Search

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-β-cyclodextrin (6- Cy Se CD) 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- Cy Se CD in cultured immortalised human keratinocyte cells ( Ha Ca T). Methods Ha Ca T cells were treated with 30 mJ/cm2 UVB to establish a damage model. The cultured Ha Ca T 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 Ha Ca T cells. UVB irradiation caused pre- G1-phase arrest in Ha Ca T cells and significantly reduced the number of HaCaT cells in the S phase. The GPx mimics 6- Cy Se CD and 2-phenyl-l,2-benzisoselenazol-3( 2H)-one (ebselen) significantly blocked UVB-induced apoptosis and changed the cell cycle of the Ha Ca T cells. The blocked effect of pretreatment 6- Cy Se CD in UVB-irradiated Ha Ca T cells was better than that of pretreatment with ebselen. Conclusion 6- Cy Se CD can relieve the damage induced by UVB irradiation in Ha Ca T cells. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×10/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]