YhbO protects cells against multiple stresses

YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1. A yhbO-disrupted mutant of Escherichia coli is highly sensitive to oxidative, thermal, UV, and pH stresses, and the putative nucleophilic cysteine C104 of YhbO is required for stress resistance. These results suggest that YhbO affects a central process in stress management.     

A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase

In Leuconostoc oenos , a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42°C), acid (pH 3) or ethanolic (12% (v/v)) shocks. Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase. Significant identity was found between the N-terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells.     

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

The physiological role of the non-homologous end-joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (DeltarecA, Deltaku, DeltaligD, Deltaku/ligD, DeltarecA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ-mutant strains (Deltaku, DeltaligD, Deltaku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis. However, there was little difference in IR sensitivity between NHEJ-mutant and wild-type strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A DeltarecA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the DeltarecA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively.     

Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence

In this study, the role of Listeria monocytogenes ferritin was investigated. The fri gene encoding the ferritin was deleted and the phenotype of the mutant was analyzed demonstrating that ferritin is necessary for optimal growth in minimal medium in both presence and absence of iron, as well as after cold- and heat-shock. We also showed that ferritin provides protection against reactive oxygen species and is essential for full virulence of L. monocytogenes. A comparative proteomic analysis revealed an effect of the fri deletion on the levels of listeriolysin O and several stress proteins. Together, our study demonstrates that fri has multiple roles that contribute to Listeria virulence.     

Decreased cellular permeability to H2O2 protects Saccharomyces cerevisiae cells in stationary phase against oxidative stress

The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown. By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary--than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase. In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2.     

NAP protects hippocampal neurons against multiple toxins

The femtomolar-acting protective peptide NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP), is broadly neuroprotective in vivo and in vitro in cerebral cortical cultures and a variety of cell lines. In the present study, we have extended previous results and examined the protective potential of NAP in primary rat hippocampal cultures, using microtubule-associated protein 2 (MAP2) as a measure for neuroprotection. Results showed that NAP, at femtomolar concentrations, completely protected against oxygen-glucose deprivation, and cyanide poisoning. Furthermore, NAP partially protected against kainic acid excitotoxicity. In summary, we have significantly expanded previous findings in demonstrating here direct neuroprotective effects for NAP on vital hippocampal neurons that are key participants in cognitive function in vivo.     

Glutathione protects cells against arsenite-induced toxicity

To understand the role of glutathione (GSH) in the protection of cells from arsenite toxicity, we studied the mechanism of apoptotic cell death in cells genetically unable to synthesize GSH (GCS-2 cells). Arsenite stimulated an increase in protein ubiquitination in GCS-2 cells while the wild-type cells were unaffected. Arsenite treatment increased lipid peroxidation and induced ubiquitination of molecular chaperone Hsp90 and impaired its ability to bind cochaperone p50(Cdc-37) and client proteins Plk-1 and Cdk-4 in GCS-2 cells. Treatment with arsenite also partially inhibited proteasome activity in GCS-2 cells. In these cells stably transfected with GFP(u) (a reporter consisting of a short degron fused to the COOH-terminus of GFP), intracellular fluorescence increased, suggesting the accumulation of GFP aggregates. GCS-2 cells underwent apoptosis accompanied by release of cytochrome c into the cytoplasm. Taken together, these data suggest that a possible mechanism of arsenite-induced apoptosis is the accumulation of ubiquitinated proteins and impairment of the protein degradative pathway. Further, protection from arsenite-induced ubiquitination is mediated by GSH and to a lesser extent by available reducing equivalents in the cells.     

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.     

Luciferase detection during stationary phase in Lactococcus lactis

The luminescence signal of luxAB-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of Lactococcus lactis, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.     

Epicatechin protects auditory cells against cisplatin-induced death

Cisplatin, a chemotherapeutic drug that is widely used to treat various cancers, promotes ototoxicity at higher doses. In this study, the effect of epicatechin (EC) on cisplatin-induced hair cell death was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1, and in vivo in zebrafish. Cisplatin promoted apoptosis and altered mitochondrial membrane potential (MMP) in HEI-OC1 cells. EC inhibited cisplatin-induced apoptosis and intracellular reactive oxygen species (ROS) generation. Labeling of zebrafish lateral line hair cells by the fluorescent dye YO-PRO1 was lost upon exposure to cisplatin, and EC protected against this cisplatin-induced loss of labeling in a dose-dependent manner. Scanning and transmission electron micrographs showed that treatment with EC protected against cisplatin-induced loss of kinocilium and stereocilia in zebrafish neuromasts. These results suggest that EC prevents cisplatin-induced ototoxicity by blocking ROS generation and by preventing changes in MMP.     

Rac1 protects epithelial cells against anoikis

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.     

Quinacrine protects neuronal cells against heat-induced injury

The effects of quinacrine (QA) on heat-induced neuronal injury have been explored, with the intention of understanding the mechanisms of QA protection. Primary cultivated striatum neurons from newborn rats were treated with QA 1 h before heat treatment at 43 °C which lasted for another 1 h, and necrosis and apoptosis were detected by Annexin-V-FITC and propidium iodide (PI) double staining. Neuronal apoptosis was determined using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) techniques. Cell membrane fluidity, activity of cytosolic phospholipase A₂ (cPLA₂) and the level of arachidonic acid (AA) were also examined. Membrane surface ultrastructure of striatum neurons was investigated by atomic force microscopy (AFM). Results showed that heat treatment induced great striatum neurons death, with many dying neurons undergoing necrosis rather than apoptosis. QA alone had little effect on the survival of striatum neurons, while QA pretreatment before heat treatment decreased necrosis. Heat treatment also resulted in decreased membrane fluidity and increased cPLA₂ activity as well as arachidonic acid level; these effects were reversed by QA pretreatment. QA pretreatment also significantly prevented damage to the membrane surface ultrastructure of heat-treated neurons. These results suggest that QA protects striatum neurons against heat-induced neuronal necrosis, and also demonstrate that inhibition of cPLA₂ activity and stabilization of membranes may contribute to protective effect of quinacrine.     

Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors.

A sublethal dose of ethanol (5%, vol/vol), acid (HCl, pH 4.5 to 5.0), H2O2 (500 ppm), or NaCl (7%, wt/vol) was added to a Listeria monocytogenes culture at the exponential phase, and the cells were allowed to grow for 1 h. Exponential-phase cells also were heat shocked at 45 degrees C for 1 h. The stress-adapted cells were then subjected to the following factors at the indicated lethal levels--NaCl (25%, wt/vol), ethanol (17.5%, vol/vol), hydrogen peroxide (0.1%, wt/vol), acid (pH 3.5), and starvation on 0.1 M phosphate buffer at pH 7.0 (up to 300 h). Viable counts of the pathogen, after the treatment, were determined on Trypticase soy agar-yeast extract, and survivor plots were constructed. The area (h.log10 CFU/ml) between the control and treatment curves was calculated to represent the protective effect resulting from adaptation to the sublethal stress factor. Adaptation to pH 4.5 to 5.0 or 5% ethanol significantly (P < 0.05) increased the resistance of L. monocytogenes to lethal doses of acid, ethanol, and H2O2. Adaptation to ethanol significantly (P < 0.05) increased the resistance to 25% NaCl. When L. monocytogenes was adapted to 500 ppm of H2O2, 7% NaCl, or heat, resistance of the pathogen to 1% hydrogen peroxide increased significantly (P < 0.05). Heat shock significantly (P < 0.05) increased the resistance to ethanol and NaCl. Therefore, the occurrence of stress protection after adaptation of L. monocytogenes to environmental stresses depends on the type of stress encountered and the lethal factor applied. This "stress hardening" should be considered when current food processing technologies are modified or new ones are developed.     

Nitrate respiration protects hypoxic Mycobacterium tuberculosis against acid- and reactive nitrogen species stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection.     

mRNA translation in yeast during entry into stationary phase

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5′-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5′-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions. Received: 31 March 1998 / Accepted: 4 May 1998     

NOX4 inhibition protects enteric glial cells against Clostridium difficile toxin B toxicity via attenuating oxidative and Endoplasmic reticulum stresses

Enteric glial cells (EGCs), one main cell population of the enteric nervous system (ENS), play a major role in regulating intestinal barrier function. Clostridium difficile toxin B (TcdB) is the major virulence factor produced by C. difficile and estimated to be toxic to EGCs by inducing cell death, cell cycle arrest, and inflammatory cytokine production; however, the detailed mechanism for such effect is still unclear. In this study, we further evaluated the toxic effect of TcdB on EGCs and the involvement of NADPH oxidases in such process using the rat-transformed EGCs (CRL-2690). The results showed that NOX4 was activated by TcdB in EGCs and functioned as the major factor causing cytotoxicity and cell apoptosis. Mechanically, NOX4-generated H₂O₂ was the inducer of oxidative stress, Ca²⁺ homeostasis disorder, and ER stress in EGCs upon TcdB treatment, and NOX4 inhibition protected EGCs against TcdB toxicity via attenuating these dysfunctions. These findings contribute to our understanding of the mechanism by which TcdB affects EGCs and suggest the potential value of NOX4 inhibition for treatment against C. difficile infection.     

Thymoquinone protects renal tubular cells against tubular injury

In this work the effect of angiotensin II (AT II) on proximal tubular epithelial cells (pTECs) in vitro was studied. AT II was found to activate the nuclear factor kappaB (NF-kappaB) and its controlled genes, for example, interleukin 6 (IL-6) of pTECs in a time-dependent manner. Two points with maximum NF-kappaB activation were found, the first after 12 h and the second after 3.5 days. The first point may be due to activation of NF-kappaB in pTECs in response to AT II while the second may be due to activation of the advanced glycation end product (AGE)/receptor of the AGE (RAGE) system. Thymoquinone (TQ) was found to decrease NF-kappaB activation in a dose-dependant manner with maximum inhibitory effect at a concentration of 500 nM. Also, pre-incubation of pTECs with TQ leads to disappearance of the second peak of NF-kappaB. These data are consistent with results obtained from IL-6 enzyme-linked immunosorbent assay (ELISA) and transient transfection experiments. The results explain the therapeutic value of TQ which can be used to delay end stage renal diseases in diabetics.