Differential role of microenvironment in microencapsulation for improved cell tolerance to stress

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.     

Oxidative stress response in eukaryotes: effect of glutathione,superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Abstract

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H₂O₂ did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H₂O₂ adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H₂O₂ when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H₂O₂ achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H₂O₂ was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H₂O₂ promoted an increase in catalase activity.     

Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli.

Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.     

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52°C for 4.5 min), ethanol (20% v/v for 30 min), H₂O₂ (0.3 M for 60 min), rapid freezing (−196°C for 20 min, cooling rate 200°C min⁻¹), slow freezing (−20°C for 24 h, cooling rate 3°C min⁻¹), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H₂O₂ stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H₂O₂ and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H₂O₂ tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast. Received 10 October 1995/ Accepted in revised form 10 September 1996     

<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

Erwinia amylovora catalases KatA and KatG are virulence factors and delay the starvation‐induced viable but non‐culturable (VBNC) response

The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H₂O₂) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H₂O₂‐neutralizing enzymes, such as catalases, during plant–pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non‐host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H₂O₂ and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant–pathogen interactions; (iii) the induction of EPSs by H₂O₂ despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation‐induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non‐culturable (VBNC) response.     

Exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> improves the chilling tolerance of manilagrass and mascarenegrass by activating the antioxidative system

The effect of foliar pretreatment by hydrogen peroxide (H₂O₂) at low concentrations of 0, 5, 10, and 15 mM on the chilling tolerance of two Zoysia cultivars, manilagrass (Zoysia matrella) and mascarenegrass (Zoysia tenuifolia), was studied. The optimal concentration for H₂O₂ pretreatment was 10 mM, as demonstrated by the lowest malondialdehyde (MDA) content and electrolyte leakage (EL) levels and higher protein content under chilling stress (7°C/2°C, day/night). Prior to initiation of chilling, exogenous 10 mM H₂O₂ significantly increased catalase (CAT), ascorbate peroxidase (APX), glutathione-dependent peroxidases (GPX), and glutathione-S-transferase (GST) activities in manilagrass, and guaiacol peroxidase (POD), APX, and glutathione reductase (GR) activities in mascarenegrass, suggesting that H₂O₂ may act as a signaling molecule, inducing protective metabolic responses against further oxidative damage due to chilling. Under further stress, optimal pretreatments alleviated the increase of H₂O₂ level and the decrease of turfgrass quality, and improved CAT, POD, APX, GR, and GPX activities, with especially significant enhancement of APX and GPX activities from the initiation to end of chilling. These antioxidative enzymes were likely the important factors for acquisition of tolerance to chilling stress in the two Zoysia cultivars. Our results showed that pretreatment with H₂O₂ at appropriate concentration may improve the tolerance of warm-season Zoysia grasses to chilling stress, and that manilagrass had better tolerance to chilling, as evaluated by lower MDA and EL, and better turfgrass quality, regardless of the pretreatment applied.     

Genome-wide identification of genes involved in tolerance to various environmental stresses inSaccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H₂O₂, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H⁺-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.     

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

The effect of hydrogen peroxide on the activities of catalase and superoxide dismutase (SOD) in S. cerevisiae has been studied under different experimental conditions: various H₂O₂ concentrations, time exposures, yeast cell densities and media for stress induction. The yeast treatment with 0.25–0.50 mM H₂O₂ led to an increase in catalase activity by 2–3-fold. At the same time, hydrogen peroxide caused an elevation by 1.6-fold or no increase in SOD activity dependently on conditions used. This effect was cancelled by cycloheximide, an inhibitor of protein synthesis in eukaryotes. Weak elevation of catalase and SOD activities in cells treated with 0.25–0.50 mM H₂O₂ found in this study does not correspond to high level of synthesis of the respective enzyme molecules observed earlier by others. It is well known that exposure of microorganisms to low sublethal concentrations of hydrogen peroxide leads to the acquisition of cellular resistance to a subsequent lethal oxidative stress. Hence, it makes possible to suggest that S. cerevisiae cells treated with low sublethal doses of hydrogen peroxide accumulate non-active stress-protectant molecules of catalase and SOD to survive further lethal oxidant concentrations.     

Hydrogen sulphide may be a novel downstream signal molecule in nitric oxide‐induced heat tolerance of maize (Zea mays L.) seedlings

Nitric oxide (NO) is a second messenger with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. In recent years, hydrogen sulphide (H₂S) has been found to have similar functions, but crosstalk between NO and H₂S in the acquisition of heat tolerance is not clear. In this study, pretreatment with the NO donor sodium nitroprusside (SNP) improved the survival percentage of maize seedlings and alleviated an increase in electrolyte leakage and a decrease in tissue vitality as well as accumulation of malondialdehyde, indicating that pretreatment with SNP improved the heat tolerance of maize seedlings. In addition, pretreatment with SNP enhanced the activity of L‐cystine desulfhydrase, which, in turn, induced accumulation of endogenous H₂S, while application of H₂S donors, NaHS and GYY4137, increased endogenous H₂S content, followed by mitigating increase in electrolyte leakage and enhanced survival percentage of seedlings under heat stress. Interestingly, SNP‐induced heat tolerance was enhanced by application of NaHS and GYY4137, but was eliminated by inhibitors of H₂S synthesis DL‐propargylglycine, aminooxyacetic acid, potassium pyruvate and hydroxylamine, and the H₂S scavenger hypotaurine. All of the above‐mentioned results suggest that SNP pretreatment could improve heat tolerance, and H₂S may be a downstream signal molecule in NO‐induced heat tolerance of maize seedlings.     

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H₂O₂, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H₂O₂ in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H₂O₂, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H₂O₂ is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H₂O₂-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H₂O₂ is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H₂O₂ was unable to induce this complex suggesting that H₂O₂ is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally     

Involvement of glutathione in heat shock– and hydrogen peroxide–induced cadmium tolerance of rice (Oryza sativa L.) seedlings

The role of reduced glutathione (GSH) in heat shock (HS)- and H₂O₂-induced protection of rice (Oryza sativa L., cv. Taichung 1) seedlings from Cd stress was investigated. HS- and H₂O₂-pretreatment resulted in an increase in GSH content in leaves of rice seedlings. Addition of exogenous GSH under non-HS conditions, which resulted in an increase in GSH in leaves, enhanced subsequent Cd tolerance of rice seedlings. Pretreatment with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, which effectively inhibited GSH content induced by HS and H₂O₂, reduced subsequent Cd tolerance. Furthermore, the effect of BSO on HS- and H₂O₂-induced GSH accumulation and toxicity by subsequent Cd stress can be reversed by the addition of GSH. The time-course analyses of HS in rice seedlings demonstrated that the accumulation of H₂O₂ preceded the increase in GSH. Based on the data obtained in this study, it could be concluded that the early accumulation of H₂O₂ during HS signals the increase in GSH content, which in turn protects rice seedlings from oxidative damage caused by Cd.     

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H₂O₂-induced DNA damage. UVC and H₂O₂ treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G₀, G₁ and S-phase. Rad18 was important for repressing H₂O₂-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G₁, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H₂O₂-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H₂O₂-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G₁. In contrast with G₁-synchronized cultures, S-phase cells were H₂O₂-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G₁ (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.