Levels Of eEF-2 Research Articles

Hsp90 and cochaperones have two genetically distinct roles in regulating eEF2 function.

Protein homeostasis relies on the accurate translation and folding of newly synthesized proteins. Eukaryotic elongation factor 2 (eEF2) promotes GTP-dependent translocation of the ribosome during translation. eEF2 folding was recently shown to be dependent on Hsp90 as well as the cochaperones Hgh1, Cns1, and Cpr7. We examined the requirement for Hsp90 and cochaperones more closely and found that Hsp90 and cochaperones have two distinct roles in regulating eEF2 function. Yeast expressing one group of Hsp90 mutations or one group of cochaperone mutations had reduced steady-state levels of eEF2. The growth of Hsp90 mutants that affected eEF2 accumulation was also negatively affected by deletion of the gene encoding Hgh1. Further, mutations in yeast eEF2 that mimic disease-associated mutations in human eEF2 were negatively impacted by loss of Hgh1 and growth of one mutant was partially rescued by overexpression of Hgh1. In contrast, yeast expressing different groups of Hsp90 mutations or a different cochaperone mutation had altered sensitivity to diphtheria toxin, which is dictated by a unique posttranslational modification on eEF2. Our results provide further evidence that Hsp90 contributes to proteostasis not just by assisting protein folding, but also by enabling accurate translation of newly synthesized proteins. In addition, these results provide further evidence that yeast Hsp90 mutants have distinct in vivo effects that correlate with defects in subsets of cochaperones.

Depression-like effects induced by chronic unpredictable mild stress in mice are rapidly reversed by a partial negative allosteric modulator of mGlu5 receptor, M-5MPEP.

Due to the numerous limitations of ketamine as a rapid-acting antidepressant drug (RAAD), research is still being conducted to find an effective and safe alternative to this drug. Recent studies indicate that the partial mGlu5 receptor negative allosteric modulator (NAM), 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP), has therapeutic potential as an antidepressant. The study aimed to investigate the potential rapid antidepressant-like effect of M-5MPEP in a mouse model of depression and to determine the mechanism of this action. Chronic unpredictable mild stress (CUMS) was used as an animal model of depression. The effects of single and four-day administration of M-5MPEP on CUMS-induced animal behaviors reflecting anhedonia, apathy, and helplessness were studied. Western blot was applied to measure the levels of proteins potentially involved in a rapid antidepressant effect, including mammalian target of rapamycin (mTOR), eukaryotic elongation factor 2 (eEF2), tropomyosin receptor kinase B (TrkB), and serotonin transporter (SERT), both in the hippocampus and the prefrontal cortex (PFC). Furthermore, excitatory synaptic transmission and long-term potentiation (LTP) were measured in the medial PFC (mPFC). We showed that M-5MPEP administration for four consecutive days abolished CUMS-induced apathy- and anhedonia-like symptoms in a mouse model of depression. We also found that these effects were accompanied by changes in hippocampal TrkB levels and mTOR and eEF2 levels in the PFC. Using electrophysiological techniques, we showed that the four-day M-5MPEP treatment reversed chronic stress-induced increases in excitatory synaptic potential and CUMS-impaired LTP in the mPFC. Partial mGlu5 receptor NAM, M-5MPEP, appears to be a potentially effective new RAAD and deserves further study.

Ribosome inactivation regulates translation elongation in neurons

Cellular plasticity is crucial for adapting to ever-changing stimuli. As a result, cells consistently reshape their translatome, and, consequently, their proteome. The control of translational activity has been thoroughly examined at the stage of translation initiation. However, the regulation of ribosome speed in cells is widely unknown. In this study, we utilized a timed ribosome runoff approach along with proteomics and transmission electron microscopy, to investigate global translation kinetics in cells. We found that ribosome speeds vary amongst various cell types, such as astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Of all cell types studied, mature cortical neurons exhibit the highest rate of translation. This finding is particularly remarkable because mature cortical neurons express eEF2 at lower levels than other cell types. Neurons solve this conundrum by inactivating a fraction of their ribosomes. As a result, the increase in eEF2 levels leads to a reduction of inactive ribosomes and an enhancement of active ones. Processes that alter the demand for active ribosomes, like neuronal excitation, cause increased inactivation of redundant ribosomes in an eEF2-dependent manner.Our data suggest a novel regulatory mechanism in which neurons dynamically inactivate ribosomes to facilitate translational remodelling. These findings have important implications for developmental brain disorders characterised by, among other things, aberrant translation.

Inositol hexaphosphate enhances chemotherapy by reversing senescence induced by persistently activated PERK and diphthamide modification of eEF2

Oxaliplatin is an important initial chemotherapy benefiting advanced-stage colorectal cancer patients. Frustratingly, acquired oxaliplatin resistance always occurs after sequential chemotherapy with diverse antineoplastic drugs. Therefore, an exploration of the mechanism of oxaliplatin resistance formation in-depth is urgently needed. We generated oxaliplatin-resistant colorectal cancer models by four representative compounds, and RNA-seq revealed that oxaliplatin resistance was mainly the result of cells’ response to stimulus. Moreover, we proved persistent stimulus-induced endoplasmic reticulum stress (ERs) and associated cellular senescence were the core causes of oxaliplatin resistance. In addition, we screened diverse phytochemicals for ER inhibitors in silico, identifying inositol hexaphosphate (IP6), whose strong binding was confirmed by surface plasmon resonance. Finally, we confirmed the ability of IP6 to reverse colorectal cancer chemoresistance and investigated the mechanism of IP6 in the inhibition of diphthamide modification of eukaryotic elongation factor 2 (eEF2) and PERK activation. Our study demonstrated that oxaliplatin resistance contributed to cell senescence induced by persistently activated PERK and diphthamide modification of eEF2 levels, which were specifically reversed by combination therapy with IP6.

ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α-kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.

Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs.

Clinical Value of Eukaryotic Elongation Factor 2 (eEF2) in Non-small Cell Lung Cancer Patients

The purpose of this study was to evaluate a new type of tumor biomarker, eukaryotic elongation factor 2 (eEF2), in serum for the early diagnosis, confirmative diagnosis as well as assessment of treatment of non-small cell lung cancer (NSCLC). 130 patients with NSCLC and 50 healthy individuals undergoing physical examination in our hospital provided the observation and healthy control groups. An enzyme linked immune sorbent assay (ELISA) method was applied to determine serum eEF2 levels. Serum neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCC) levels in the observation group were assessed with an automatic biochemical analyzer. The median levels of eEF2 in the serum of NSCLC patients was found to be significantly higher than the healthy control group (p < 0.01) and it was markedly higher in stages III, IV than stages I, II (p < 0.05). eEF2 was higher with tumor size ≥ 2 cm than <2 cm (P< 0.01). Furthermore, two weeks after surgery patients showed a significant trend for eEF2 decrease (p < 0.05). The eukaryotic elongation factor 2 (eEF2) has certain clinical values for early diagnosis, verification, and prognosis as well as classification of lung cancer patients.

Molecular control of the amount, subcellular location, and activity state of translation elongation factor 2 in neurons experiencing stress

Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery whereby it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability and by posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated, resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with nonphosphorylated (active) eEF-2, but not with its phosphorylated form. The p53–eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2 and decreased cell death after exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress.

In vitro and in vivo protection by melatonin against the decline of elongation factor‐2 caused by lipid peroxidation: preservation of protein synthesis

As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). eEF-2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF-2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF-2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4-hydroxynoenal (HNE). We have previously reported that hepatic eEF-2 forms adducts with low-molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF-2 must be specifically carried out by a compound with lipoperoxyl radical-scavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF-2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF-2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF-2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH-induced oxidative stress, which was partially or totally prevented by melatonin.

Impaired associative taste learning and abnormal brain activation in kinase-defective eEF2K mice

Memory consolidation is defined temporally based on pharmacological interventions such as inhibitors of mRNA translation (molecular consolidation) or post-acquisition deactivation of specific brain regions (systems level consolidation). However, the relationship between molecular and systems consolidation are poorly understood. Molecular consolidation mechanisms involved in translation initiation and elongation have previously been studied in the cortex using taste-learning paradigms. For example, the levels of phosphorylation of eukaryotic elongation factor 2 (eEF2) were found to be correlated with taste learning in the gustatory cortex (GC), minutes following learning. In order to isolate the role of the eEF2 phosphorylation state at Thr-56 in both molecular and system consolidation, we analyzed cortical-dependent taste learning in eEF2K (the only known kinase for eEF2) ki mice, which exhibit reduced levels of eEF2 phosphorylation but normal levels of eEF2 and eEF2K. These mice exhibit clear attenuation of cortical-dependent associative, but not of incidental, taste learning. In order to gain a better understanding of the underlying mechanisms, we compared brain activity as measured by MEMRI (manganese-enhanced magnetic resonance imaging) between eEF2K ki mice and WT mice during conditioned taste aversion (CTA) learning and observed clear differences between the two but saw no differences under basal conditions. Our results demonstrate that adequate levels of phosphorylation of eEF2 are essential for cortical-dependent associative learning and suggest that malfunction of memory processing at the systems level underlies this associative memory impairment.

Thermal Injury Activates the eEF2K‐Dependent eEF2 Pathway in Pediatric Patients

Burn induces a hypermetabolic state characterized by alterations in protein metabolism, which is associated with increased morbidity and mortality. Eukaryotic elongation factor 2 (eEF2) plays a crucial role in regulating protein synthesis in many diseases, but whether it participates in burn-induced hypermetabolism is unclear. The aim of this study was to determine the expression of eEF2 and the upstream eEF2-inactivating kinase, eEF2K, in severely burned pediatric patients. Eight pediatric patients (> 40% total body surface area) and 3 nonburned pediatric volunteers were enrolled. Muscle and skin biopsies were collected at early (0-10 days postburn [dpb]), middle (11-49 dpb), and late (50-365 dpb) time points. Resting energy expenditure (REE), body composition, and muscle protein fractional synthesis rate (FSR) were measured. Proteins were extracted and analyzed by Western blotting. To further investigate the protein synthesis pathway, microarray data from muscle and skin were examined from 22 nonburned and 20 burned children. Burn patients exhibited a profound hypermetabolic response, as seen by a significant increase in REE (P < .05) and loss of lean body mass without altered muscle FSR, indicating a shift to catabolism after thermal injury. In muscle, the phosphorylation of eEF2K-dependent eEF2 was down regulated early and middle postburn. Similar changes in eEF2K and eEF2 levels occurred in skin at the early time point. Total amounts of eEF2 and eEF2K were not altered. Burn induces prolonged activation of eEF2K and eEF2. Alterations in these mediators may contribute to profound hypermetabolism in severely burned patients.

Effect of aging and oxidative stress on elongation factor-2 in hypothalamus and hypophysis

The hypothalamic–hypophysis system (HHS) secretes peptide hormones whose synthesis requires the integrity of the translation machinery. As the organisms age, a considerable diminution of the protein synthesis takes place in several tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). We studied whether the level of this protein was affected in the HHS in old animals. The effects of aging are compared to those of an oxidant compound (cumene hydroperoxide) administered to young rats. The results indicate that oxidative stress could be involved in the alterations of eEF-2, which forms adducts with malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The alterations of eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes could contribute to the suboptimal hormone production from these tissues during aging. Besides eEF-2, proteomic analysis shows that several other proteins are affected.

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Adduct formation of 4-hydroxynonenal and malondialdehyde with elongation factor-2 in vitro and in vivo

Protein synthesis is universally affected by aging in all organisms. There is no clear consensus about the mechanism underlying the decline of translation with aging. Previous reports from our laboratory have shown that the elongation step is especially affected with aging as a consequence of alterations in elongation factor-2 (eEF-2), the monomeric protein that catalyzes the movement of the ribosome along the mRNA during protein synthesis. eEF-2 seems to be specifically affected by lipid peroxidant compounds, which concomitantly produce several reactive, toxic aldehydes, such as MDA and HNE. These aldehydes are able to form adducts with proteins that lead to their inactivation. In this paper we studied the formation of adducts between MDA or HNE and eEF-2. The study was performed both in vitro, using liver homogenates treated with cumene hydroperoxide, and in vivo using young control rats, treated with the same oxidant, and 12-and 24-month-old rats. In all cases we found a decrease in the levels of eEF-2, an increase in the amount of lipid peroxidation, and a concomitant formation of adducts between eEF-2 and MDA or HNE. The results suggest that one possible mechanism responsible for the decline of protein synthesis during aging could be the alteration in eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes.

‘In vitro’ Protective Effect of a Hydrophilic Vitamin E Analogue on the Decrease in Levels of Elongation Factor 2 in Conditions of Oxidative Stress

Background: Protein synthesis is inhibited by oxidative stress. Among the possible causes of this inhibition are the modifications of elongation factor 2 (eEF-2), the protein that catalyzes the translocation of the ribosome through mRNA. eEF-2 is extremely sensitive to oxidative stress caused mainly by lipid peroxidant compounds such as cumene hydroperoxide (CH). Objective: The purpose of this study was to determine whether the antioxidant Trolox prevents the effect of CH on the levels of hepatic eEF-2. Methods: The effect was determined in liver homogenates treated with both compounds. Lipid peroxides and carbonyl content were also measured. Results: The results show that Trolox at certain doses prevents the decrease in the level of eEF-2 caused by CH. Conclusion: Under oxidative stress circumstances, vitamin E can prevent the effect of oxidations on relevant biological processes such as protein synthesis.

218 POSTER Phase I/II study of CNTO 95, a fully human monoclonal antibody (mAb) to alpha-v integrins, in patients with metastatic melanoma

Protein synthesis is universally affected by aging in all organisms. There is no clear consensus about the mechanism underlying the decline of translation with aging. Previous reports from our laboratory have shown that the elongation step is especially affected with aging as a consequence of alterations in elongation factor-2 (eEF-2), the monomeric protein that catalyzes the movement of the ribosome along the mRNA during protein synthesis. eEF-2 seems to be specifically affected by lipid peroxidant compounds, which concomitantly produce several reactive, toxic aldehydes, such as MDA and HNE. These aldehydes are able to form adducts with proteins that lead to their inactivation. In this paper we studied the formation of adducts between MDA or HNE and eEF-2. The study was performed both in vitro, using liver homogenates treated with cumene hydroperoxide, and in vivo using young control rats, treated with the same oxidant, and 12-and 24-month-old rats. In all cases we found a decrease in the levels of eEF-2, an increase in the amount of lipid peroxidation, and a concomitant formation of adducts between eEF-2 and MDA or HNE. The results suggest that one possible mechanism responsible for the decline of protein synthesis during aging could be the alteration in eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes.

Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast

The eukaryotic translation elongation factor 2 (eEF2), a member of the G-protein superfamily, catalyzes the post-peptidyl transferase translocation of deacylated tRNA and peptidyl tRNA to the ribosomal E- and P-sites. eEF2 is modified by a unique post-translational modification: the conversion of His699 to diphthamide at the tip of domain IV, the region proposed to mimic the anticodon of tRNA. Structural models indicate a hinge is important for conformational changes in eEF2. Mutations of V488 in the hinge region and H699 in the tip of domain IV produce non-functional mutants that when co-expressed with the wild-type eEF2 result in a dominant-negative growth phenotype in the yeast Saccharomyces cerevisiae. This phenotype is linked to reduced levels of the wild-type protein, as total eEF2 levels are unchanged. Changes in the promoter, 5′-untranslated region (5′-UTR) or 3′-UTR of the EFT2 gene encoding eEF2 do not allow overexpression of the protein, showing that eEF2 levels are tightly regulated. The H699K mutant, however, also alters translation phenotypes. The observed regulation suggests that the cell needs an optimum amount of active eEF2 to grow properly. This provides information about a new mechanism by which translation is efficiently maintained.

Does phosphorylation of eukaryotic elongation factor eEF2 regulate protein synthesis in ischemic preconditioning?

Ischemia/reperfusion-associated translation inhibition in the hippocampus is attenuated significantly at reinitiation and elongation steps by ischemic preconditioning (Burda et al. [2003] Neurochem. Res. 28:1237-1243). To address potential regulation of the elongation step by changes in eukaryotic elongation factor 2 (eEF2) phosphorylation with and without acquired ischemic tolerance (IT), Wistar rats were preconditioned by 5-min sublethal ischemia and 2 days later, 30-min lethal ischemia was induced. Given the important role that oxidative stress plays in the ischemic process, eEF2 phosphorylation was also studied in a model of oxidative stress in vitro. Three blocks of our results support a lack of correlation between eEF2 phosphorylation status and protein synthesis rate. First, eEF2 was dephosphorylated significantly (activated) after transient cerebral ischemia in rats with and without IT or H2O2-treated cells; however, protein synthesis was significantly inhibited under these three conditions. Second, after 30-min reperfusion, the protein synthesis rate was maintained below control levels in cortex and hippocampus of rats without IT. Eukaryotic EF2 phosphorylated levels were notably low only in the cortex, whereas levels in the hippocampus were close to that of sham controls. In rats with IT, protein synthesis was virtually restored in both brain regions, but phosphorylated eEF2 levels were even higher than in rats without IT. Third, after 4-hr reperfusion, the protein synthesis rate in cortex and hippocampus was observed to be below sham control values in rats with and without IT. Conversely, phosphorylated eEF2 levels were below sham control in rats with IT and reached sham control values in rats without IT.