RESUMEN
BACKGROUND: The generation of harmful reactive oxygen species (ROS) induced by UVB irradiation could induce cell apoptosis and change the cell cycle. 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-ß-cyclodextrin (6-CySeCD) is a novel glutathione peroxidase (GPx; EC 1.11.1.9) mimic. The aim of this study was to investigate the anti-oxidative effects of 6-CySeCD in cultured immortalised human keratinocyte cells (HaCaT). METHODS: HaCaT cells were treated with 30 mJ/cm(2) UVB to establish a damage model. The cultured HaCaT cells were randomly assigned to the control, UVB and treatment groups. The treatment group was incubated with 20 µmol/L of GPx mimics before UVB irradiation. Cell viability was detected by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the level of lipid peroxidation was determined by the formation of malondialdehyde (MDA), DNA fragmentation was observed using agarose gel electrophoresis and the levels of intracellular ROS and cell cycle progression were measured by flow cytometry. RESULTS: The levels of cytotoxicity, intracellular ROS, lipid peroxidation and oxidative DNA damage significantly increased after UVB irradiation in the HaCaT cells. UVB irradiation caused pre-G1 -phase arrest in HaCaT cells and significantly reduced the number of HaCaT cells in the S phase. The GPx mimics 6-CySeCD and 2-phenyl-l,2-benzisoselenazol-3(2H)-one (ebselen) significantly blocked UVB-induced apoptosis and changed the cell cycle of the HaCaT cells. The blocked effect of pretreatment 6-CySeCD in UVB-irradiated HaCaT cells was better than that of pretreatment with ebselen. CONCLUSION: 6-CySeCD can relieve the damage induced by UVB irradiation in HaCaT cells.
Asunto(s)
Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , Compuestos de Organoselenio/farmacología , Protectores contra Radiación/farmacología , beta-Ciclodextrinas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Azoles/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de la radiación , Humanos , Isoindoles , Queratinocitos/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase S del Ciclo Celular/efectos de la radiaciónRESUMEN
Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.
Asunto(s)
Glutatión Peroxidasa/genética , Glutatión Transferasa/química , Proteínas del Helminto/química , Proteínas Recombinantes de Fusión/química , Schistosoma japonicum/enzimología , Superóxido Dismutasa/química , Animales , Glutatión Peroxidasa/química , Glutatión Transferasa/genética , Proteínas del Helminto/genética , Especies Reactivas de Oxígeno/química , Proteínas Recombinantes de Fusión/genética , Schistosoma japonicum/genética , Superóxido Dismutasa/genéticaRESUMEN
As the twenty-first amino acid, selenocysteine can be co-translationally incorporated into the polypeptide chain at UGA codon in the coding region of selenoprotein mRNA. The incorporation of selenocysteine needs a cis-acting element SECIS and four gene products: SelA, SelB, SelC and SelD. The position of SECIS in the mRNA of prokaryote and its structural features are greatly different from that of eukaryote. The researchers have made exploration in selenoprotein engineering by virtue of the mechanism of selenocysteine incorporation in Escherichia coli.
Asunto(s)
Proteínas/metabolismo , ARN Mensajero/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/química , ARN Mensajero/metabolismo , SelenoproteínasRESUMEN
The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.