OBJECTIVEPERK/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.

METHODSThe 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.

RESULTSOx-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.

CONCLUSIONThis 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.

Apoptosis ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Signal Transduction ; Transcription Factor CHOP ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

Cells can adapt to environment and development by reconstructing their transcriptional networks to regulate diverse cellular processes without altering the underlying DNA sequences. These alterations, namely epigenetic changes, occur during cell division, differentiation and cell death. Numerous evidences demonstrate that epigenetic changes are governed by various types of determinants, including DNA methylation patterns, histone posttranslational modification signatures, histone variants, chromatin remodeling, and recently discovered chromosome conformation characteristics and non-coding RNAs (ncRNAs). Here, we highlight recent efforts on how the two latter epigenetic factors participate in the sophisticated transcriptional process and describe emerging techniques which permit us to uncover and gain insights into the fascinating genomic regulation.
Cell Death ; Cell Differentiation ; Cell Division ; Chromatin ; chemistry ; metabolism ; Chromatin Assembly and Disassembly ; DNA Methylation ; Epigenesis, Genetic ; Eukaryotic Cells ; cytology ; metabolism ; Histones ; genetics ; metabolism ; Humans ; Protein Processing, Post-Translational ; RNA, Untranslated ; genetics ; metabolism ; Transcription, Genetic

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.
Bacillus subtilis ; Biotin ; chemistry ; Eukaryotic Cells ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; Promoter Regions, Genetic ; Trans-Activators

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.
Animals ; Escherichia coli ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; HEK293 Cells ; Humans ; Insecta ; cytology ; genetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; metabolism ; Yeasts ; genetics

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke.
Acrolein ; Animals ; Biomarkers ; Brain Infarction ; C-Reactive Protein ; Diacetyl ; DNA ; Early Detection of Cancer ; Eukaryotic Cells ; Humans* ; Hydrogen Peroxide ; Inflammation ; Interleukin-6 ; Lysine ; Metabolism ; Mice ; Oxidative Stress ; Oxidoreductases ; Phospholipids ; Plasma ; Polyamines* ; Putrescine ; Renal Insufficiency ; RNA ; Sensitivity and Specificity ; Spermidine ; Spermine ; Stroke

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calciumregulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule superresolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.
Active Transport, Cell Nucleus ; physiology ; Animals ; Calcium ; metabolism ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Diffusion ; Endoplasmic Reticulum ; metabolism ; Eukaryotic Cells ; metabolism ; Humans ; Ion Transport ; physiology ; Microscopy, Fluorescence ; Molecular Conformation ; Nuclear Pore ; chemistry ; metabolism ; Nuclear Pore Complex Proteins ; chemistry ; metabolism ; Oocytes ; cytology ; metabolism ; Signal Transduction ; Xenopus laevis

Eukaryotic DNA is hierarchically packaged into chromatin to fit inside the nucleus. Dynamics of the chromatin structure plays a critical role in transcriptional regulation and other biological processes that involve DNA, such as DNA replication and DNA repair. Many factors, including histone variants, histone modification, DNA methylation and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. Identifying the structural details and dynamics of higher-order chromatin fibers is therefore very important for understanding the organization and regulation of gene activities. Here, we review studies on the dynamics of chromatin higher order structure and its relationship with gene transcription.
Animals ; Chromatin ; chemistry ; metabolism ; Chromatin Assembly and Disassembly ; Eukaryotic Cells ; chemistry ; metabolism ; Gene Expression Regulation ; Humans ; Models, Molecular

Activity-dependent dendritic translation in CNS neurons is important for the synapse-specific provision of proteins that may be necessary for strengthening of synaptic connections. A major rate-limiting factor during protein synthesis is the availability of eukaryotic translation initiation factor 4E (eIF4E), an mRNA 5'-cap-binding protein. In this study we show by fluorescence in situ hybridization (FISH) that the mRNA for eIF4E is present in the dendrites of cultured rat hippocampal neurons. Under basal culture conditions, 58.7 +/- 11.6% of the eIF4E mRNA clusters localize with or immediately adjacent to PSD-95 clusters. Neuronal activation with KCl (60 mM, 10 min) very significantly increases the number of eIF4E mRNA clusters in dendrites by 50.1 and 74.5% at 2 and 6 h after treatment, respectively. In addition, the proportion of eIF4E mRNA clusters that localize with PSD-95 increases to 74.4 +/- 7.7% and 77.8 +/- 7.6% of the eIF4E clusters at 2 and 6 h after KCl treatment, respectively. Our results demonstrate the presence of eIF4E mRNA in dendrites and an activity-dependent increase of these clusters at synaptic sites. This provides a potential mechanism by which protein translation at synapses may be enhanced in response to synaptic stimulation.
Animals ; Cells, Cultured ; Dendrites/*metabolism ; Eukaryotic Initiation Factor-4E/genetics/*metabolism ; Hippocampus/*cytology ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Microscopy, Confocal ; Neurons/cytology/*physiology ; Potassium Chloride/pharmacology ; Protein Biosynthesis ; RNA, Messenger/genetics/*metabolism ; Rats ; Rats, Sprague-Dawley ; Synapses ; *Up-Regulation

OBJECTIVETo construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products (RAGE) mutants.

METHODSSite-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE(S391A), pcDNA3-HA-RAGE(S399A), pcDNA3-HA-RAGE(S400A), and pcDNA3-HA-RAGE(T401A). After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody.

RESULTSThe HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells.

CONCLUSIONThe success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.

Cell Line ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; genetics

OBJECTIVETo clone human intestinal trefoil factor (hITF/hTFF3) gene into an eukaryotic expression vector for its expression in eukaryotic cells.

METHODSThe total RNA was extracted from normal human colon mucosa, and transcribed into cDNAs using RT-PCR. hTFF3 gene was amplified by PCR and ligated into pGEMT vector by TA cloning method. After sequencing, the hTFF3 gene was transfered into the eukaryotic expression vector pCMV5-myc. The recombinant vector was transfected into 293-T cells, and the expression of the recombinant protein was detected by Western blotting.

RESULTS AND CONCLUSIONhTFF3 gene was successfully cloned from normal human colon mucosa. The vector pCMV5-myc-hTFF3 was reconstructed, and in 293-T cells transfected with the vector, hTFF3 expression was detected by Western blotting.

Blotting, Western ; Cell Line ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; Humans ; Intestinal Mucosa ; metabolism ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; methods ; Trefoil Factor-2 ; Trefoil Factor-3