Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H₂O₂ induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H₂O₂ in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.     

Proteomic analysis of glutamate-induced toxicity in HT22 cells

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.     

Proteasome inhibition protects HT22 neuronal cells from oxidative glutamate toxicity

Oxidative stress caused by glutathione depletion after prolonged exposure to extracellular glutamate leads to a form of neuronal cell death that exhibits morphologically mixed features of both apoptosis and necrosis. However, specific downstream executioners involved in this form of cell death have yet to be identified. We report here that glutamate exposure does not activate caspase-3 in the HT22 neuronal cell line. Furthermore, no cytoprotection was achieved with either the pan-caspase inhibitor Z-VAD-fmk or the caspase-3-specific inhibitor DEVD-CHO. In contrast, inhibition of the proteasome by lactacystin protected both HT22 cells and rat primary neuronal cells against cell lysis. In parallel, oxidatively altered and ubiquitinated proteins accumulated in the mitochondrial fraction of cells after proteasome inhibition. These findings suggest that caspases can be decoupled from oxidative stress under some conditions, and implicate the ubiquitin/proteasome pathway in neuronal cell death caused by oxidative glutamate toxicity.     

Accumulation of ubiquitinated proteins in mouse neuronal cells induced by oxidative stress

Ubiquitin protein conjugates are commonly detected in neuronal brain inclusions of patients with neurodegenerative disorders. The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result from changes in the activity of the ubiquitin/ATP-dependent proteolytic pathway. This proteolytic pathway plays a major role in the degradation of short lived, abnormal and denatured proteins. Cadmium is a potent cell poison and is known to affect the ubiquitin pathway and to cause oxidative stress. Increases in protein mixed-disulfides (Pr-SSG) and decreases in glutathione (GSH) are often used as markers of oxidative stress. To investigate the relationship between the ubiquitin pathway and cellular glutathione (GSH), we treated HT4 cells (a mouse neuronal cell line) and rat mesencephalic primary cultures with different concentrations of the heavy metal. We observed marked increases in Pr-SSG as well as decreases in GSH, after exposure of HT4 cells or primary mesencephalic cultures to Cd²⁺. Furthermore, our results show that Cd²⁺ induced the accumulation of ubiquitinated proteins. Detection was by Western blotting of total cell extracts probed with antibodies that recognize ubiquitin-protein conjugates. These results suggest that the ubiquitin-pathway is closely involved in the cell response to cadmium-mediated oxidative stress. Abbreviations: GSH – glutathione; GSSG – glutathione disulfide; Pr-SSG – protein mixed disulfides.     

Response of a DNA-binding protein to radiation-induced oxidative stress

The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis.Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins.When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently.The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.     

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein

Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.     

Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage

SIRT6 is a NAD+-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stressinduced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.     

DSSylation,a novel protein modification targets proteins induced by oxidative stress,and facilitates their degradation in cells

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.     

星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激

星形胶质细胞维持神经元微环境,给予营养和代谢支持,并调节其对损伤的反应。鱼藤酮特异阻断线粒体复合物Ⅰ,长期暴露于鱼藤酮可能增加患帕金森病的几率,并引起帕金森综合征。然而,星形胶质细胞在鱼藤酮所致多巴胺能神经元损伤过程中的作用尚无报道。本研究采用多巴胺能神经元细胞系MN9D细胞模型,将经过或未经过星形胶质细胞条件培养基处理的MN9D细胞暴露于不同浓度的鱼藤酮中,用计数法测生长曲线,MTT法测细胞活性,DCFH染色流式细胞仪测氧化应激水平,比色法测还原型谷胱甘肽含量。结果显示,MN9D细胞在条件和普通培养基培养条件下生长曲线无明显差别;鱼藤酮浓度依赖性地降低细胞活性;不同浓度鱼藤酮作用24、48h后,经条件培养基处理的细胞其活性显著高于普通培养基培养的细胞：不同浓度的条件培养基都有保护作用,纯的条件培养基保护作用稍弱：预先24h条件培养基处理或同时给予鱼藤酮和条件培养基处理都有保护作用,鱼藤酮作用12h后再给予条件培养基则无保护作用;经条件培养基处理的细胞氧化应激水平降低：另外,条件培养基提高了细胞内还原型谷胱甘肽含量,缓解了鱼藤酮所致的谷胱甘肽耗竭。结果提示,星形胶质细胞可保护MN9D细胞抵抗鱼藤酮所致的氧化应激,还原型谷胱甘肽可能参与了该保护过程。     

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimer's disease is the deposition of the amyloid beta peptide (Abeta) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Abeta aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Abeta(1-40) peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Abeta(1-40) for 48 h caused a significant increase in the content and activity of Ca2+ dependent and Ca2+ independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down-regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Abeta(1-40) treatment. A combination of Abeta(1-40) and 30-min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and by enhanced 4',6-diamidino-2-phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the myristoyl alanine-rich C-kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Abeta(1-40). Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Abeta(1-40), phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylatability was diminished possibly because of protein kinase C down-regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Abeta(1-40) combined with anoxic stress may play a role in Alzheimer's disease pathology.     

A proteasomal stress response: pre-treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.     

Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins

Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H(2)O(2), FeSO(4)) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition.     

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.     

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.     

APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells

Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer’s disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.     

tRNA cleavage is a conserved response to oxidative stress in eukaryotes

Recent results have identified a diversity of small RNAs in a wide range of organisms. In this work, we demonstrate that Saccharomyces cerevisiae contains a small RNA population consisting primarily of tRNA halves and rRNA fragments. Both 5′ and 3′ fragments of tRNAs are detectable by Northern blot analysis, suggesting a process of endonucleolytic cleavage. tRNA and rRNA fragment production in yeast is most pronounced during oxidative stress conditions, especially during entry into stationary phase. Similar tRNA fragments are also observed in human cell lines and in plants during oxidative stress. These results demonstrate that tRNA cleavage is a conserved aspect of the response to oxidative stress.