyanin and allophycocyanin, which are important PBS pigments, decreased significantly in response to H(2)O(2). Further, electron transport assays revealed that ROS cause damage primarily to PSII, whereas they do not significantly affect PSI in comparison; siROS induced by 0.01 mmol H(2)O(2)/(g cell) led to a 15% inhibition of PSII, and increase in siROS by 9-, 40-, and 70-fold led to 22%, 36%, and 46% inhibition, respectively.     

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

Exposure of Bacillus subtilis to a shear rate of 1,482/s leads to a rapid loss of cell viability after 10 h of growth. Biochemical and molecular evidences provided below strongly suggest that cell death under high shear results from an apoptosis-like process similar to that described in eukaryotes, with activation of a caspase-3-like protease (C(3)LP) followed by DNA fragmentation. Shear stress leads to an increase in specific intracellular reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS precedes the activation of C(3)LP and DNA fragmentation, thus establishing siROS as the molecular link between shear stress and apoptosis-like cell death. A model is proposed in which NOX is viewed as being strategically placed on the plasma membrane of B. subtilis that senses and converts a mechanical force arising from shear stress into a chemical signal leading to activation of C(3)LP, DNA fragmentation, and thus, apoptosis-like cell death.     

The general stress protein Ctc of Bacillus subtilis is a ribosomal protein

Cells respond to stress conditions by synthesizing general or specific stress proteins. The Ctc protein of Bacillus subtilis belongs to the general stress proteins. The synthesis of Ctc is controlled by an alternative sigma factor of RNA polymerase, sigmaB. Sequence analyses revealed that Ctc is composed of two domains, an N-terminal domain similar to the ribosomal protein L25 of Escherichia coli, and a C-terminal domain. The similarity of the N-terminal domain of Ctc to L25 suggested that Ctc might be a ribosomal protein in B. subtilis. The function of the C-terminal domain is unknown. We purified Ctc to homogeneity and used the pure protein to raise antibodies. Western blot analyses demonstrate that Ctc is induced under stress conditions and can be found in ribosomes of B. subtilis. As observed for its E. coli counterpart L25, Ctc is capable of binding 5S ribosomal RNA in a specific manner. The stress-specific localization of Ctc in B. subtilis ribosomes and the sporulation defect of ctc mutants at high temperatures suggest that Ctc might be required for accurate translation under stress conditions.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

ROS, mitochondria and the regulation of autophagy

Accumulation of reactive oxygen species (ROS) is an oxidative stress to which cells respond by activating various defense mechanisms or, finally, by dying. At low levels, however, ROS act as signaling molecules in various intracellular processes. Autophagy, a process by which eukaryotic cells degrade and recycle macromolecules and organelles, has an important role in the cellular response to oxidative stress. Here, we review recent reports suggesting a regulatory role for ROS of mitochondrial origin as signaling molecules in autophagy, leading, under different circumstances, to either survival or cell death. We then discuss the relationship between mitochondria and autophagosomes and propose that mitochondria have an essential role in autophagosome biogenesis.     

D-Glyceraldehyde causes production of intracellular peroxide in pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial pathways

D-Glyceraldehyde (D-GLYC) is usually considered to be a stimulator of insulin secretion but theoretically can also form reactive oxygen species (ROS), which can inhibit beta cell function. We examined the time- and concentration-dependent effects of D-GLYC on insulin secretion, insulin content, and formation of ROS. We observed that a 2-h exposure to 0.05-2 mM D-GLYC potentiated glucose-stimulated insulin secretion (GSIS) in isolated Wistar rat islets but that higher concentrations inhibited GSIS. A 24-h exposure to 2 mm D-GLYC inhibited GSIS, decreased insulin content, and increased intracellular peroxide levels (2.14 +/- 0.31-fold increase, n = 4, p < 0.05). N-Acetylcysteine (10 mM) prevented the increase in intracellular peroxides and the adverse effects of d-GLYC on GSIS. In the presence of 11.1 but not 3.0 mm glucose, koningic acid (10 microM), a specific glyceraldehyde-3-phosphate dehydrogenase inhibitor, increased intracellular peroxide levels (1.88 +/- 0.30-fold increase, n = 9, p < 0.01) and inhibited GSIS (control GSIS = p < 0.001; koningic acid GSIS, not significant). To determine whether oxidative phosphorylation was the source of ROS formation, we cultured rat islets with mitochondrial inhibitors. Neither rotenone or myxothiazol prevented D-GLYC-induced increases in islet ROS. Adenoviral overexpression of manganese superoxide dismutase also failed to prevent the effect of D-GLYC to increase ROS levels. These observations indicate that exposure to excess D-GLYC increases reactive oxygen species in the islet via non-mitochondrial pathways and suggest the hypothesis that the oxidative stress associated with elevated D-GLYC levels could be a mechanism for glucose toxicity in beta cells exposed chronically to high glucose concentrations.     

Senescence-induced increases in intracellular oxidative stress and enhancement of the need for ascorbic acid in human fibroblasts

Many studies have suggested that there is a close correlation among declines in internal ascorbic acid (AsA) levels, various disorders, and senescence. To clarify the relationships between age-associated changes in intracellular AsA levels and the effects of AsA administration on intracellular reactive oxygen species (ROS) levels, we investigated aging-related changes in AsA uptake, ROS levels, and the effects of AsA administration on intracellular ROS levels in young and old (senescent) human fibroblasts. Our results demonstrated that AsA uptake was increased in old cells compared with young cells, although mRNA and protein expression of sodium-dependent vitamin C transporter 2 was barely altered between the young and old cells. We also demonstrated that the intracellular superoxide anion level was higher in young cells, whereas the level of intracellular peroxides was significantly increased in old cells under both normal and oxidative stress conditions. Moreover, AsA administration markedly decreased the augmentation of intracellular peroxides in old cells, whereas there was no effect of AsA treatment in young cells under both normal and oxidative stress conditions. Therefore, our results also indicate that AsA could play an important role in regulating the intracellular ROS levels in senescent cells and that the need for AsA is enhanced by cellular senescence.     

DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae

Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA-alkylating agent methyl methanesulfonate. Our results indicate that the DNA damage-induced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways.     

Modulation of intracellular ROS levels by TIGAR controls autophagy

The p53‐inducible TIGAR protein functions as a fructose‐2,6‐bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy—a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.     

褪黑素对非生物胁迫下雨生红球藻中虾青素积累的影响

以雨生红球藻Haematococcus pluvialis LUGU株为研究对象, 研究在高光照和缺氮胁迫条件下, 添加不同浓度褪黑素(melatonin, MLT)对雨生红球藻生长、虾青素积累、活性氧(ROS)、信号分子及dxs基因表达量的影响。结果表明, 外源添加10 μmol/L MLT可有效提高藻细胞中虾青素的含量, 最高可达31.32 mg/g, 是对照组(13.27 mg/g)的2.36倍; 抑制了细胞内ROS水平, 上调了信号分子一氧化氮(NO)和水杨酸(SA)的含量; 此外, dxs基因表达水平比对照组明显提高, 最高达11.3倍。研究表明, 在非生物胁迫条件下, 雨生红球藻中虾青素的大量积累可能与外源MLT调控细胞内ROS、信号分子及基因表达有关。     

Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance

Salt imposes immediate problems for plant cells, such as osmotic stress, impaired ion homeostasis and sodium toxicity, followed by a secondary oxidative stress caused by generation of reactive oxygen species (ROS). Here, we analyzed the production of ROS during salt stress. We show that salt stress triggered plasma membrane internalization, resulting in the production of ROS within endosomes. The intracellular ROS were produced by NADPH oxidase in response to the ionic but not the osmotic stress. Both endocytosis and ROS production were suppressed in phosphatidylinositol (PtdIns) 3-kinase (PI3K) mutants, PI3K being a key regulator of vesicle trafficking in animals and plants, and by wortmannin, which is a specific inhibitor of PI3K and PI4K. Endocytosis and the production of ROS were rescued by supplementation of seedlings with exogenous PtdIns 3-phosphate (PtdIns3P), less with PtdIns4P, but not with PtdIns(4,5)P(2). Surprisingly, despite reduced oxidative stress, the mutants and the wortmannin-treated plants exhibited a phenotype overly sensitive to salt, as also resulted from treatment with diphenyleneiodonium, a suicide inhibitor of NADPH oxidase, suggesting a positive role for ROS in salt tolerance. In summary, our results show that salt stress responses, such as increased plasma membrane endocytosis and the intracellular production of ROS, are coordinated by phospholipid-regulated signaling pathways, and suggest that ROS act in the signal transduction of the salt tolerance response.     

Stress induced lipid production in Chlorella vulgaris: Relationship with specific intracellular reactive species levels

Microalgae have significant potential to be an important alternative energy source, but the challenges to the commercialization of bio‐oil from microalgae need to be overcome for the potential to be realized. The application of stress can be used to improve bio‐oil yields from algae. Nevertheless, the understanding of stress effects is fragmented due to the lack of a suitable, direct quantitative marker for stress. The lack of understanding seems to have limited the development of stress based strategies to improve bio‐oil yields, and hence the commercialization of microalgae‐based bio‐oil. In this study, we have proposed and used the specific intracellular reactive species levels (siROS) particularly hydroxyl and superoxide radical levels, separately, as direct, quantitative, markers for stress, irrespective of the type of stress induced. Although ROS reactions are extremely rapid, the siROS level can be assumed to be at pseudo‐steady state compared to the time scales of metabolism, growth and production, and hence they can be effective stress markers at particular time points. Also, the specific intracellular (si‐) hydroxyl and superoxide radical levels are easy to measure through fluorimetry. Interestingly, irrespective of the conditions employed in this study, that is, nutrient excess/limitation or different light wavelengths, the cell concentrations are correlated to the siROS levels in an inverse power law fashion. The composite plots of cell concentration (y) and siROS (x) yielded the correlations of y = k₁ · x^?0.7 and y = k₂ · x^?0.79, for si‐hydroxyl and si‐superoxide radical levels, respectively. The specific intracellular (si‐) neutral lipid levels, which determine the bio‐oil productivity, are related in a direct power law fashion to the specific hydroxyl radical levels. The composite plot of si‐neutral lipid levels (z) and si‐hydroxyl radical level (x) yielded a correlation of z = k₃ · x^0.65. More interestingly, a nutrient shift caused a significant change in the sensitivity of neutral lipid accumulation to the si‐hydroxyl radical levels. Biotechnol. Bioeng. 2013; 110: 1627–1636. © 2013 Wiley Periodicals, Inc.     

Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species

Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H(2)O(2) compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment with N-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H(2)O(2) in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H(2)O(2) observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis.