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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]

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]

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) 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. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

Aim: The reperfusion following liver ischemia results in the damage and apoptosis of hepatocytes. The aim of this study was to investigate the possible effects and mechanism of a new synthesized glutathione peroxidase (GPX) mimic, 2-selenium-bridged beta-cyclodextrin (2-SeCD), on rat liver ischemia-reperfusion (I/R) injury., Methods: Male Wistar rats (n = 32) were randomly divided into four groups: I. sham-operated group, II. I/R group, III. I/R +2-SeCD group, IV. I/R + Ebselen group. Hepatic I/R was administered by 90 min of ischemia and 12 h of reperfusion. Liver tissues were collected at the end of reperfusion period for measurement of various biochemical parameters., Results: The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and tissue malondialdehyde, myeloperoxidase levels were increased in I/R group, while the increase was significantly reduced by 2-SeCD treatment. The glutathione level, depressed by I/R, was elevated back to normal levels by treatment with 2-SeCD. Severe hepatic damage were observed by light and transmission electron microscopy whilst pretreatment with 2-SeCD resulted in tissue and cellular preservation. Furthermore, 2-SeCD reduced cytochrome c release from mitochondria and subsequent DNA fragmentation by regulating Bcl-2/Bax expression ratio. RESULTS suggested that 2-SeCD was more effective than ebselen in the reversal of the alteration in tissue structural and biochemical parameters caused by I/R injury., Conclusion: 2-selenium-bridged beta-cyclodextrin playes an important role in the protection of liver against I/R injury and this treatment may be a novel pharmacological agent for liver surgery.

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.

To elucidate the relationships between molecular recognition and catalytic ability, we chose three assay systems using three different thiol substrates, glutathione (GSH), 3-carboxyl-4-nitrobenzenethiol (CNBSH), and 4-nitrobenzenethiol (NBSH), to investigate the glutathione peroxidase (GPx) activities of 2,2'-ditellurobis(2-deoxy-beta-cyclodextrin) (2-TeCD) in the presence of a variety of structurally distinct hydroperoxides (ROOH), H2O2, tert-butyl peroxide (tBuOOH), and cumene peroxide (CuOOH), as the oxidative reagent. A comparative study of the three assay systems revealed that the cyclodextrin moiety of the GPx mimic 2-TeCD endows the molecule with selectivity for ROOH and thiol substrates, and hydrophobic interactions are the most important driving forces in 2-TeCD complexation. Furthermore, in the novel NBSH assay system, 2-TeCD can catalyze the reduction of ROOH about 3.4 x 10(5) times more efficiently than diphenyl diselenide (PhSeSePh), and its second-order rate constants for thiol are similar to some of those of native GPx. This comparative study confirms that efficient binding of the substrate is essential for the catalytic ability of the GPx mimic, and that NBSH is the preferred thiol substrate of 2-TeCD among the chosen thiol substrates. Importantly, the proposed mode of action of 2-TeCD imitates the role played by several possible noncovalent interactions between enzymes and substrates in influencing catalysis and binding.

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.

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.