Inhibition of endoplasmic reticulum stress in high-fat-diet-induced obese C57BL/6 mice: Efficacy of a novel extract from mulberry (Morus alba) leaves fermented with Cordyceps militaris / 한국실험동물학회지

A few clues about correlation between endoplasmic reticulum (ER) stress and mulberry (Morus alba) leaves were investigated in only the experimental autoimmune myocarditis and streptozotocin-induced diabetes. To investigate whether a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) could suppress ER in fatty liver, alterations in the key parameters for ER stress response were measured in high fat diet (HFD)-induced obese C57L/6 mice treated with EMfC for 12 weeks. The area of adipocytes in the liver section were significantly decreased in the HFD+EMfC treated group as compared to the HFD+Vehicle treated group, while their level was higher in HFD+Vehicle treated group than No treated group. The level of the eukaryotic initiation factor 2 alpha (eIF2α) and inositol-requiring enzyme 1 beta (IRE1α) phosphorylation and CCAAT-enhancer-binding protein homologous protein (CHOP) expression were remarkably enhanced in the HFD+Vehicle treated group. However, their levels were restored in the HFD+EMfC treated group, although some differences were detected in the decrease rate. Similar recovery was observed on the ER stress-induced apoptosis. The level of Caspase-3, Bcl-2 and Bax were decreased in the HFD+EMfC and HFD+orlistat (OT) treated group compared to the HFD+Vehicle treated group. The results of the present study therefore provide first evidence that EMfC with the anti-obesity effects can be suppressed ER stress and ER stress-induced apoptosis in the hepatic steatosis of HFD-induced obesity model.

Celecoxib induces cell death on non-small cell lung cancer cells through endoplasmic reticulum stress / 대한해부학회지

Cyclooxygenase-2 (COX-2) is an enzyme induced by various proinflammatory and mitogenic stimuli. Celecoxib is a selective inhibitor of COX-2 that have been shown to affect cell growth and apoptosis. Lung cancer cells expressing COX-2 is able to be a target of celecoxib, this study focuses on investigating that celecoxib induces apoptosis via endoplasmic reticulum (ER) stress on lung cancer cells. We investigated whether celecoxib induced apoptosis on non-small cell lung cancer cell line, A549 and H460. The 50 µM of celecoxib increased apoptotic cells and 100 µM of celecoxib significantly induced apoptosis. To check involvement of caspase cascade, pretreatment of z-VAD-fmk blocked celecoxib-induced apoptosis. However, caspase-3, -8, and -9 were not activated, but cleavage of non-classical caspase-4 was detected using western blot. As checking ER stress associated molecules, celecoxib did not increase expressions of growth arrest and DNA damage inducible protein 34, activating transcription factor 4, and spliced X-box binding protiens-1, but increase of both glucose-regulated protein 78 (GRP78) and C/EBP homologous transcription factor were detected. Salubrinal, inhibitor of eIF2 and siRNA for IRE1 did not alter celecoxib-induced apoptosis. Instead, celecoxib-induced apoptosis might be deeply associated with ER stress depending on GRP78 because siRNA for GRP78 enhanced apoptosis. Taken together, celecoxib triggered ER stress on lung cancer cells and celecoxib-induced apoptosis might be involved in both non-classical caspase-4 and GRP78.

Correlation between laxative effects of uridine and suppression of ER stress in loperamide induced constipated SD rats / 한국실험동물학회지

A correlation between endoplasmic reticulum (ER) stress and laxative effects was first reported in a constipation model treated with an aqueous extract of Liriope platyphylla (AEtLP) roots. To investigate the correlation between the laxative effect of uridine (Urd) and ER stress response, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with Urd. The efficacy of the laxative effect of Urd was notable on the symptoms of chronic constipation, including alteration of stool parameters and structure of the transverse colon, in Lop induced constipated SD rats. In the PERK/eIF2-ATF4 pathway of ER stress response, the levels of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and DNA damage-inducible protein (GADD34) transcripts were significantly enhanced in the Lop+Vehicle treated group. However, the levels were restored in the Lop+Urd treated group, although few differences were detected in the decrease rate. Similar changes were observed for levels of inositol-requiring enzyme 1 beta (IRE1β) phosphorylation and X-box binding protein 1 (XBP-1) transcript in the IRE1α/XBP pathway. Furthermore, the number of ER stress-induced apoptotic cells and Bax and Bcl-2 expression were recovered in the Lop+Urd treated group compared to the Lop+Vehicle treated group. The results of the present study therefore provide first evidence that the laxative effects of Urd may be tightly correlated with the recovery of ER stress response in constipation models.

Role of PERK/eIF2α/CHOP Endoplasmic Reticulum Stress Pathway in Oxidized Low-density Lipoprotein Mediated Induction of Endothelial Apoptosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>PERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.</p><p><b>METHODS</b>The effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.</p><p><b>RESULTS</b>Ox-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.</p><p><b>CONCLUSION</b>This study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.</p>

Laxative effects of Liriope platyphylla are tightly correlated with suppression of endoplasmic reticulum stress in loperamide-induced constipation of SD rats / 한국실험동물학회지

A dysfunction of endoplasmic reticulum (ER) stress response can result in various diseases, including cancer, inflammation, diabetes and neurodegenerative disorders. To investigate whether ER stress response can play an essential role in the induction and treatment of chronic constipation, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with aqueous extracts of Liriope platyphylla (AEtLP), which has been shown to have a laxative effect. Symptoms of chronic constipation including alteration of stool parameters and the transverse colon's structure were successfully induced by Lop treatment. Laxative effects such as enhancement of stools parameters, recovery of the mucosa thickness, increased muscle thickness and recovery of flat luminal surface were also observed in the Lop+AEtLP treated group. Furthermore, enhancement of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and inositol-requiring enzyme 1 beta (IRE1β) expression, key indicators for ER stress, that were observed in the Lop+vehicle treated group were significantly recovered in the Lop+AEtLP treated group, although the phosphorylation level of c-Jun N-terminal protein kinase (JNK) remained constant. Moreover, alterations in the transcription level of the marker genes X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) were similar to those of eIF2α and IRE1β. However, their level was slightly or completely recovered after AEtLP treatment. Overall, this study provides the first evidence that ER stress response may be tightly correlated with chronic constipation induced by Lop treatment, as well as the laxative effects of AEtLP.

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway / 中国天然药物

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

Protective effects of salubrinal on liver injury in rat models of brain death / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Previous studies have indicated that endoplasmic reticulum stress participates in and mediates liver injury and apoptosis in brain-dead (BD) rats. In this study, we observed the effect of salubrinal (Sal, Sigma, USA) on liver cells in BD rats and explored its relevant mechanisms.</p><p><b>METHODS</b>Thirty Sprague-Dawley rats were equally randomized into three groups: BD group, Sal group, and DMSO group. The BD models were established by increasing intracranial pressure in a modified, slow, and intermittent way. In the drug groups, Sal was administered 1 h before the induction of BD. After modeling was completed, the blood and liver samples were harvested. CHOP and Caspase-12 mRNA expression was detected using quantitative polymerase chain reaction. PKR-like ER kinase (PERK), P-eukaryotic translation initiation factor 2α (eIF2α), eIF2α, CHOP and caspase-12 expression was detected using western blotting (WB). CHOP and caspase-12 distribution and expression in liver tissues were determined using immunohistochemistry (IHC). Alanine aminotransferase and aspartate aminotransferase level were detected using an automatic biochemical analyzer. Hepatic cell apoptosis was detected using TUNEL. The results were analyzed using Quantity-one v4.62 software (Bio-Rad, USA).</p><p><b>RESULTS</b>CHOP and caspase-12 expression and PERK, eIF2α, and P-eIF2α protein expression showed no significant difference between BD group and DMSO group. Compared with BD group, Sal group had a significantly higher P-eIF2C level and a lower P-PERK level 2 h and 6 h after BD (P < 0.05). However, eIF2α expression showed no significant difference (P > 0.05). After the Sal treatment, CHOP and caspase-12 mRNA expression significantly decreased 4 h after BD (P < 0.05). WB and IHC indicated that CHOP and caspase-12 expression also significantly decreased after Sal treatment. Sal was associated with improved liver function and decreased hepatic cell apoptosis.</p><p><b>CONCLUSIONS</b>Sal can significantly reduce apoptosis in hepatic cells of BD rats. This protective effect may be achieved via the PERK-eIF2α signaling pathway.</p>

Phosphorylation of eukaryotic initiation factor 2-alpha inhibits cisplatin-mediated apoptosis of hepatocellular carcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test.</p><p><b>RESULTS</b>Cisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05).</p><p><b>CONCLUSION</b>Phosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.</p>

Attenuation and mechanism of endoplasmic reticulum stress-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by qinggan huoxue recipe and its disassembled formulas / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore attenuation and mechanism of endoplasmic reticulum stress (ERS)-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by Qinggan Huoxue Recipe (QGHXR) and its disassembled formulas (Qinggan Recipe and Huoxue Recipe respectively).</p><p><b>METHODS</b>A rat model of chronic alcoholic liver injury was successfully established using a compound reagent of alcohol, corn oil, and pyrazol. The modeled rats were randomly divided into the model group, the QGHXR group, the Qinggan Recipe (QGR) group, and the Huoxue Recipe group (HXR). The CCl4 control group and the normal control group were also set up. There were ten rats in each group. All rats of modeled groups were gastrogavaged with alcohol compound reagent every morning. Rats in the QGHXR group (at the daily dose of 9. 5 g/kg, QGR group (at the daily dose of 3.0 g/kg), and HXR group (at the daily dose of 6.5 g/kg) were administered with corresponding medicines by gastrogavage every afternoon. Equal volume of normal saline was given to rats of the model group by gastrogavage. CCl4 was intraperitoneally injected at the dose of 0.3 mL/kg to rats in the CCl4 control group, once per week. Normal saline was given to rats in the normal control group by gastrogavage. The treatment was lasted for two weeks. Pathological changes of the liver were observed by histopathology. Serum total homocysteine (tHCY) level was detected by ELISA. The hepatocyte apoptosis rate was detected using flow cytometry. The gene and protein expressions of eukaryotic translation initiation factor 2 alpha (elF-2alpha), phosphorylation elF-2alpha (pelF-2alpha), glucose-regulated protein 78 (GRP78), and Caspase-3 in the liver were examined using Real-time PCR and Westen blot respectively.</p><p><b>RESULTS</b>Compared with the normal control group, typical pathological changes of chronic alcoholic liver injury such as steatosis, inflammation, and even fibrosis occurred in model rats. The hepatocyte apoptosis obviously increased, with the apoptosis rate reaching the five-fold of that in normal rats. Besides, early apoptosis dominated. The serum tHCY level significantly increased. The expressions of p-elF-2alpha, GRP78, and Caspase-3 protein obviously increased (P < 0.01). Expressions of GRP78 and Caspase-3 mRNA significantly increased (P < 0.05, P < 0.01). Compared with the model group, the degrees of the liver injury and the hepatocyte apoptosis in the QGHXR group, the QGR group, and the HXR group were significantly alleviated. The serum tHCY level was significantly lowered. The protein expressions of p-elF-2a, GRP78, and Caspase-3 obviously decreased (P < 0.01). mRNA expressions of GRP78 and Caspase-3 obviously decreased in the QGHXR group (P < 0.05, P < 0.01). Only GRP78 mRNA expression obviously decreased in the QGR group (P < 0.05).</p><p><b>CONCLUSION</b>QGHXR and its disassembled formulas could attenuate ERS-mediated hepatocyte apoptosis in alcohol-induced liver injury rats by lowering the serum tHCY level and expressions of ERS apoptosis correlated factors.</p>

The Effects of Glyburide on Apoptosis and Endoplasmic Reticulum Stress in INS-1 Cells in a Glucolipotoxic Condition

BACKGROUND: beta-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of beta-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes. METHODS: Apoptosis was induced by adding various concentrations of GB (0.001 to 200 microM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated. RESULTS: Addition of any concentration of GB in 150 microM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2alpha) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly. CONCLUSION: GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2alpha may attenuate ER stress for adaptation to increased ER protein load.

Role of PERK/eIF2a signaling pathway in hepatocyte apoptosis of alcoholic liver injury rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of PERK/eIF2alpha signaling pathway in hepatocyte apoptosis of alcoholic liver injury rats.</p><p><b>METHODS</b>Rat models with ethanol-induced liver injury were successfully developed by gastric gavage with ethanol-corn oil mixtures for 12 weeks. At different time points (4, 6, 10, 12 week), liver pathology was dynamically observed. The hepatocyte apoptosis was quantitatively analyzed by Annexin V-FITC/PI double-labeled flow cytometry, the serum total homocysteine (tHCY) level was detected by ELISA and the expressions of eIF2a, p-eIF2a, GRP78/Bip, GRP94, caspase-3 and caspase-12 in liver were examined using Real-time PCR and Western blot.</p><p><b>RESULTS</b>Typical acute liver injury and chronic liver injury were observed at week 4 and week 12 respectively. The hepatocyte apoptosis rates in 6-week model rats significantly increased compared with normal rats (P value less than 0.05), and the degree of hepatocyte apoptosis continued to increase with the modeling time, and the percentages of early and total apoptosis reached 26% and 29% at week 12. From week 6 to week 12, the serum tHCY levels in model rats were obviously higher than in normal rats (P value less than 0.01). Since week 4, eIF2a protein phosphorylation in model rat livers remarkably elevated compared with that in normal rat livers (P value less than 0.01), and at week 12 the peIF2a protein expression in model rat livers increased by 2.81-fold. Since week 4 the expressions of GRP78/Bip, GRP94, caspase-12 and caspase-3 mRNA and protein in model rat livers showed a significant increase as compared to normal rat livers, and at week 12, these gene and protein levels increased 4.70, 12.95, 3.83, 4.05 fold and 3.93, 6.93, 9.88, 3.31 fold, respectively (P value less than 0.01).</p><p><b>CONCLUSION</b>Activation of PERK/eIF2a signaling pathway contributes to the occurrence and development of hepatocyte apoptosis in alcoholic liver injury rats and it might be as a potential target for therapeutic applications in alcoholic liver diseases.</p>

Genetic polymorphisms of MxA protein and eIF-2a-reg2 and their responses to interferon treatment in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To identify the host single nucleotide polymorphisms (SNP) of myxovirus resistance A (MxA) protein and eukaryote initiation factor 2alfa regulatory region 2(eIF-2a-reg2) and to predict interferon (IFN) treatment responses in patients with chronic hepatitis B.</p><p><b>METHODS</b>Two hundred sixty-two patients with chronic hepatitis B (CHB) were treated with interferon alfa (IFN ) for 12 months. Six months later the therapeutic effectiveness was evaluated. All the patients had signed a formal consent form. The patients were grouped into a sustained response (SR) group and a non-sustained response (NSR) group according to their responses to the IFNa treatment. Single nucleotide polymorphisms of the antiviral protein MxA promoter -88,-123 and protein kinase(PKR) activated eIF-2a-reg2 sites were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and were compared with the responsiveness to IFN treatment of these CHB patients.</p><p><b>RESULTS</b>Among the 262 patients, 212 (80.9%) were non-sustained responders to IFNa and 50 (19.1%) were sustained responders. The rate of sustained responders with GT heterozygote at MxA promoter -88 was higher than that of the GG genotype (OR: 5.3, 95% CI: 2.46-11.43, P less than 0.01) and also higher than that of the TT genotype (OR: 4.1, 95% CI: 1.86-9.09, P less than 0.01). There were no statistically significant differences in IFN therapeutic effectiveness among the patients with different genotypes at MxA promoter -123, eIF-2a-reg2 and haplotypes made by MxA promoter -88 G/T, and -123 C/A alleles (P more than 0.05).</p><p><b>CONCLUSION</b>Patients with GT genotype at MxA promoter -88 responded well to IFN treatment. SNP as a potential marker could be used to predict IFN treatment responses of patients with chronic hepatitis B.</p>

HIV-1 Infection Causes Intracellular Expression of p53, Which Induces PKR Expression, Followed by Inhibition of HIV-1 Tat Activity

Few papers have reported that the HIV-1 replication was inhibited by p53 in the infected cells. However, the detail mechanism for the p53-medicated HIV-1 suppression has not yet been clearly demonstrated. In our previous report, we addressed that p53-mediated Tat suppression is very likely associated with PKR. In the present study, we found that the amounts of p53 in the HIV-1 infected cells increased over 10 times in the early stages of infection as much as those in normal cells. Particularly noteworthy is that the both exogenous p53 and endogenous p53 enhanced PKR expression in the transformed or treated cells, and the amounts of PKR induced by p53 were almost equivalent to those induced by interferon. In the PKR promoter studies using Ppkr-CAT (CAT reporter system under the control of PKR promoter), CAT activity induced by p53 was stronger than that by interferon, suggesting that the p53-mediated PKR expression might be more efficient than interferon under the control of PKR promoter. Co-immunoprecipitation experiments showed that PKR directly binds to Tat protein. We established eIF-2alpha dominant negative (S51A) Jurkat cells (JK/eIF2alpha-51A) to block the PKR-mediated cell cycle arrest or apoptosis. In the JK/eIF2alpha-51A cells, not only p53 but also PKR inhibited the Tat activity. Taken together, our results demonstrate that the HIV-1 infection induces p53, which enhances PKR expression by promoter activation, followed by the inhibition of the Tat activity, finally resulting in the inhibition of HIV-1 replication. Detail mechanisms for the PKR-mediated Tat inactivation are under investigation.

Up-regulation of prothymosin alpha in THP-1 cells infected with Mycobacterium tuberculosis

Mycobacterium tuberculosis is capable of growing and survival within macrophage. The purpose of this study was to identify the genes regulated by infection of mycobacteria in human monocytic THP-1 cells. We used the differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and nothern blot analysis to confirm the differentially expressed genes from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv, heat-kille Mycobacterium tuberculosis H37Rv and live Mycobacterium bovis BCG. Among many up or down-regulated clones, 27 clones were sequenced and compared with known genes on GenBank. Thirteen of over-expressed clones from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv were identical to human prothymosin alpha, eight were novel clones and six clones showed homology with Human ferritin H chain, Escherichia coli bgl, Mouse RNA-dependent EIF-2 alpha kinase, E. coli htrL, Hyaluronan receptor and T cell receptor. Our result suggests that Mycobacterium tuberculosis might regulate prothymosin alpha gene transcription in monocytic THP-1 cell.

Análise mutacional da região amino-terminal da subunidade beta do fator 2 de ínicio de tradução (elF2) / Mutational analysis of de N-terminal region of the beta subunit of elF2

elF2 está envolvido na seleção do codon AUG para inicio de tradução em eucariotos, uma vez que em levedura, mutações em suas três subunidades foram identificadas por permitirem o reconhecimento de um codon UUG pelo anti-codon do Met-tRNAiMet. A subunidade beta de elF2 contém um motivo que poderia mediar interações com RNA compreendido por três blocos de sete resíduos de lisina, os quais são altamente conservados evolutivamente. Neste trabalho foi realizada uma análise de deleções nesta proteína em Saccharomyces cerevisiae, a qual indicou que somente um bloco de lislnas é suficiente para manter sua função "in vivo", provavelmente devido à carga, uma vez que a troca destes aminoácidos por arglnina, mas não por alanina, manteve a viabilidade celular. A remoção de qualquer um dos bloco de lisinas não afeta a expressão de GCN4. Entre as duplas deleções, somente aquela em que permanece o segundo bloco levou a uma marcada desrepressão de GCN4. A tripla deleção resultou em altos níveis de expressão de GCN4, correspondendo a um fenótipo dominante negativo, indicando que este alelo pode ainda estar mantendo uma função parcial, competindo com a proteína selvagem e proporcionando um defeito na síntese protélca. Esta proteína deletada associa-se com elF2a e elF2g e é encontrada no complexo de pré-iniciação. A ligação de mRNA "in vitro" a proteínas de fusão GST-elF2b é dependente dos blocos de lisinas, sendo mantlda na presença de um único bloco de lisinas ou argininas, mas não de alaninas. A ligação de elF2 ao mRNA é dependente da presença dos blocos de lisinas na subunidadeb. A ligação de elF2 a Met-tRNAiMet de forma GTP-dependente não é afetada pela ausência das repetições de lisinas. Os resultados obtidos aqui fornecem fortes evidências do papel dos blocos de lisinas de elF2b em manter a interação com mRNA "in vivo"

Mechanism of action of an eukaryotic initiation factor-2 (eIF-2) associated 67 kDa glycoprotein (p67) and an eIF-2 kinase (dsI).

Mechanism of regulation of eIF-2 alpha-subunit phosphorylation by dsI and p67 was studied. The results are as follows: (1) At low dsI concentration, p67 protected equimolar concentration of eIF-2. (2) At high dsI concentration, dsI efficiently phosphorylated eIF-2 alpha-subunit even when equimolar concentrations of both p67 and eIF-2 were present. Significantly increased p67 concentration was necessary to protect eIF-2 alpha-subunit at high dsI concentration. (3) dsI was also phosphorylated as it phosphorylated eIF-2 alpha-subunit. p67 inhibited both eIF-2 alpha-subunit and dsI phosphorylation similarly. (4) Although the [32P]-labelled dsI formed during the reaction could be effectively chased upon subsequent addition of excess unlabelled eIF-2 and ATP, the [32P] labelled eIF-2 formed under identical conditions, retained most of the radioactivity. (5) dsI coimmunoprecipitated with three subunit eIF-2 and p67 inhibited this coimmunoprecipitation reaction. It has been proposed: Three subunit eIF-2 and free p67 are in equilibrium with eIF-2 bound to p67 and, eIF-2.p67 complex is resistant to dsI phosphorylation. Activated dsI is already phosphorylated. At high concentration, dsI(P) can bind to free three subunit eIF-2 and form eIF-2.dsI(P) complex. dsI(P) in this complex then transfers its phosphoryl residue to eIF-2 and forms eIF-2 alpha(P) in an irreversible reaction. In a subsequent reaction, unphosphorylated dsI is autophosphorylated using [gamma 32P]-ATP and the cycle continues. Inhibition of eIF-2 alpha-subunit phosphorylation by p67 blocks this phosphorylation cycle and consequent dsI phosphorylation.

Genetic and molecualr analysis of initiation of protein synthesis in eukaryotes

Control of gene expression operating at the level of initiation of protein synthesis has been increasingly considered to play a major role in eukaryotic cells. The complexity of the translational machinery involved in the efficient utilization of a message is reflected in the number of initiation factors present in eukaryotes. Their functions are far from being completely understood at the molecular level. This review is centered particularly in the translation initiation factor eIF-2, and in how it may play a role in the recognition of the initiator AUG codon.