Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein

AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analysis,sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells.Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR).Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins.In vivo 32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation,eIF2α phosphorylation,and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus.In a sucrose gradient,approxi-mately 30% of the overexpressed p67 was bound with ribosomes.p67 interacted with the kinase domain,butnot the dsRNA-binding domains of PKR.Only the glycosylated p67 was associated with the ribosome,and p67 did not compete with PKR for ribosome binding.In breast cancer cells,there was increased autophosphorylation of PKR but no phosphorylation of eIF2α,compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.

Neuroprotective role of edaravone and the effects of endoplasmic reticulum stress in an adult rat model of focal cerebral ischemia/reperfusion

BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.

An ongoing search for potential targets and therapies for lethal sepsis

Sepsis, which refers to a systemic inflammatory response syndrome resulting from a microbial infection, represents the leading cause of death in intensive care units. The pathogenesis of sepsis remains poorly understood although it is attributable to dysregulated immune responses orchestrated by innate immune cells that sequentially release early(e.g., tumor necrosis factor(TNF), interleukin-1(IL-1), and interferon-γ(IFN-γ) and late(e.g., high mobility group box 1(HMGB1)) pro-inflammatory mediators. As a ubiquitous nuclear protein, HMGB1 can be passively released from pathologically damaged cells, thereby converging infection and injury on commonly dysregulated inflammatory responses. We review evidence that supports extracellular HMGB1 as a late mediator of inflammatory diseases and discuss the potential of several Chinese herbal components as HMGB1-targeting therapies. We propose that it is important to develop strategies for specifically attenuating injury-elicited inflammatory responses without compromising the infection-mediated innate immunity for the clinical management of sepsis and other inflammatory diseases.

DNA repair genes BRCA1 and DNA-PKcs have great potential in radiation therapy

Radiotherapy is a part of the front-line treatment regime for many cancers. The mechanisms of radiation-induced effects in cancers mainly involves double-strand breaks (DBS) which plays very important role in maintaining the stability of gene. As DNA repair gene breast cancer 1 (BRCA1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can act to maintain genetic stability though two distinct and complementary mechanisms for DNA DSB repair-homologous recombination (HR) and non-homologous end joining (NHEJ). Therefor, BRCA1 and DNA-PKcs are closely associated with radiation sensitivity, which means that they may be used as a useful tool to predict radio sensitivity in human tumour cells.

Endoplasmic reticulum stress and liver diseases

Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1 a(IRE1 a),double-stranded RNA-dependent protein kinase(PKR)-like ER kinase(PERK)and activating transcription factor 6(ATF6)signaling pathways,is a protective cellular response activated by ER stress.However,UPR activation can also induce cell death upon persistent ER stress.The liver is susceptible to ER stress given its synthetic and other biological functions.Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and the UPR signaling pathways in the pathogenesis of liver diseases,including non-alcoholic fatty liver disease(NAFLD),alcoholic liver disease(ALD),alpha-1 antitrypsin(AAT)deficiency(AATD),cholestatic liver disease,drug-induced liver injury,ischemia/reperfusion(I/R)injury,viral hepatitis and hepatocel-lular carcinoma(HCC).Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of the UPR pathways during the development of the diseases.Moreover,ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.