Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced HO production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.
Antioxidants ; pharmacology ; Benzopyrans ; pharmacology ; Caesalpinia ; chemistry ; Humans ; Hydrogen Peroxide ; toxicity ; Keratinocytes ; cytology ; drug effects ; enzymology ; radiation effects ; Oxidative Stress ; drug effects ; radiation effects ; Peroxidases ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Ultraviolet Rays

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
Amino Acid Sequence ; Catalysis ; Coloring Agents ; metabolism ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peroxidases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Zymomonas ; enzymology

Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heme ; biosynthesis ; genetics ; Peroxidases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic

OBJECTIVETo analysis the effects of water deficit on the transcript level of SOD, APX, DHAR and MDHAR genes in Scutellaria baicalensis.

METHODThree-month-old S. baicalensis was in glasshouse under water deficit stress, and the transcript level of SOD, APX, DHAR and MDHAR genes were analysis utilized semi-quantitative RT-PCR.

RESULTCompared with the control group, a significant decline of the transcriptional level of APX gene was observed at 70 days after water deficit. The transcript level of DHAR gene was reduced at 30 and 50 days after water deficit. And MDHARI gene was significant declined at 50 days.

CONCLUSIONAsA which is an important antioxidant plays a major role in hydrogen peroxide clear system under water deficit, and maybe have an antagonistic effect to the accumulation of baicalein.

Ascorbate Peroxidases ; genetics ; metabolism ; Ascorbic Acid ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Plant ; drug effects ; Hydrogen Peroxide ; metabolism ; NADH, NADPH Oxidoreductases ; genetics ; metabolism ; Oxidoreductases ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scutellaria baicalensis ; enzymology ; genetics ; metabolism ; Superoxide Dismutase ; genetics ; metabolism ; Time Factors ; Water ; metabolism ; pharmacology

Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.
Animals ; Antibodies ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Peroxidases ; biosynthesis ; genetics ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Leaf senescence is often caused by water deficit and the chimeric gene P(SAG12)-IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of P(SAGl2)-IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6000 nutrient solution for 20 h under continuous light [130 micromol/(m(2) x s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in P(SAG12)-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.
Alkyl and Aryl Transferases ; genetics ; Antioxidants ; metabolism ; Arabidopsis Proteins ; genetics ; Ascorbate Peroxidases ; Asteraceae ; genetics ; metabolism ; Carotenoids ; metabolism ; Catalase ; metabolism ; Chlorophyll ; metabolism ; Cysteine Endopeptidases ; genetics ; Genes, Bacterial ; Genes, Plant ; Lipid Peroxidation ; Osmotic Pressure ; Oxidoreductases ; metabolism ; Peroxidase ; metabolism ; Peroxidases ; metabolism ; Plant Leaves ; metabolism ; Plant Proteins ; metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Solubility ; Superoxide Dismutase ; metabolism

To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.
Cell Line, Transformed ; Cloning, Molecular ; Embryo, Mammalian ; Eukaryotic Cells ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Kidney ; cytology ; metabolism ; Peroxidases ; biosynthesis ; genetics ; Peroxiredoxin III ; Peroxiredoxins ; Plasmids ; genetics ; Transfection

To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.
Cell Line, Transformed ; Cloning, Molecular ; Embryo ; Eukaryotic Cells/*metabolism ; Gene Expression ; Genetic Vectors ; Kidney/cytology ; Kidney/*metabolism ; Peroxidases/*biosynthesis ; Peroxidases/genetics ; Plasmids/*genetics ; Transfection

Peroxiredoxin II (Prx II) is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to endow cancer cells with resistance to both H2O2 and cisplatin and to grant them radioresistance. In this study, we examined whether Prx II antisense could enhance cisplatin-induced cell death. When gastric cancer cells were transfected with various concentrations of Prx II antisense plasmid, pPrxII/AS, and then treated with the same concentrations of cisplatin, Prx II antisense enhanced cisplatin-induced cell death. The combination index (CI) at all doses of the combination was below 1, indicating that Prx II antisense sensitized cisplatin-induced cell death. This synergism was also observed in the cells transfected with a Prx II antisense oligomer. Our present results, therefore, suggest that Prx II antisense would be a very good sensitizer for cisplatin, and that Prx II as a target for chemosensitizers constitutes a promising avenue for future research.
Antineoplastic Agents/*pharmacology ; Antioxidants/metabolism ; Apoptosis/drug effects ; Cell Death/*drug effects ; Cisplatin/*pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Genetic Vectors ; Human ; Oligonucleotides, Antisense/*metabolism ; Peroxidases/*metabolism ; Plasmids/genetics/metabolism ; Stomach Neoplasms/*metabolism ; Tumor Cells, Cultured

Peroxiredoxin II (Prx II) is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to endow cancer cells with resistance to both H2O2 and cisplatin and to grant them radioresistance. In this study, we examined whether Prx II antisense could enhance cisplatin-induced cell death. When gastric cancer cells were transfected with various concentrations of Prx II antisense plasmid, pPrxII/AS, and then treated with the same concentrations of cisplatin, Prx II antisense enhanced cisplatin-induced cell death. The combination index (CI) at all doses of the combination was below 1, indicating that Prx II antisense sensitized cisplatin-induced cell death. This synergism was also observed in the cells transfected with a Prx II antisense oligomer. Our present results, therefore, suggest that Prx II antisense would be a very good sensitizer for cisplatin, and that Prx II as a target for chemosensitizers constitutes a promising avenue for future research.
Antineoplastic Agents/*pharmacology ; Antioxidants/metabolism ; Apoptosis/drug effects ; Cell Death/*drug effects ; Cisplatin/*pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Genetic Vectors ; Human ; Oligonucleotides, Antisense/*metabolism ; Peroxidases/*metabolism ; Plasmids/genetics/metabolism ; Stomach Neoplasms/*metabolism ; Tumor Cells, Cultured