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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]

A 6A,6A′-dicyclohexylamine-6B,6B′-diselenide-bis-β-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, β-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6′-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H2O2, tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application. [ABSTRACT FROM AUTHOR]

Abstract: Glutathione peroxidase (GPX) is an important antioxidant enzyme, which plays an important role in scavenging reactive oxygen species. To obtain humanized GPX catalytic antibodies, the phage displayed human antibody library on the surface of the filamentous bacteriophage was used to select novel antibodies by repetitive screening. Phage antibodies B8, H6 and C1 with the GSH-binding site were obtained from the library by enzyme-linked immunosorbent assay (ELISA) analysis with four rounds of selection against three haptens, S-2,4-dinitrophenyl t-butyl ester [GSH-S-DNP-Bu (B)], S-2,4-dinitrophenyl t-hexyl ester [GSH-S-DNP-He (H)] and S-2,4-dinitrophenyl cycle-hexyl ester [GSH-S-DNP-cHe (C)], and characterized using surface plasmon resonance (SPR) biosensor. The gold layer was modified by dithiodiglycolic acid (DDA) and three haptens were easily attached to DDA by self-assembling to form a biosensor membrane. The membrane bounds specifically corresponding antibodies. The kinetic process of the reaction between phage antibodies and their haptens was studied by SPR biosensor. In order to improve selectivity, chemical modification was used to incorporate directly catalytic group selenocysteine (Sec) into selected phage clone B8, H6 and C1 to form Se-B8, Se-H6 and Se-C1, respectively. The GPX activities of Se-B8, Se-H6 and Se-C1 were found to be 3000, 2000 and 700units/μmol, respectively. Compared with conventional ELISA analysis, the proposed method based on SPR biosensor is much more rapid and simpler. [Copyright &y& Elsevier]