The separation and enrichment of cell membrane proteins was achieved by the construction of bi-functional magnetic fluorescent nanoprobes.

A 76-mer selenium-containing peptide (Se-CuZn-76P)with double glutathione peroxidase (GPx)and superoxide dismutase (SOD)activity,which was embedded in a sequence of membrane-penetrating domain,was designed according to the principles of module enzyme,and was expressed by the method of cysteine auxotrophic expression in a single protein production (SPP)system,then the cell-penetrating effects of Se-CuZn-76P were identified. Experimental results show that the method can successfully express the high purity of Se-CuZn-76P,and demonstrate GPx activity (109 μmol/min)and SOD activity (1218 μmol/min)per mg of protein.The GPx activity and the SOD activity in hepatic L02 cells are increased by 1.65 and 4.12 times respectively,when L02 cells are cultured for a period of time in the presence of Se-CuZn-76P,and Se-CuZn-76P can be successfully entered into L02 cells.

Using fluorescence staining of 4,6-diamidino-2-phenylindole dihyodrochloride and flow cytometry we studied protective effect of an artificial mimic 2-tellurium bridged cyclodextrin (2-TeCD)with the activity of glutathione peroxidase (GPx)on UVB-induced damage in NIH3T3 cells.The results show that 2-TeCD can affect DNA lysis rate,the level of lipid peroxidation,the volume fraction of ROS,cell cycle,P53 protein expression and other indicators in NIH3T3 cells caused by UVB.The 2-TeCD can play a protective role against UVB damage by removing free radicals.

OBJECTIVE:To investigate the effect of selenopeptide on phagocytosis, NO and H2O2 secretion of mouse peritoneal macrophages.METHODS:Mouse peritoneal macrophages induced by lipopolysaccharide (LPS) were cultured for 24 hours by various concentrations of Se-ZnCu-65P, which is a selenopeptide with double antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Then, the relative cell activity was determined by methyl thiazolyl tetrazolium (MTT) assays, the phagocytic ability of macrophages was evaluated by neutral red uptake assay, nitric oxide (NO) content was examined by nitrate reductase method, and hydrogen peroxide (H2O2) content was detected by molybdate colorimetric method.RESULTS:When Se-ZnCu-65P acted on macrophages alone, the relative cell viability was enhanced, the phagocytic ability was not affected, H2O2 content was reduced, and NO level was almost unchanged. However, the phagocytic ability of macrophages induced by LPS was raised, meanwhile, secretion of NO and H2O2 was promoted. When Se-ZnCu-65P acted on macrophages induced by LPS, the phagocytic ability were further enhanced, and secretion of NO and H2O2 was inhibited significantly.CONCLUSION:Se-ZnCu-65P can effectively improve the relative cell activity of mouse peritoneal macrophages induced by LPS, enhance the phagocytic ability of macrophages, and reduce the secretion levels of NO and H2O2.

目的:在大肠杆菌中进行含硒抗病毒多肽的可溶性半胱氨酸缺陷型融合蛋白的表达,并对其纯化蛋白进行生物学活性的初步鉴定.方法:将含硒抗病毒多肽Se-GBVA10基因克隆到GST融合表达载体pGEX-2T中,转化至半胱氨酸缺陷型大肠杆菌BL21 cysE51后进行半胱氨酸缺陷型表达,通过谷胱甘肽Sepharose 4B亲和柱纯化目的蛋白质,并对其含硒量、抗病毒活力等性质进行初步鉴定.结果:利用半胱氨酸缺陷型表达法成功地可溶性表达了sjGST-Se-GBVA10和sjGSTGBVA10融合蛋白,并经凝血酶切后获得了纯化的Se-GBVA10和GBVA10抗病毒多肽.测定SeGBVA10的含硒量为0.974 mol/mol peptide,GPX活力为47.52 U/μmol,其EC50为21.73 μmol/L,CC50为849.41 μmol/L.结论:Se-GBVA10具有与GBVA10相同的抗病毒活性,并具有一定的抗氧化活力.

In this paper, we constructed a novel bifunctional superoxide dismutase(SOD)/glutathione peroxidase(GPx) mimic, a selenium-, copper-containing 35-mer peptide conjugate(Se-Cu-35P) in which a three-amino acid linker(Gly-Asn-Gly) connects the C-terminus of 17-mer polypeptide SOD mimic with the N-terminus of 15-mer polypeptide GPx mimic. The SOD and GPx activities of Se-Cu-35P are two orders of magnitude lower than those of natural SOD and GPx, respectively. It provides a GPx activity 56-fold higher than Ebselen(a well-known GPx mimic). The glutathione(GSH) binding constant is 5.6×102 L·mol−1. Se-Cu-35P synergistically resists against the inactivation by H2O2 and protects the mitochondria from oxidative damage in a dose dependent manner. These results highlight the challenge of generating an efficient SOD/GPx synergism mimic. It could facilitate the studies of the cooperation of GPx and SOD and could be a potential therapeutic agent for the treatment of ROS-mediated diseases.

A 65-mer peptide (Se–CuZn–65P) is an enzyme mimic with antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX). The metallic selenopeptide imitate was obtained through a single protein production system and cysteine auxotrophic expression technique, and it enters cells in the assistance of the trans-activator of transcription peptide. Because Se–CuZn–65P was provided with the antioxidant activities of SOD and GPX, it can be a potential drug for the prevention of alcoholic liver diseases. Therefore, the protective effects of Se–CuZn–65P on ethanol-induced L02 cells were evaluated in vitro. Our results revealed that Se–CuZn–65P significantly protected the hepatocytes against ethanol-induced cell cytotoxicity which displayed by increase of cell viability, migration and decrease of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH), aspartate aminotransferase (AST). Furthermore, the experiments of TUNEL assay, flow cytometry assay, expression levels determination of apoptosis-related gene ( Bad , Bax , Bcl - xL , Bcl - 2 ) and protein (pro-caspase3, PARP cleavage) indicated that Se–CuZn–65P inhibited ethanol-induced L02 cell apoptosis. Mechanically, Se–CuZn–65P could attenuate the oxidative damage of mitochondria in ethanol-induced L02 cells. Se–CuZn–65P prevented ethanol-triggered ROS and MDA generation in L02 cells and inhibited the release of the AST and LDH. Se–CuZn–65P also restored the equilibrium of proapoptotic and antiapoptotic genes including Bad , Bax , Bcl - xL , and Bcl - 2 . And the reduction of procaspase-3 and the cleavages of PARP were attenuated by Se–CuZn–65P. Thereby, the L02 cell apoptosis were significantly prevented by treatment with Se–CuZn–65P, mainly due to attenuation of oxidative stress. In summary, Se–CuZn–65P could prevent ethanol-induced liver injury through enhancing hepatocyte antioxidant abilities in intracellular.

In Escherichia coli , four gene products( selA, selB, selC and selD) and a selenocysteine(Sec) insertion sequence( SECIS) are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression of selC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L -arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L -arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the efficiency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.

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 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).

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-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.

As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxidative stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(H2O2) challenged rat cardiomyocytes, we examined malondialdehyde(MDA), lactate dehydrogenase(LDH), superoxide dismutase(SOD) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H2O2 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H2O2 with the decreases of cell apoptosis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocytes.

The two soybean peptides (SP), SP-1 and SP-2, were obtained after hydrolyzing the Soybean Isolated Protein (SIP) by alcalase protease. And then, the SPs were converted into selenium-containing SPs (Se-SP) by chemically modification. The Se-SPs showed higher GPX activity than Ebselen, the first mimic of GPX, but they were much lower than native GPX. The characters of Se-SPs were also determined such as the selenium content, binding constant of active for GSH. In addition, antioxidant activity experiment indicated that Se-SPs has potential application. The Se-SPs possessed high scavenging ability to super-oxide anion, and they can protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-(Fe 2+ /Vc) induced mitochondria damage model. This not only increases the practical value of SPs, but also increased value-added products to market was provided.

Objective To observe the influence of microwave radiation in the apoptosis of human neuroblastoma SH-SY5Y cells and explore its mechanism.Methods Regarding 0 mW·cm-2 as sham radiation group,the SH-SY5Y cells were radiated for 5 minutes by 10,30 and 50 mW·cm-2 of microwave.The morphological changes of cells were observed under light microscope;the cell nucleus stained by DAPI were observed under fluorescence microscope;the cell genomic DNA was extracted by CTAB and electrophoresed to observe DNA ladder;the cell viability was detected by trypan blue exclusion assay;the apoptotic rate was detected by flow cytometry with annexin V-FITC/PI double staining;the relative activity of cells was detected by MTT assay;the expressions of apoptosis-related proteins were detected by Western blotting.Results After microwave radiation,the morphology of SH-SY5Y cells changed immediately,the nucleus and cytoplasm structure were less clear,and cell shrinkage or even off the wall were observed;chromatin in the nucleus appeared irregular condensation,and fragmentated into 2 to 5 micronuclei;cell DNA ladder was apparent.The survival rate of SH-SY5Y cells was reduced gradually with the increasing of microwave radiation intensity,there was no significant difference between 10 mW·cm-2 radiation group and sham radiation group(P0.05);however,the cell survival rates in 30 and 50 mW·cm-2 radiation group were lower than that in sham radiation group(P0.05).The apoptotic rate of SH-SY5Y cells was gradually increased with the microwave radiation intensity in 6 h after microwave radiation,and the apoptotic rates in each radiation group were higher than that in sham radiation group(P0.05).Compared with sham radiation group,the cell relative activities of SH-SY5Y cells in each radiation group were markedly decreased 24 and 48 h after microwave radiation(P0.05),and they were decreased with the increasing of microwave radiation intensity.After microwave radiation,the expressions of apoptosis related proteins Bcl-2 and survivin were decreased,the expressions of Bax,Caspase-3 and Caspase-7 were increased gradually,but the expressions of Caspase-8 and Caspase-9 changed little.Conclusion The apoptosis of SH-SY5Y cells can be induced by microwave radiation in a obvious dose-dependent manner,which may be related to the increase of Survivin and Bcl-2 proteins,the decrease of Bax protein and the action of the Caspase-3 and Caspase-7,but with little relationship of the activation of Caspase-3 mediated by caspase-8 and Caspase-9.

In order to create a new mimic of glutathione peroxidase(GPx), bioimprinting was used to generate glutathione(GSH) binding site and chemical modification was used to incorporate catalytic group selenocystine(Sec). Human serum albumin(HSA) and S-substituted dinitrophenyl glutathione(GSH-S-DNP) were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein(GSH-HSA) by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GPx mimic(Se-GSH-HSA). Se-GSH-HSA displayed considerably higher GPx activity than non-printed HSA(Se-HSA). The enzymic properties and kinetics of Se-GSH-HSA were studied. Moreover, Se-GSH-HSA was confirmed to have stronger antioxidant ability to protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-induced mitochondria damage model, indicating that Se-GSH-HSA has potential application in medicine.

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(See) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

In order to create a new mimic of glutathione peroxidase(GPx), bioimprinting was used to generate gluta- thione(GSH) binding site and chemical modification was used to incorporate catalytic group selenocystine(Sec). Human serum albumin(HSA) and S-substituted dinitrophenyl glutathione(GSH-S-DNP) were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein(GSH-HSA) by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GPx mimic(Se-GSH-HSA). Se-GSH-HSA displayed considerably higher GPx activity than non-printed HSA(Se-HSA). The enzymic properties and kinetics of Se-GSH-HSA were studied. Moreover, Se-GSH-HSA was confirmed to have stronger antioxidant ability to protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-induced mitochondria damage model, indicating that Se-GSH-HSA has potential application in medicine. Keywords Antioxidant; Enzyme mimic; Glutathione peroxidase; Molecular imprinting Article ID 1005-9040(2011)-02-258-06

The 2-hydroxyl of -cyclodextrins can be selenized,producing an artificial simulation GPX enzyme of selenium which has wide application prospects.In order to carry on the further research,a lot of products were needed.Middle scale preparation of 2-SeCD was done based on the experiments of the original research and the amount was expanded for original 50 times.7 experimental results showed that the technologies were perfect and a large number of products were obtained.The structure of product obtained by middle scale preparation was verified by a series of characterization experiments such as carbon and hydrogen analysis,IR,NMR mass spectrometry analysis,etc.GPX activity of 2-SeCD was measured as 5.2 U/mol.The results of the biological effect test showed that low concentrations of 2-SeCD can reduce cell death but high concentrations of 2-SeCD inhibit cell growth.Apoptosis of Hela cells induced by 2-SeCD was found through morphological observation and DNA ladder detection,moreover,its mechanism was identified as the intrinsic mitochondrial apoptosis pathway by relative activity of caspase-3 assay.

Reactive oxygen species (ROS) are involved in cell growth, differentiation, and death. Excessive amounts of ROS (e.g., O2−, H2O2, and HO) play a role in aging as well as in many human diseases. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are critical antioxidant enzymes in living organisms. SOD catalyzes the dismutation of O2− to H2O2, and GPx catalyzes the reduction of H2O2 and other harmful peroxides by glutathione (GSH). They not only function in catalytic processes but also protect each other, resulting in more efficient removal of ROS, protection of cells against injury, and maintenance of the normal metabolism of ROS. To imitate the synergism of SOD and GPx, a 65-mer peptide (65P), containing sequences that form the domains of the active center of SOD and the catalytic triad of GPx upon the incorporation of some metals, was designed on the basis of native enzyme structural models; 65P was expressed in the cysteine auxotrophic expression system to obtain Se-65P. Se-65P was converted into Se–CuZn-65P by incorporating Cu2+ and Zn2+. Se–CuZn-65P exhibited high SOD and GPx activities because it has a delicate dual-activity center. The synergism of the enzyme mimic was evaluated by using an in vitro model and a xanthine/xanthine oxidase/Fe2+-induced mitochondrial damage model system. We anticipate that the peptide enzyme mimic with synergism is promising for the treatment of human diseases and has potential applications in medicine as a potent antioxidant.