Correspondence to: David Schubert, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel:(858) 453-4100, ext
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Abstract |
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Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stressinduced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2 and express a low amount of eIF2
. Sensitivity is restored when the clones are transfected with full-length eIF2
; transfection of wild-type cells with the truncated eIF2
gene confers resistance. The phosphorylation of eIF2
also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca2+. In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca2+ when stressed, and the GSH synthetic enzyme
-glutamyl cysteine synthetase (
GCS) is elevated. The change in
GCS is regulated by a translational mechanism. Therefore, eIF2
is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.
Key Words:
oxidative stress, glutathione, eIF2, resistance, glutamate
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Introduction |
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Although programmed cell death (PCD)1 is a widely used mechanism for sculpturing the developing nervous system, its inappropriate activation leads to premature nerve cell death in neuropathological disorders such as Alzheimer's disease (AD) (-glutamyl cysteine synthetase (
GCS), the rate-limiting step in GSH synthesis, results in the selective degeneration of dopaminergic neurons (
There are several ways in which the concentration of intracellular GSH and the oxidative burden of cells can be regulated. One of these is through extracellular glutamate. Although glutamate is generally thought of as both a neurotransmitter and an excitotoxin, extracellular glutamate can also kill neurons through a nonreceptor-mediated pathway which involves the glutamate-cystine antiporter, system Xc- ( subunit of the translation initiation factor 2 (eIF2
) as a gene whose expression is involved in oxidative stressinduced cell death and the regulation of intracellular GSH. eIF2 is a trimeric complex involved in the initiation of translation (
subunit dictates whether protein synthesis will or will not take place and is often referred to as the control point for protein synthesis. The eIF2 complex brings the 40S ribosomal subunit together with the initiating tRNAmet when eIF2
is bound to GTP. Upon hydrolysis of GTP to GDP, the complex is no longer active and protein synthesis is not initiated. GDP/GTP exchange takes place readily with the assistance of a guanine nucleotide exchange factor, eIF2B. However, when the
subunit of eIF2 is phosphorylated on serine 51, a change in the conformation enables it to bind and sequester eIF2B, thus inhibiting GDP/GTP exchange and protein synthesis. eIF2
phosphorylation takes place during ischemia (
may have significant roles in the cell death process after oxidative stress that are separate from its known function as a regulator of protein synthesis. The experiments described below show that the downregulation or phosphorylation of eIF2
protects nerve cells from oxidative stressinduced cell death by inhibiting GSH depletion and the increase in both ROS and intracellular Ca2+ that are normally seen in cells exposed to oxidative stress. These data demonstrate a unique role of eIF2
in oxidative stressinduced programmed nerve cell death, acting as a translational switch which dictates whether a cell activates a survival response or follows a cell death pathway. eIF2
may therefore play a central role in neuropathologies involving nerve cell death which are associated with oxidative stress.
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Materials and Methods |
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The following chemicals were purchased from Sigma-Aldrich: puromycin, TCA, formic acid, GSH, GSH reductase, triethanolamine, sulfosalicylic acid, NADPH, BSA, glutaraldehyde, and L-glutamic acid (glutamate). The fluorescent probes 2',7'-dichlorofluorescein (DCF) diacetate and indoacetoxymethylester (Indo-1), pluronic F-127, and propidium iodide were obtained from Molecular Probes. The Coomassie Plus protein assay reagent and the SuperSignal substrate were both purchased from Pierce Chemical Co. Immobilon P was purchased from Millipore.
Infection with the Retroviral cDNA Library
HT22 cells were infected with the retroviral vector pcLXSN containing a cDNA library derived from the human embryonic lung cell line, MRC-5 (107 virus particles. The cDNA library contains both sense and antisense sequences. The retrovirus stably integrates into the host cell's genomic DNA and expresses the cDNA inserted between its long terminal repeats. Clones containing genes that confer glutamate resistance were identified by selecting cells that survived in 10 mM glutamate. Genomic DNA from each clone was analyzed by PCR using primers that straddle the cDNA insert in the retroviral vector. The cDNA inserts were then subcloned and sequenced. Viral vectors were rescued from the clones by transfection with an ecotropic helper plasmid. These viral particles were collected from the media and used to infect the packaging cell line, PA317, which amplified the virus (
Immunoblotting and Northern Blot Procedures
Cells were plated at 5 x 105 cells per 100-mm dish 1216 h before use and lysed in sample buffer containing 3% SDS. Lysates were sonicated, protein concentrations were normalized using the Coomassie Plus protein assay reagent from Pierce Chemical Co., and 25 µg protein was loaded per lane on 12% Tris-glycine SDS-PAGE gels (Novex). Gels were transferred onto Immobilon P membrane (Millipore) and blocked with 5% milk in TBS for 1 h at room temperature. An antibody against eIF2 (Research Genetics) was shown previously to recognize only phosphorylated eIF2
. However, in our hands the antibody recognized both phosphorylated and unphosphorylated protein when the Western blots and lysates were dephosphorylated with a mixture of bovine and calf intestine alkaline phosphatase. Blots were also probed with antibodies against both phosphorylated and total mitogen-activated protein kinase to confirm that proteins were completely dephosphorylated after treatment with the phosphatases. Therefore, this anti-eIF2
antibody was used to determine the levels of total eIF2
in the HT22 cells and the resistant clones 8 and 15. The anti-eIF2
primary antibody was diluted into 5% BSA in TBS plus Tween 20 (TTBS) at 1:250 and placed on the blot overnight at 4°C. Blots were incubated with the secondary antibody, goat antirabbit IgG HRP conjugated (Bio-Rad Laboratories), for 1 h at room temperature at a dilution of 1:20,000 in 5% milk in TTBS. Blots were exposed to Eastman Kodak Co. X-OMAT Blue film for chemiluminescence using the SuperSignal substrate from Pierce Chemical Co.
Northern blots of the GCS catalytic subunit were done as described in the original paper in which cDNA clones were isolated (
3.7 kb.
Transfection of Full-Length eIF2 into Clones 8 and 15
The full-length cDNA for eIF2 was obtained from Dr. Miyamoto (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) and was cloned into the pCLBABEpuro retroviral vector, a modified version of the pBABEpuro vector (
Production of Retrovirus Expressing the Dominant Negative Mutants of eIF2a
The cDNA constructs for two mutants of eIF2 (S51A and S51D) were obtained from Dr. Kaufman (University of Michigan, Ann Arbor, MI) and subcloned into pCLBABEpuro. Retroviral vectors were made as described (
Translation and Degradation Assays
For translation assays, cells were labeled in 60-mm dishes with 500,000 cpm of [3H]leucine diluted in DME supplemented with 10% FBS for 30 min. The cells were then washed with ice-cold serum-free DME and lysed on the dish using 1 ml ice-cold 10% TCA plus 1 mM DTT and 1 mM cold leucine. Cellular protein was precipitated, dissolved in formic acid, and the [3H]leucine incorporation was determined by scintillation counting. The protein concentration was determined using the Coomassie blue plus protein reagent (Pierce Chemical Co.). The total counts per minute of [3H]leucine incorporated per milligram of protein for 30 min was calculated for each sample. Samples were prepared in triplicate. Protein degradation assays were done exactly as described elsewhere (
Growth Assays
Five sets of triplicate dishes of cells were plated at 5 x 104 in 35-mm dishes. The triplicate sets of each cell type were counted at 12, 24, 48, and 72 h after plating. Cells were dissociated using pancreatase (GIBCO BRL) for 15 min, resuspended in DME, and placed in Eppendorf tubes. Cells were counted directly on a Beckman Coulter counter after dilution in isotonic saline. The data are plotted as cell counts versus time in order to compare the growth rates for the different clones.
GSH Assay
Total intracellular reduced GSH and oxidized GSH (GSSG) were measured as described previously (
Flow Cytometric Studies
Cells were plated on 60-mm dishes at 2 x 105 cells per dish 12 h before adding 25 mM glutamate for 10 h. Samples were then labeled with the fluorescent dyes DCF and Indo-1 to determine ROS production and Ca2+ influx, respectively. Samples were prepared as described previously (
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Results |
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eIF2a Is Involved in the Oxidative Glutamate Toxicity Pathway
Although a mechanistic outline of oxidative glutamate toxicity-mediated PCD has been developed ( subunit of eIF2 (eIF2
) were identified in two separate clones. This gene was chosen for further study because of the requirement for protein synthesis in this form of cell death (
, anti-FAS antibody, serum starvation, and glucose deprivation (data not shown).
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Clones 8 and 15 Cause Glutamate Resistance by Lowering eIF2 Expression
As outlined previously, the introduction of the eIF2 gene fragment into clones 8 and 15 with the retroviral cDNA library could lead to stress resistance by one of several mechanisms. It is unlikely that the eIF2
gene fragment is causing glutamate resistance by disrupting or upregulating a gene whose expression is involved in cell death because the same sequence generates glutamate resistance upon reinfection. This leaves the possibility that the eIF2
cDNA fragment is altering eIF2
expression. Therefore, the two resistant clones and wild-type cells were assayed for eIF2
expression by Western blotting. Although the antibody used for these studies can identify the phosphorylated form of eIF2
(
in HT22 cells (see Materials and Methods). Using this antibody, it was found that both clones 8 and 15 express lower levels of eIF2
protein (Fig 1B and Fig C). Similar results were obtained with another antibody against eIF2
(
Since the retroviral expression library contained cDNAs in both the sense and antisense orientations as well as partial fragments of cDNAs, it is likely that an antisense fragment was expressed to downregulate eIF2 expression. The gene fragments that were rescued from clones 8 and 15 are identical and contain a fragment of the eIF2
cDNA from the 3' end of the full sequence (728941 bp). Antisense gene fragments from cDNA libraries in retroviral vectors have been used previously to identify physiologically relevant genes (
in the resistant clones is responsible for the resistance of the cells to glutamate, then the expression of full-length eIF2
should restore the sensitivity to glutamate. Transfection of full-length eIF2
human cDNA into both clones 8 and 15 restored glutamate sensitivity to both of the clones, whereas the empty vector had no effect (Fig 2B and Fig C). The restoration of glutamate sensitivity is not, however, up to the level of wild-type cells at the highest glutamate concentrations, probably because it was only possible to elevate eIF2
to 8090% of its original level (Fig 1B and Fig C). Wild-type HT22 cells remained sensitive to glutamate after being transfected with the full-length eIF2
cDNA (Fig 2 A). This demonstrates that modulation of eIF2
expression has significant effects on glutamate toxicity in HT22 cells.
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eIF2 Phosphorylation Also Mediates Glutamate Resistance
To confirm that the loss of eIF2 activity is linked to glutamate resistance, a second method was employed which utilizes a dominant negative approach to regulate eIF2
function. The phosphorylated form of eIF2
sequesters the guanine nucleotide exchange factor, eIF2ß, resulting in a decrease in protein translation (
mimics constitutive phosphorylation when serine 51 in eIF2
is replaced with an aspartic acid (
is replaced with alanine (
. To assay the effect of eIF2
phosphorylation on glutamate sensitivity, wild-type HT22 cells were infected with virus that contained either the S51D or S51A mutant or an empty vector, and the cells were tested for glutamate resistance. HT22 cells infected with virus containing the mutant S51D become more resistant to glutamate (Fig 3). The S51A mutant of eIF2
did not have any effect on the response of the cells to glutamate relative to empty vector (Fig 3). These data show that the downregulation of eIF2
activity by protein phosphorylation can lead to glutamate resistance and that eIF2
phosphorylation may play an important role in cell death or survival after glutamate exposure. However, we could not directly assay eIF2
phosphorylation after glutamate exposure because none of the available antibodies immunoprecipitate or distinguish phosphorylated from unphosphorylated eIF2
in HT22 cells.
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Changes in eIF2 Expression Do Not Affect Translation Rates but Do Slow Growth
To determine if eIF2 downregulation in the glutamate-resistant clones causes a decrease in protein synthesis, protein translation rates were measured in clones 8 and 15 as well as in cells expressing mutants S51A and S51D. By inhibiting translation with cycloheximide, HT22 cells are able to survive in the presence of glutamate for short periods of time (
mutants (S51A and S51D) or empty vector (Fig 4 B). Similarly, exposure of HT22 cells to glutamate during a 10-h time course does not lead to any significant changes in overall protein translation (data not shown). These data indicate that the inhibition of overall protein synthesis is not the mechanism underlying protection by eIF2
. However, the translation rates do not reflect the growth rates for each clone, as the growth rate of the wild-type HT22 cell line is more than twofold faster than either clone 8 or 15 (Fig 4 C). HT22 cells infected with the eIF2
mutant S51D also have a slower growth rate than wild-type HT22 cells (Fig 4 D) even though the protein translation rate of this mutant is the same as that in the wild-type cells (Fig 4 B). In contrast, the S51A mutant has no significant effect on the translation rate (Fig 4 B) or the growth rate (Fig 4 D). These data show that changes in eIF2
expression or activation by phosphorylation may lead to alterations in cell growth but not necessarily translation rates. However, it is possible that although the bulk of protein synthesis is not altered, the synthesis of specific proteins required for cell proliferation and cell death is regulated by altered eIF2
expression or phosphorylation.
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eIF2 Expression Alters Glutathione, ROS, and Ca2+ Responses to Glutamate
To understand the role of eIF2 in oxidative glutamate toxicity, several parameters of the glutamate response were measured in the resistant clones and the S51A and S51D mutant-expressing cell lines and compared with the wild-type HT22 cells. HT22 cells undergo a rapid depletion of GSH upon exposure to glutamate (
50% of the original level compared with the 70% decrease in the wild-type and empty vectorinfected cells. On the other hand, the S51A mutant cell line shows a decrease in GSH to
20% of control levels (Fig 5 B). This pattern of GSH depletion is consistent with the survival data which demonstrate that although the S51D-expressing HT22 cells are still healthy and dividing after 24 h of glutamate exposure, the other cell lines are dead (Fig 3). HT22 cells exposed to glutamate for 10 h show a very large increase in ROS which follows the drop in GSH (
in clones 8 and 15 and the expression of the dominant negative phosphorylation mutant S51D all prevent the decrease in GSH and the increases in ROS and Ca2+ normally associated with oxidative stressinduced cell death.
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The Inactivation of eIF2 Upregulates
GCS Expression by a Translational Mechanism
Resistant clones 8 and 15 have decreased eIF2 activity and increased basal levels of GSH. Furthermore, the resistant clones and the cells expressing the phosphorylation mutant, S51D, maintain GSH levels 50% of their basal levels after glutamate exposure. To determine if there is a causal relationship between eIF2
protein levels and GSH production, the expression of the rate-limiting enzyme for GSH synthesis,
GCS, was examined in the wild-type cells and the resistant clones. Protein expression and mRNA levels of the catalytic subunit of
GCS were measured by Western and Northern blotting, respectively. Western blotting shows that the level of the catalytic subunit of
GCS is threefold higher in the resistant clones than in the wild-type HT22 cells (Fig 7A and Fig B). In contrast, when both
GCS and actin mRNA were quantitated and their ratio normalized to cells expressing the empty pCLBABE retroviral vector, the amount of
GCS mRNA remained relatively constant (Fig 7A and Fig B). To rule out the possibility that eIF2
activity changes the rate of
GCS breakdown, resistant clone 15 and wild-type cells were treated with cycloheximide and the rate of protein loss followed by Western blotting. This method gives values of protein turnover identical to pulsechase experiments (
GCS was degraded more slowly but at the same rate in resistant and wild-type cells. These results indicate that a decrease in eIF2
wild-type protein levels leads to an increase in production of the catalytic subunit of
GCS by a translational mechanism, resulting in significantly higher levels of GSH.
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If eIF2 directly regulates
GCS expression, then its expression should be upregulated in wild-type cells made resistant by the S51D phosphorylation mutant and downregulated in the resistant cells which were transfected with wild-type eIF2
to render them more sensitive to oxidative stress. Fig 7 D shows that the levels of
GCS increased
60% in cells transfected with S51D relative to wild-type cells. In contrast, the expression of
GCS decreased between 20 and 40% in the resistant clones 8 and 15 which already have a high level of
GCS protein when these clones were transfected with normal
IF2
(Fig 7 B). These data, along with those presented above, strongly suggest that eIF2
expression and activity can directly modulate
GCS protein levels. It is also likely that the expression of additional proteins involved in the resistance to oxidative stress is regulated by eIF2
.
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Discussion |
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The above data show that eIF2 plays a central role in programmed nerve cell death initiated by oxidative stress. Alterations in either the level of eIF2
or its phosphorylation protect cells from glutamate-induced oxidative stress as well as other prooxidant agents. We will first discuss the evidence for the involvement of eIF2
in glutamate-induced cell death, followed by possible mechanisms that eIF2
could use to signal this type of cell death. The potential relevance of eIF2
nerve cell death in PD will also be discussed.
eIF2 Is Specifically Involved in Oxidative Glutamate Toxicity
HT22 glutamate-resistant clones 8 and 15 were derived from a genetic screen after infection with a retrovirus-based cDNA expression library and selection with a high concentration of the prooxidant glutamate. Both clones contain an identical fragment of the gene for eIF2 from the retroviral library. The following evidence shows that eIF2
activity is required for cells to die via oxidative glutamate toxicity and other forms of oxidative stress: (a) eIF2
fragments rescued from the glutamate-resistant cells make wild-type cells resistant to glutamate upon reinfection; (b) Western blotting demonstrates that the eIF2
protein levels in the resistant clones are lower than in wild-type HT22 cells; and (c) eIF2
downregulation alone causes resistance to glutamate since clones 8 and 15, when transfected with full-length human eIF2
, become glutamate sensitive. Since eIF2
regulates the rate of protein translation and cell death requires protein synthesis, it is possible that the inhibition of cell death simply reflects a decrease in the rate of protein synthesis in the resistant cells. However, the decrease of eIF2
in the resistant cells did not necessarily lead to a slower rate of protein synthesis. Although clones 8 and 15 are equally resistant to glutamate, only clone 8 has a rate of protein synthesis which is lower than that in the wild-type cells. In addition, cells infected with the eIF2
phosphorylation mutant S51D, which also induces glutamate resistance, synthesize protein at a rate that is equal to that of the wild-type cells. These results indicate that a decrease in the rate of translation per se does not lead to glutamate resistance. Further evidence that eIF2
phosphorylation plays a key role in determining the fate of the glutamate-exposed HT22 cells is evident when the S51D mutant of eIF2
is expressed in the HT22 cells, resulting in glutamate resistance. The S51D mutant mimics a constitutively phosphorylated form of eIF2
that cannot be dephosphorylated, such that it is able to sequester the guanine nucleotide exchange factor, eIF2B, and inhibit the initiation of protein synthesis (
or the phosphorylation mutants leads to overexpression of their respective transcripts but does not alter the overall levels of eIF2
protein (data not shown), the amount of eIF2
protein that is synthesized must be highly regulated. In contrast to our data, the S51D mutant causes apoptosis when transiently transfected into another cell line (
eIF2 Downregulation and the Constitutively Phosphorylated Form of eIF2
Alter the Same Intermediates in the Cell Death Pathway
The observation that the two glutamate-resistant clones selected by expression cloning and the overexpression of the phosphorylation mutant, S51D, produce similar changes in cell physiology during glutamate exposure further supports the critical role of eIF2 in the toxicity cascade. These cell lines all exhibit higher GSH levels than controls after glutamate exposure and lower levels of ROS and intracellular Ca2+. GSH levels in wild-type HT22 cells decline to <20% of controls after glutamate exposure, whereas GSH levels in both the resistant clones and the cells expressing the dominant negative S51D mutant drop to <50% of their basal levels. In contrast to control levels, this level of GSH is sufficient to maintain cell viability (
The above results suggest that the downregulation or phosphorylation of eIF2 during times of stress signals the translation of specific proteins that increase cell survival. Since decreases in either eIF2
activity or protein levels both lead to an increase in GSH, we asked if the rate-limiting enzyme in GSH production,
GCS, was increased in the resistant cells compared with the wild-type HT22 cells. Fig 7 shows that although the amount of
GCS is increased in the original resistant clones, the
GCS mRNA level remains constant and there is no difference in the rates of
GCS breakdown. In addition,
GCS is upregulated by the phosphorylation mutant, S51D, and downregulated by the introduction of additional eIF2
into the glutamate-resistant clones 8 and 15 (Fig 7). These data show that eIF2
regulates
GCS expression by a translational mechanism. Amino acid starvation in Saccharomyces cerevisiae also causes eIF2
phosphorylation and leads to the selective translation of one specific transcription factor that signals the synthesis of amino acids so that the yeast can survive starvation (
activity is low, leading to an increased production of
GCS to promote cell survival. In addition, it was recently shown that another form of stress, the unfolded protein response, causes the phosphorylation of
IF2
and the increased translation of activating transcription factor 4 (
eIF2 Plays a Unique Role in Programmed Cell Death
There have been several reports that positively link eIF2 to apoptosis: eIF2
phosphorylation by double-stranded RNAactivated protein kinase is the cause of cell death in TNF-
stimulated cells (
is cleaved by caspases after an increase in PKR kinase activity induced by TNF-
or poly(I):poly(C) (
, indicating that they utilize a survival mechanism that is unique to oxidative stress. Ischemia and reperfusion in the rat brain also lead to eIF2
phosphorylation and cell death (
phosphorylation, protein synthesis shutdown, and cell lysis. In contrast, our data show that eIF2
phosphorylation protects cells from death. HT22 cells treated with thapsigargin, a substance shown to cause eIF2
phosphorylation (
phosphorylation determine whether eIF2
will be used to prevent or promote cell death. The above experiments link oxidative stress, GSH depletion, and the regulation of
GCS directly to eIF2
and programmed nerve cell death. Markers for both oxidative stress and the depletion of intracellular GSH are found in areas of central nervous system nerve cell death in PD (
, can regulate the ability of a nerve cell to deal with oxidative stress. This appears to be primarily done through the regulation of GSH levels, as sustained GSH depletion is the initial event which triggers downstream events such as peroxide accumulation and ultimately cell death. Cells with low amounts of eIF2
or phosphorylated eIF2
maintain high levels of GSH when stressed and do not die. These results point to a central role of eIF2
as a translational switch in the control of oxidative stress within the nervous system. They also suggest a possible therapeutic target for manipulating intracellular GSH levels.
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Footnotes |
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1 Abbreviations used in this paper: AD, Alzheimer's disease; DCF, dichlorofluorescein; eIF, translation initiation factor; GSH, glutathione; GCS, gamma-glutamyl cysteine synthetase; MTT, 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; PCD, programmed cell death; PD, Parkinson's disease; ROS, reactive oxygen species.
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Acknowledgements |
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The authors would like to thank Dr. S. Miyamoto for giving us the human cDNA for eIF2, Dr. R. Kaufman for the S51A and S51D mutants of eIF2
, and Dr. J. Hershey for sending us a polyclonal antibody against eIF2
. We are also grateful to Dr. H.J. Forman for supplying us with his antibody against the catalytic subunit of
GCS. We thank R. Dargusch, T. Soucek, and Dr. M. Pando for their constructive comments and reading of the manuscript. Finally, we thank Dr. Inder Verma for his contributions and for his support of the work done by N. Somia.
This work was supported by the Edward C. Johnson Fund, the National Institutes of Health, and Department of Defense grants to D. Schubert, and the Bundy Foundation fellowship and the American Association of University Women dissertation fellowship to S. Tan.
Submitted: 28 November 2000
Revised: 11 January 2001
Accepted: 18 January 2001
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References |
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