Diminished ERK 1/2 and p38 MAPK phosphorylation in skeletal muscle during sepsis

Sepsis induces weight loss and the loss of skeletal muscle proteins, in part through an inhibition of protein synthesis secondary to an inhibition of the key steps controlling mRNA translation in skeletal muscle. We have previously shown that sepsis decreases the phosphorylation of eIF4E. The present study examines the phosphorylation of Erk 1/2 MAPK and p38 MAPK in skeletal muscle of rats with a chronic (5-day) intra-abdominal septic abscess. Mnk1 catalyzes the phosphorylation of eIF4E, and Mnk1 is activated by phosphorylation via Erk1/2 MAPK and p38 MAPK. Sepsis resulted in a significant decrease in the steady-state phosphorylation of Erk 1/2 and p38 MAPKs compared with sterile inflammation. To examine the mediators responsible for decreased phosphorylation of Erk 1/2 and p38 MAPKs, rats were treated with TNF binding protein (TNFbp) or infused for 24 h with TNF. Treatment of septic rats with TNFbp resulted in an increase in the phosphorylation of both Erk 1/2 and p38 MAPKs in skeletal muscle. This was associated with enhanced phosphorylation of eIF4E. In contrast, constant intravenous infusion of TNF-alpha for 24 h resulted in a complete inhibition of p38 MAPK phosphorylation while Erk 1/2 MAPK phosphorylation was increased. The net effect was a modest increase in eIF4E phosphorylation. The results suggest altered regulation of Erk 1/2 and p38 MAPK signal translation pathways by endogenously produced TNF, or some compound dependent on TNF may modulate, in part, the phosphorylation state of eIF4E in skeletal muscle during sepsis.     

Inhibition of eukaryotic initiation factor 4E phosphorylation by cercosporamide selectively suppresses angiogenesis,growth and survival of human hepatocellular carcinoma

Mnk kinase is required for the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), which regulates translation of proteins involve in important aspects of hepatocellular carcinoma (HCC). Here we investigated whether an antifungal agent, cercosporamide, which had been recently identified as a potent Mnk inhibitor, is active against HCC and angiogenesis. We showed that cercosporamide significantly inhibited growth and induced caspase-dependent apoptosis on numerous HCC cell lines, while sparing normal liver cells. In addition, cercosporamide impaired HCC angiogenesis via inhibiting HCC-endothelial cells (HCC-EC) capillary network formation, migration, proliferation and survival. Importantly, cercosporamide sensitized HCC cells to cisplatin in in vitro cell culture and in vivo HCC xenograft mouse model. Cercosporamide blocked the phosphorylation of eIF4E but not Erk or p38 in a dose- and time-dependent manner in HCC and HCC-EC cells, suggesting that suppression of eIF4E phosphorylation was the result of inhibition of Mnk but not Mnk upstream pathways. Overexpression of constitutively active eIF4E (S209D) but not the nonphosphorylatable eIF4E (S209A) abolished the inhibitory effects of cercosporamide in HepG2 cells. Altogether, our work demonstrates that cercosporamide acts as a Mnk inhibitor through blockage of eIF4E phosphorylation and selectively exhibits anti-HCC activities. Our work suggests that targeting MNK-eIF4E pathway represents a therapeutic strategy to overcome chemo-resistance for HCC treatment.     

Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients

Activation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2alpha. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins - eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function - and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2alpha activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2alpha phosphorylation. Therefore, high expression of PKR and subsequent eIF2alpha phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.     

eIF4E knockdown decreases breast cancer cell growth without activating Akt signaling

Activation of translation initiation is essential for the malignant phenotype and is emerging as a potential therapeutic target. Translation is regulated by the expression of translation initiation factor 4E (eIF4E) as well as the interaction of eIF4E with eIF4E-binding proteins (e.g., 4E-BP1). Rapamycin inhibits translation initiation by decreasing the phosphorylation of 4E-BP1, increasing eIF4E/4E-BP1 interaction. However, rapamycin also inhibits S6K phosphorylation, leading to feedback loop activation of Akt. We hypothesized that targeting eIF4E directly would inhibit breast cancer cell growth without activating Akt. We showed that eIF4E is ubiquitously expressed in breast cancer cell lines. eIF4E knockdown by small interfering RNA inhibited growth in different breast cancer cell subtypes including triple-negative (estrogen receptor/progesterone receptor/HER-2-negative) cancer cells. eIF4E knockdown inhibited the growth of cells with varying total and phosphorylated 4E-BP1 levels and inhibited rapamycin-insensitive as well as rapamycin-sensitive cell lines. eIF4E knockdown led to a decrease in expression of cyclin D1, Bcl-2, and Bcl-xL. eIF4E knockdown did not lead to Akt phosphorylation but did decrease 4E-BP1 expression. We conclude that eIF4E is a promising target for breast cancer therapy. eIF4E-targeted therapy may be efficacious in a variety of breast cancer subtypes including triple-negative tumors for which currently there are no targeted therapies. Unlike rapamycin and its analogues, eIF4E knockdown is not associated with Akt activation.     

Enhanced DNA damage-induced p53 peptide phosphorylation and cell-cycle arrest in Sjögren's syndrome cells

Background Cells from primary Sjögren’s syndrome (SS) patients have been reported to show alterations in DNA repair and p53 expression. The DNA‐dependent protein kinase (DNA‐PK) autoantigen may be involved in both of these alterations in relation to cellular DNA damage responses. We conducted this study of cell‐cycle kinetics and p53 to find additional evidence for an abnormal stress response role in the pathogenesis of SS. Design DNA‐dependent protein kinase activity, p53 peptide phosphorylation and p53 protein levels were determined in gamma‐irradiated long‐term T lymphocyte cultures. Cell‐cycle progression of peripheral blood mononuclear cells was analysed with flow cytometry. Results No significant differences in the DNA‐PK activities or p53 protein levels appeared between the SS patients and the healthy individuals. However, patients with the SS hallmark Ro/SS‐A and La/SS‐B autoantibodies showed enhancement of both p53 peptide phosphorylation (P = 0·036) and G1 cell‐cycle arrest (P = 0·015) in response to gamma radiation. Conclusions Sjögren’s syndrome cells express an enhanced G1 checkpoint function which may be mediated partly by p53 phosphorylation, suggesting that an abnormal stress response in SS is of relevance for the development of this autoimmune disease.     

Leucine acutely reverses burn-induced alterations in translation initiation in heart

Myocardial dysfunction is a common manifestation of thermal injury, the etiology of which appears to be multifactorial. We have previously demonstrated that burn injury impairs cardiac protein synthesis at the level of translation initiation. The purpose of the present study was to determine whether oral administration of leucine, which is known to stimulate translation initiation in skeletal muscle, can ameliorate burn-induced changes in signal transduction pathways known to regulate protein synthesis in cardiac muscle. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied in the fasted condition 24 h later; appropriate time-matched nonburned control rats were also included. Separate groups of control and burn rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined using immunoprecipitation and immunoblotting techniques. Hearts from burned rats demonstrated a redistribution of eIF4E as evidenced by the increased binding of the translational repressor 4E-BP1 with eIF4E, a decreased amount of eIF4E bound with eIF4G, and a decreased amount of the hyperphosphorylated gamma-isoform of 4E-BP1. Furthermore, constitutive phosphorylation of mTOR, the ribosomal protein S6, and eIF4G was also decreased in hearts from burned rats. In control rats, leucine failed to alter eIF4E distribution but did increase the phosphorylation of S6K1 and S6. However, in hearts from burn rats, leucine acutely reversed the alterations in eIF4E distribution as well as the changes in S6, eIF4G, and mTOR phosphorylation. These data suggest that oral administration of leucine can acutely reverse multiple defects in cardiac translation initiation produced by thermal injury.     

Interaction of Pdcd4 with eIF4E inhibits the metastatic potential of hepatocellular carcinoma

Oxidative stress can contribute to the development of hepatocellular carcinoma (HCC) ability of the carcinoma. It has been found that oxidative stress stimulates the phosphorylation of eIF4E primarily through mitogen-activated protein kinase (MAPK) pathways resulting in increased protein translation. Utilizing specific inhibitors of MAPK pathways (SP600125 for c-Jun amino-terminal kinases [JNKs], PD098059 for extracellular signal-regulated kinases [ERKs], and SB203580 for p38 MAPK), we determined that it is primarily the inhibition of JNK that results in the suppression of the increase of p-eIF4E. We also found that PDCD4 inhibits JNK activity resulting in inhibition of the phosphorylation of c-Jun, one isoform of AP-1. We demonstrated that transfection with PDCD4 or inhibition of JNK by SP600125 alters the expression and phosphorylation of eIF4E in the presence of H₂O₂. PDCD4 results in a stronger inhibitory effect than SP600125.     

Modulation of 4E-BP1 function as a critical determinant of enzastaurin-induced apoptosis

Enzastaurin (LY317615.HCl) is currently in a phase III registration trial for diffuse large B-Cell lymphoma and numerous phase II clinical trials. Enzastaurin suppresses angiogenesis and induces apoptosis in multiple human tumor cell lines by inhibiting protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K)/AKT pathway signaling. PI3K/AKT pathway signaling liberates eukaryotic translation initiation factor 4E (eIF4E) through the hierarchical phosphorylation of eIF4E binding proteins (4E-BP). When hypophosphorylated, 4E-BPs associate with eIF4E, preventing eIF4E from binding eIF4G, blocking the formation of the eIF4F translation initiation complex. Herein, we show that enzastaurin treatment impacts signaling throughout the AKT/mTOR pathway leading to hypophosphorylation of 4E-BP1 in cancer cells of diverse lineages (glioblastoma, colon carcinoma, and B-cell lymphoma). Accordingly, enzastaurin treatment increases the amount of eIF4E bound to 4E-BP1 and decreases association of eIF4E with eIF4G, thereby reducing eIF4F translation initiation complex levels. We therefore chose to evaluate whether this effect on 4E-BP1 was involved in enzastaurin-induced apoptosis. Remarkably, enzastaurin-induced apoptosis was blocked in cancer cells depleted of 4E-BP1 by siRNAs, or in 4EBP1/2 knockout murine embryonic fibroblasts cells. Furthermore, eIF4E expression was increased and 4E-BP1 expression was decreased in cancer cells selected for reduced sensitivity to enzastaurin-induced apoptosis. These data highlight the importance of modulating 4E-BP1 function, and eIF4F complex levels, in the direct antitumor effect of enzastaurin and suggest that 4E-BP1 function may serve as a promising determinant of enzastaurin activity.     

MAP Kinase cross talks in oxidative stress‐induced impairment of insulin secretion. Involvement in the protective activity of quercetin

Insulin secretion preservation is a major issue for the prevention or treatment of type 2 diabetes. We previously showed on β‐cells that quercetin (Q), but not resveratrol (R) or N‐acetyl cysteine (NAC), amplified glucose‐induced insulin secretion in a calcium‐ and ERK1/2‐dependent manner. Quercetin, but not resveratrol or NAC, also protected β‐cell function and hyperamplified ERK1/2 phosphorylation in oxidative stress conditions. As quercetin may interfere with other stress‐activated protein kinases (JNK and p38 MAPK), we further explored MAPK cross talks and their relationships with the mechanism of the protective effect of quercetin against oxidative stress. In INS‐1 insulin‐secreting β‐cells, using pharmacological inhibitors of MAPK pathways, we found that under oxidative stress (50 μm H₂O₂) and glucose‐stimulating insulin secretion conditions: (i) p38 MAPK phosphorylation was increased and regulated by ERK1/2 (positively) and JNK (negatively), although p38 MAPK activation did not seem to play any significant role in oxidative stress‐induced insulin secretion impairment; (ii) the JNK pathway appeared to inhibit both ERK1/2 activation and insulin secretion, although JNK phosphorylation was not significantly changed in our experimental conditions; (iii) the functionality of β‐cell in the presence of oxidative stress was closely linked to the level of ERK1/2 activation, (iv) quercetin, resveratrol, or NAC inhibited H₂O₂‐induced p38 MAPK phosphorylation. The preservation of β‐cell function against oxidative stress appears dependent on the balance between ERK1/2 and JNK activation. The protecting effect of quercetin appears due to ERK1/2 hyperactivation, possibly induced by L‐type calcium channel opening as we recently showed (Br. J. Pharmacol. 2013, 169, 1102–1113).     

Expression of Ac-PK2 protein from AcMNPV improved the progeny virus production via regulation of energy metabolism and protein synthesis

Baculovirus encoded PK2 protein can increase viral fitness through inhibition of the eIF2α family kinases activity. Previous studies indicated that the virus might take over the control of cellular machinery post-infection, which would impose a high metabolic burden to infected insect cells. Here we showed that eIF2α phosphorylation decreased, with concomitant up-regulation of total and heterologous protein synthesis in AcMNPV-PK2-EGFP infected Sf9 cells and the larvae of Spodoptera exigua. Simultaneously, the lactic acid accumulation decreased and the uptake of glucose increased in AcMNPV-PK2-EGFP infected Sf9 cells. We proposed a model that Ac-PK2 protein overexpression would help protein synthesis by inhibiting eIF2α phosphorylation, which provided a more favorable scenario to support the efficient replication of the virus by re-directing the cellular metabolism toward ATP production. Finally, we confirmed that AcMNPV-PK2-EGFP could improve the production of progeny virus in infected Sf9 cells and enhance insecticidal activity against Spodoptera exigua larvae.

Baculovirus encoded PK2 protein can increase viral fitness through inhibition of the eIF2α family kinases activity.     

Cover Image,Volume 13, Issue 6

The purpose of this study is to evaluate the effect of extracorporeal shock wave therapy (ESWT) according to treatment timing in rabbits with 10% dextrose‐induced carpal tunnel syndrome (CTS); 0.1‐ml 10% dextrose solution was injected under ultrasound guidance twice weekly to the left forepaw subsynovial connective tissue (SSCT) within the carpal tunnel of 36 New Zealand white rabbits to induce CTS. The rabbits were randomly allocated into four groups: G1‐S (sham ESWT), G2‐E4 (ESWT at 4 weeks), G3‐E8 (at 8 weeks), and G4‐E16 (at 16 weeks). Radial ESWT (500 pulses, 0.08 mJ/mm², 2 Hz) was repeated thrice weekly. Median nerve distal motor latency (DML) was measured before injection and at 4, 8, 12, 16, and 20 weeks after the first injection. All rabbits were sacrificed 20 weeks after injection. The median nerve cross‐sectional area (CSA) and SSCT thickness were measured with light microscopy. The mean median nerve DML at 4 weeks after the first dextrose injection did not differ from that at preinjection in all groups. The mean median nerve DML significantly increased before ESWT in all groups (p < .05); however, it did not increase in G2‐E4 and G3‐E8 for 12 weeks after ESWT and in G4‐E16 for 4 weeks (p > .05). Mean CSA of the median nerve and mean SSCT thickness in G2‐E4 were significantly lower than those in the other groups (p < .05). ESWT may prevent the progression of CTS for 12 weeks in rabbits with dextrose‐induced CTS regardless of treatment timing, and early application results in superior outcomes.     

MiR-202-5p attenuates neurological deficits and neuronal injury in MCAO model rats and OGD-induced injury in Neuro‐2a cells by targeting eIF4E-mediated induction of autophagy and inhibition of Akt/GSK-3β pathway

Ischemic stroke is a common cerebrovascular disease caused by insufficient blood supply to the brain. In recent years, studies have demonstrated that microRNAs (miRNAs) are involved in a variety of biological processes in the nervous system. However, the effects of miR-202-5p on cerebral ischemic stroke injury have not been completely elucidated. In our study, N2a cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, and middle cerebral artery occlusion (MCAO) rat models were constructed. Our results indicated that decreased miR-202-5p expression was connected to N2a cells after OGD/R-induced injury and rats after MCAO. In addition, high miR-202-5p expression increased proliferation and prevented apoptosis and autophagy of OGD/R-treated N2a cells, while also effectively decreasing the infarct volume in MCAO model rats. We validated the interplay between miR-202-5p and eukaryotic translation initiation factor 4E (eIF4E), and found that miR-202-5p downregulated eIF4E by targeted combination. Moreover, we demonstrated that miR-202-5p accelerated proliferation and suppressed autophagy of OGD/R-induced N2a cells by targeting eIF4E. Meanwhile, our other results suggest that upregulation of miR-202-5p may activate the Akt/GSK-3β pathway in ischemic brain injury. Our findings suggest that miR-202-5p may serve as a protective agent for ischemia-reperfusion injury in stroke via eIF4E.     

MAPK Signal-integrating Kinase Controls Cap-independent Translation and Cell Type-specific Cytotoxicity of an Oncolytic Poliovirus

Many animal viruses exhibit proficient growth in transformed cells, a property that has been harnessed for the development of novel therapies against cancer. Despite overwhelming evidence for this phenomenon, understanding of the molecular mechanisms enabling tumor-cell killing is rudimentary for most viruses. We report here that growth and cytotoxicity of the prototype oncolytic poliovirus (PV), PVSRIPO, in glioblastoma multiforme (GBM) is promoted by mitogen-activated protein kinases (MAPKs) converging on the MAPK signal-integrating kinase 1 (Mnk1) and its primary substrate, the eukaryotic initiation factor (eIF) 4E. Inducing Mnk1-catalyzed eIF4E phosphorylation through expression of oncogenic Ras substantially enhanced PVSRIPO translation, replication, and cytotoxicity in resistant cells. This effect was mimicked by expression of constitutively active forms of Mnk1 and correlated with enhanced translation of subgenomic reporter RNAs. Our findings implicate Mnk1 activity in stimulation of PVSRIPO cap-independent translation, an effect that can be synergistically enhanced by inhibition of the phosphoinositide-3 kinase (PI3K).     

电子克隆结合RT-PCR获得两条eIF4E剪接变异体的全长cDNA

本研究旨在克隆急性白血病复发相关的新EST片段全长cDNA并进行生物信息学分析。电子克隆结合RT—PCR克隆新EST片段的全长cDNA,利用生物信息学技术分析所获得的cDNA。结果获得全长序列为1904bp和3393bp的两条真核细胞翻译起始因子4E（eukaryotic initiation factor4E,eIF4E）新的剪接变异体,命名为真核细胞翻译起始因子剪接变异体1（splicing variant 1 of eIF4E）和真核细胞翻译起始因子剪接变异体2（splicing variant 2 of eIF4E）。编码的蛋白产物分别为245和132个氨基酸。二者编码的蛋白产物与eIF4E蛋白有所不同。结论：获得了两条eIF4E剪接变异体的全长cDNA,这为进一步探讨与急性白血病复发的相关性提供基础。     

Apolipoprotein-B subclasses as acceptors of cholesteryl esters transferred by CETP

Background Five apolipoprotein (apo)‐defined apoB‐containing lipoprotein (Lp) subclasses designated LpB, LpB:C, LpB:E, LpB:C:E and LpA‐II:B:C:D:E are present in human plasma. This study was to determine whether these subclasses functioned equally as acceptors of cholesteryl esters (CE) transferred from high‐density lipoproteins (HDL) by CE transfer protein in healthy subjects with normal and mildly increased plasma triglyceride (TG) levels. Materials and methods After 4 h incubation of plasma from 14 subjects at 37 °C, apoB‐containing lipoproteins were separated from HDL by heparin‐Mn⁺⁺ precipitation and fractionated by immunochemical methods into these five subclasses. The neutral lipid (NL) composition for each subclass was measured by gas chromatography (GC) and compared between 0 h and 4 h. A subclass was considered to be a CE acceptor if its CE content increased more than 5% at 4 h and a non‐acceptor if no change was observed. Results Employing the above definition, TG‐rich LpB:C and LpB:E + LpB:C:E functioned as CE acceptors and TG‐poor LpB and LpA‐II:B:C:D:E as non‐acceptors. Both LpB:C and LpB:E + LpB:C:E could only actively accept CE as long as they retained their TG‐rich character and displayed neutral lipid profiles similar to those of very low‐density lipoproteins (VLDL) and intermediate density lipoproteins (IDL). When, as a result of lipolysis their TG content dropped below 25%, they ceased to function as CE acceptors. In subjects with elevated plasma TG, LpB:C was the dominant CE acceptor, a condition that may have pro‐atherogenic consequences. Conclusions Among the apoB‐containing particles, LpB:C and LpB:C:E + LpB:E functioned as CE acceptors while LpB and LpA‐II:B:C:D:E did not.     

苦参碱抑制宫颈癌HeLa细胞增殖作用及机制

目的探讨苦参碱对人宫颈癌HeLa细胞体外增殖的抑制作用及其分子机制。方法以不同浓度（0.5、1.0、1.5、2.0g/L）的苦参碱分别处理HeLa细胞24、48、72h后,应用MTT法检测细胞增殖抑制率,WesternBlot方法检测培养48h细胞中真核细胞翻译起始因子4E（eIF4E）、4E结合蛋白1（4E-BP1）蛋白表达情况;1.0g/L的苦参碱作用HeLa细胞1、3、6、12h后,Western Blot方法测其eIF4E、4E-BP1蛋白磷酸化水平。结果苦参碱明显抑制HeLa细胞增殖,且呈时间和剂量依赖性：在相同时间内,随着苦参碱浓度的增加,HeLa细胞的增殖抑制率明显升高（F=235.035,P〈0.05）;在同一浓度时,随着苦参碱作用时间的延长,HeLa细胞的增殖抑制率也明显升高（F=320.207,P〈0.05）;2.0g/L苦参碱作用72h对HeLa细胞增殖抑制率最大,为（65.30±2.17）%。不同浓度苦参碱处理HeLa细胞48h后,eIF4E、4E-BP1蛋白的表达均无明显变化（F=0.171、0.932,P〉0.05）。1.0g/L苦参碱作用于HeLa细胞不同时间后,细胞中eIF4E、4E-BP1蛋白磷酸化水平均降低,且呈时间依赖性（F=43.48、107.23,P〈0.01）。结论苦参碱可以明显抑制体外培养宫颈癌HeLa细胞的增殖,其作用可能是通过抑制eIF4E以及4E-BP1的磷酸化,从而抑制蛋白翻译来实现的。     

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m⁷GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry–based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.     

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT‐PCR using degenerate primers based on the N‐terminal amino acid sequence of purified hJHBP and a conserved region near the C‐terminus of other lepidopteran hJHBPs. 5′‐ and 3′‐ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP‐mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS‐PAGE with a M_r of 32 kDa and showed a K_d of 4.5 × 10^?7 M with JH III, both similar to those of native hJHBP.     

Ubiquitin-dependent degradation of p53 protein despite phosphorylation at its N terminus by acetaminophen

We previously reported that acetaminophen (APAP, 4-hydroxyacetanilide) caused apoptosis of C6 glioma cells. Therefore, we hypothesized that the level of p53, which usually stimulates apoptosis, might be increased after APAP exposure. However, APAP exposure for 24 h markedly decreased the p53 content and its downstream target p21 in a concentration-dependent manner. Reduction of p53 was not accompanied by a decrease in p53 mRNA in C6 glioma cells, suggesting that p53 was mainly affected at the protein level. Unexpectedly, APAP stimulated phosphorylation of p53 at Ser15, Ser20, and Ser37, which usually elevates p53 content. However, phosphorylation of these residues did not prevent APAP-induced decrease in p53. The p53 reduction was independent from the level of phospho-Akt, which is known to promote p53 degradation. Immunoblot analysis of the immunoprecipitated p53 revealed that increased amounts of murine double minute 2 (mdm2) and ubiquitin were bound to p53 during its degradation. Lactacystin and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), inhibitors of proteasomal proteolysis, prevented the decrease, supporting the proteasomal degradation of p53 upon APAP exposure. Pretreatment with chlormethiazole, an inhibitor of ethanol-inducible CYP2E1, significantly lowered the CYP2E1 enzyme activity and the rate of APAP-induced cell death while it prevented the reduction of p53 and p21 in C6 glioma cells. A nontoxic analog of APAP, 3-hydroxyacetanilde, did not reduce p53 and p21 contents in C6 glioma cells and LLC-PK1 porcine kidney cells. Taken together, our results show that APAP or its reactive metabolite(s) can directly reduce the p53 content through mdm2-mediated ubiquitin conjugation, despite phosphorylation of p53 at its N terminus.