A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.
Adsorption ; Animals ; Antibodies ; COS Cells ; DNA ; Helper Viruses ; HIV-1* ; Membranes ; Plasmids ; Product Packaging ; RNA ; Virion

OBJECTIVETo evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection.

METHODSIron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet.

RESULTSForeign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold.

CONCLUSIONIONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Animals ; COS Cells ; Ferric Compounds ; administration & dosage ; Genetic Vectors ; Magnetics ; Polylysine ; administration & dosage ; Transfection ; methods

The purpose of this research was to construct a lentiviral vector containing pir-b gene, and to detect the expression of pir-b gene in 293T cells. The open reading frame (ORF) of pir-b gene from the mRNA of mouse was cloned, then inserted into a sequencing vector. The pir-b gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells by Lipofectin 2000, thereafter, the supernatant was collected and concentrated to transfect 293T cells. Western blot was used to detect the expression of the exogenous PIR-B after 293T cells were infected by the virus, while the lentivirus harboring egfp gene was packaged as the control group. The result indicated that the ORF of the pir-b gene was successfully cloned, the sequence of which was consistent to that was expected and the PIR-B protein could be expressed in 293T cells normally. It is concluded that the lentiviral vector containing pir-b gene was constructed successfully, which would contribute to illustrating the important role of pir-b gene in the immunological regulation.
Animals ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; RNA, Messenger ; genetics ; Receptors, Immunologic ; genetics

OBJECTIVETo investigate the expression and analysis of its activity of anti-bacterial peptide gloverin in COS-7 cells.

METHODSThe appearance frequency of all genetic codes in the cDNA sequence from the same species of protein Attacin A was analyzed, and its cDNA sequence was synthesized by PCR overlapping extension method in conjunction with the designation of the known protein sequence of gloverin. The genes were inserted into pCDSI, an eukaryotic vector, after being identified correctly. As a result, the vector pBZHG was constructed. Thereafter, the liposome FuGENE( trade mark ) 6 was employed as the vector, and the COS-7 cells were transfected with liposome pBZHG and blank vector pCDSI. The normal cells were taken as the control. The supernatant was collected for the detection of its bactericidal activity after 72 PBHs.

RESULTSThe gloverin cDNA sequence designed artificially was expressed in COS-7 cells. The supernatant of the cells transfected by pBZHG exhibited bactericidal activity to E. coli J5 when compared with that from normal cells and in cells transfected with blank vectors.

CONCLUSIONThe designed cDNA sequence of gloverin was proved to be genuine, and it provided the basis for future study of its antibiotic and anti-endotoxin activities.

Animals ; Anti-Bacterial Agents ; pharmacology ; COS Cells ; Cercopithecus aethiops ; Proteins ; genetics ; pharmacology ; Sequence Analysis, DNA ; Transfection

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.
Animals ; Arachidonic Acid ; Chimera ; COS Cells ; Dithionitrobenzoic Acid ; Dithiothreitol ; Membranes ; Oxidants

This study inquired about the influences on the gene delivery efficiency of polyethylenimine. pSVbeta plasmids were transferred into COS-7 and NIH3T3 cells with polyethylenimine. Influences of plasmids factor, albumin, serum, cell density, operation methods and polyethylenimine/DNA preserve factors on transfection efficiency were investigated. Inhibitors of biological activities in plasmids could be removed by ultrafiltration with cutoff molecular weight of 3000 or 10000. Linear plasmids lowered transfection efficiency. Serum and albumin in the culture medium decreased the transfection efficiency of polyethylenimine/DNA. Cell density was associated with PEI/DNA transfection efficiency. Incubation of PEI/DNA complexes with the cells for 8 h and then aspiration for removal of the complexes could obtain an optimal transfection effect. Freezing of PEI/DNA complexes significantly decreased transfection efficiency. In conclusion, polyethylenimine could obtain optimal and reduplicate transfection results by controlling related factors.
3T3 Cells ; Animals ; COS Cells ; Cercopithecus aethiops ; DNA ; genetics ; Genetic Vectors ; genetics ; Mice ; Plasmids ; genetics ; Polyethyleneimine ; chemistry ; Transfection ; Ultrafiltration

BACKGROUND: This study investigated the effect of remifentanil pretreatment on Cos-7 cells exposed to oxidative stress, and the influence of remifentanil on intracellular autophagy and apoptotic cell death. METHODS: Cells were divided into 4 groups: (1) Control: non-pretreated cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂). (2) H₂O₂: non-pretreated cells were exposed to H₂O₂ for 24 h. (3) RPC+H₂O₂: cells pretreated with remifentanil were exposed to H₂O₂ for 24 h. (4) 3-MA+RPC+H₂O₂: cells pretreated with 3-Methyladenine (3-MA) and remifentanil were exposed to H₂O₂ for 24 h. We determined the cell viability of each group using an MTT assay. Hoechst staining and FACS analysis of Cos-7 cells were performed to observe the effect of remifentanil on apoptosis. Autophagy activation was determined by fluorescence microscopy, MDC staining, and AO staining. The expression of autophagy-related proteins was observed using western blotting. RESULTS: Remifentanil pretreatment increased the viability of Cos-7 cells exposed to oxidative stress. Hoechst staining and FACS analysis revealed that oxidative stress-dependent apoptosis was suppressed by the pretreatment. Additionally, fluorescence microscopy showed that remifentanil pretreatment led to autophagy-induction in Cos-7 cells, and the expression of autophagy-related proteins was increased in the RPC+H₂O₂ group. CONCLUSIONS: The study showed that remifentanil pretreatment stimulated autophagy and increased viability in an oxidative stress model of Cos-7 cells. Therefore, we suggest that apoptosis was activated upon oxidative stress, and remifentanil preconditioning increased the survival rate of the cells by activating autophagy.
Animals ; Apoptosis* ; Autophagy* ; Blotting, Western ; Cell Death ; Cell Survival ; COS Cells* ; Hydrogen* ; Microscopy, Fluorescence ; Oxidative Stress ; Survival Rate

BACKGROUND: In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (H₂O₂)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂) for 24 h without propofol; H₂O₂, cells were exposed to H₂O₂ (400 µM) for 2 h; PPC + H₂O₂, cells pretreated with propofol were exposed to H₂O₂; and 3-methyladenine (3-MA) + PPC + H₂O₂, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H₂O₂. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis. RESULTS: Cell viability decreased more significantly in the H₂O₂ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased H₂O₂-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + H₂O₂ group than that in the H2O2 group. CONCLUSION: PPC has a protective effect on H₂O₂-induced COS-7 cell apoptosis, which is mediated by autophagy activation.
Animals ; Apoptosis* ; Autophagy* ; Blotting, Western ; Cell Survival ; COS Cells* ; Hydrogen Peroxide ; Methods ; Microscopy, Fluorescence ; Oxidative Stress ; Propofol* ; Reactive Oxygen Species

PURPOSE: Cefodizime is a new third-generation cephalosporin which has a structure and immunomodutation properties similar to cefotaxime. Various studies on cefodizime have demonstrated the direct eradication of bacteria in cooperation with the host defense mechanism, particularly with phagocytosis. We evaluated the effects of cefodizime on the phagocytosis of COS-1 cells transfected with FcgammaRI/gammagamma or FcgammaRIIA cDNA. METHODS: Phagocytosis was measured using the in vitro COS-1 cell modeling system according to Schreiber's method. COS-1 cells, which lack endogenoous Fcgammareceptors but have phagocytic potential, were transfected with either FcgammaRI/gammagammaor FcgammaRIIA cDNA. COS-1 cells, as target cells, were treated with antibiotics for 1 or 24 hours and incubated for 30 min with IgG coated sheep RBCs. Adhered IgG coated sheep RBCs were removed after brief exposure to hypotonic phosphate buffered saline. Phagocytosis index (PI) was calculated as the number of ingested RBCs per 100 phagocytic cells after wright-Giemsa staining. RESULTS: COS-1 cells tranfected with FcgammaR (either FcgammaRI/gammagamma or FcgammaRIIA cDNA) showed the phagocytic activity against IgG coated sheep RBC, while untransfected COS-1 cells did not. After treatment with cefodizime, phagocytic activity of FcgammaRI/gammagammacDNA transfected COS-1 cells was significantly increased, while that of FcgammaRIIA cDNA transfected COS-1 cells did not. Marked enhancement of phagocytosis of COS-1 cells was observed after treatment with cefodizime, but was not observed with ceftriaxone or moxalactam. CONCLUSION: Cefodizime showed marked enhancement of phagocytic activity of FcgammaR transfected COS-1 cells. FcgammaRI seems to play an important role in the enhancement of phagocytosis. Further studies will be required.
Animals ; Anti-Bacterial Agents ; Bacteria ; Cefotaxime ; Ceftriaxone ; COS Cells ; DNA, Complementary ; Immunoglobulin G ; Moxalactam ; Phagocytes ; Phagocytosis* ; Sheep

In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.
Animals ; Base Sequence ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Molecular Sequence Data ; Mucin-1 ; genetics ; Multiple Myeloma ; genetics ; Plasmids ; Transfection