Although superoxide anions (O2.-) and H2O2 are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. In mammalian cells, a variety of extracellular stimuli have recently been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of stimulant-dependent signaling. In the next few years, therefore, a flurry of research activity is expected in relation to the elucidation of ROS production in response to receptor stimulation, identification of ROS target molecules, and investigation of ROS elimination. The goal of this report is to review our current knowledge of ROS-regulated signal transduction and propose future directions.
Animal ; Eukaryotic Cells/metabolism ; Human ; Hydrogen Peroxide/metabolism* ; Oxidation-Reduction ; Prokaryotic Cells/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction*

BACKGROUNDTo construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection.

METHODSThe DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell.

RESULTSThe psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA.

CONCLUSIONSThe gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.

3T3 Cells ; Animals ; Eukaryotic Cells ; metabolism ; Gene Expression ; Genetic Vectors ; Mice ; Plasmids ; genetics ; RNA, Antisense ; genetics ; Receptors, CCR5 ; genetics ; Transfection

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

OBJECTIVETo express human soluble CD14 (sCD14) in eukaryotic cells.

METHODSHuman sCD14 cDNA was amplified from U937 cells with RT-PCR method. The recombinant expression plasmid pEF1/HisC/sCD14(348aa) was constructed and the expression in COS-7 cells was carried out using liposome transfection method. The yield was examined with scanning map identification. The expressed product was purified by immuno-affinity chromatography.

RESULTSSequence analysis demonstrated that the amplified gene sequence and those reported by documents were completely identical. sCD14 was expressed with high-yield. The expressed product was purified to above 90%. Recombinant sCD14, specifically combinable with endotoxins, had a natural biological activity.

CONCLUSIONSHuman sCD14 was expressed in COS-7 cells, which laid a foundation for further study.

Animals ; COS Cells ; metabolism ; Chromatography, Affinity ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Humans ; Lipopolysaccharide Receptors ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells ; metabolism

OBJECTIVETo investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells.

METHODSEukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity.

RESULTSThe Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction.

CONCLUSIONThe IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.

Animals ; Eukaryotic Cells ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation ; Hepatitis B ; metabolism ; Interferon-alpha ; biosynthesis ; genetics ; physiology ; Marmota ; metabolism ; Prokaryotic Cells ; metabolism ; Signal Transduction ; Transcription Factors

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

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
Animals ; CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins ; biosynthesis ; genetics

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells/metabolism ; Genetic Vectors ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins/*biosynthesis ; alpha-Fetoproteins/genetics

OBJECTIVETo investigate the effects of exogenous wild PTEN gene stably transfection on growth of breast cancer cells in vitro.

METHODSAt first, a recombinant eukaryotic expression plasmid pcDNA3.1-PTEN was constructed. Human breast cancer cell line MDA468 was transfected with pcDNA3.1-PTEN or mock transfected plasmid pcDNA3.1(-) with lipofectamine. RT-PCR, immunohistochemical staining and Western blot were used to determine target gene expression. Cell viability was tested by MTT assay. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI.

RESULTSThe PTEN stably transfected cells demonstrated the integration of the exogenous target gene and corresponding mRNA and protein over-expression. There was a significant decline in cell viability of pcDNA3.1-PTEN transfected MDA468 cells in comparison with the mock-transfected ones (P < 0.01). The PTEN-trasfected MDA468 cells also showed an increase in the rate of apoptosis, compared with parental and mock-trasfected cells (P < 0.001).

CONCLUSIONStable expression of exogenous PTEN can suppress the malignant phenotypes of the human breast cancer cell line MDA468.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Eukaryotic Cells ; metabolism ; Humans ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; Phenotype ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection