Silent information regulator sirtuin 1 ameliorates acute liver failure via the p53/glutathione peroxidase 4/gasdermin D axis

BACKGROUND Acute liver failure(ALF)has a high mortality with widespread hepatocyte death involving ferroptosis and pyroptosis.The silent information regulator sirtuin 1(SIRT1)-mediated deacetylation affects multiple biological processes,including cellular senescence,apoptosis,sugar and lipid metabolism,oxidative stress,and inflammation.AIM To investigate the association between ferroptosis and pyroptosis and the upstream regulatory mechanisms.METHODS This study included 30 patients with ALF and 30 healthy individuals who underwent serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)testing.C57BL/6 mice were also intraperitoneally pretreated with SIRT1,p53,or glutathione peroxidase 4(GPX4)inducers and inhibitors and injected with lipopolysaccharide(LPS)/D-galactosamine(D-GalN)to induce ALF.Gasdermin D(GSDMD)^(-/-)mice were used as an experimental group.Histological changes in liver tissue were monitored by hematoxylin and eosin staining.ALT,AST,glutathione,reactive oxygen species,and iron levels were measured using commercial kits.Ferroptosis-and pyroptosis-related protein and mRNA expression was detected by western blot and quantitative real-time polymerase chain reaction.SIRT1,p53,and GSDMD were assessed by immunofluorescence analysis.RESULTS Serum AST and ALT levels were elevated in patients with ALF.SIRT1,solute carrier family 7a member 11(SLC7A11),and GPX4 protein expression was decreased and acetylated p5,p53,GSDMD,and acyl-CoA synthetase long-chain family member 4(ACSL4)protein levels were elevated in human ALF liver tissue.In the p53 and ferroptosis inhibitor-treated and GSDMD^(-/-)groups,serum interleukin(IL)-1β,tumour necrosis factor alpha,IL-6,IL-2 and C-C motif ligand 2 levels were decreased and hepatic impairment was mitigated.In mice with GSDMD knockout,p53 was reduced,GPX4 was increased,and ferroptotic events(depletion of SLC7A11,elevation of ACSL4,and iron accumulation)were detected.In vitro,knockdown of p53 and overexpression of GPX4 reduced AST and ALT levels,the cytostatic rate,and GSDMD expression,restoring SLC7A11 depletion.Moreover,SIRT1 agonist and overexpression of SIRT1 alleviated acute liver injury and decreased iron deposition compared with results in the model group,accompanied by reduced p53,GSDMD,and ACSL4,and increased SLC7A11 and GPX4.Inactivation of SIRT1 exacerbated ferroptotic and pyroptotic cell death and aggravated liver injury in LPS/D-GalNinduced in vitro and in vivo models.CONCLUSION SIRT1 activation attenuates LPS/D-GalN-induced ferroptosis and pyroptosis by inhibiting the p53/GPX4/GSDMD signaling pathway in ALF.

Effects of Hg^(2+) on Isozymes of Peroxidase,Catalase and Superoxide Dismutase in Wheat Seedlings

[Objective] This work was aimed to explore the mechanism of Hg2+ toxicity on plants.[Method]Activities of peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)were investigated in wheat(Triticum aestivum L.)seedlings under Hg2+ stress at different concentrations.[Result]① There were no obvious effects on the growth of seedlings when the concentration of Hg2+ was lower than 0.10 mmol/L.However,toxic effects on the growth of seedling were observed when the concentration of Hg2+ was higher than 0.10 mmol/L.② Different tissues showed different resistant ability in response to Hg2+ stress.The leaves and roots of wheat seedlings were more insensitive to Hg2+ toxicity.③ CAT was more sensitive to Hg2+ stress compared to POD and SOD.[Conclusion]The toxic effect was related to the concentration of Hg2+(0.10 mmol/L).The higher concentration of Hg2+ could affect the expression of POD,CAT,and SOD isozymes in the leaves,roots of wheat seedlings and germinated seeds,which further affect the normal metabolism of membrane lipid and inhibit the growth of wheat seedlings at last.

Effects of Catalase and Ascorbate Peroxidase on the Root Growth of Rice under Cadmium Stress

[Objective] The research aimed to study the correlations between catalase（CAT） and ascorbate peroxidase（ASP） and the growth and development of rice roots under cadmium stress.[Method] Taking rice variety Zhonghua No.11 as materials,the changes of rice seedlings under the treatment conditions of Cd,Cd＋CAT inhibitor,Cd＋APX inhibitor were studied.[Result] Under Cd stress,inhibition of CAT activity caused the significant inhibition on the growth of aerial parts,decreased the number of adventitious roots and lateral roots,but it can significant promote the elongation growth of adventitious roots and lateral roots.Moreover,the length of the first lateral root from root tip on the primary roots and adventitious roots was also increased than control.When APX activity was inhibited,the growth changes of rice were similar with that treated by CAT inhibitor.[Conclusion] CAT and APX may play important roles in the regulation of rice root system growth in both non-stress and Cd-stressed rice

Regulation of Heat Shock Factor AtHsfA1a on Ascorbate Peroxidase under Heat Stress in Arabidopsis thaliana

[Objective] This study was conducted to investigate the regulation of heat shock factor AtHsfA1a on ascorbate peroxidase under heat stress in Arabidopsis thaliana. [Method] After heat stress treatment on transgenetic A. thaliana with silenced endogenetic AtHsfA1a gene and wild A. thaliana plants as materials, the change in activity of APX enzyme was analyzed by spectrophotometry, the expression level of APX gene was investigated by real-time fluorescent quantitative PCR, and the binding condition of AtHsfAla with the promoter region of APX gene was analyzed by chromatin immunoprecipitation assay. [Result] The activity and mRNA level of APX in plants with silenced endogenetic AtHsfAla gene were higher than those in wild plants. Fragments of the promoter region of APX gene were not screened from the plants with silenced endogenetic AtHsfA1a gene, but found in wild plants. [Conclusion] This study provides a theoretical basis for the understanding of the important role of AtHsfAla in resistance to stress in plant, and is of great significance to the revealing of mechanism of resistance to stress in plant.

Enhanced Fenton,photo-Fenton and peroxidase-like activity and stability over Fe_3O_4/g-C_3N_4 nanocomposites

We prepared the Fe3O4/g‐C3N4nanoparticles(NPs)through a simple electrostatic self‐assembly method with a3:97weight ratio to investigate their Fenton,photo‐Fenton and oxidative functionalities besides photocatalytic functionality.We observed an improvement of the Fenton and photo‐Fenton activities of the Fe3O4/g‐C3N4nanocomposites.This improvement was attributed to efficient charge transfer between Fe3O4and g‐C3N4at the heterojunctions,inhibition of electron‐hole recombination,a high surface area,and stabilization of Fe3O4against leaching by the hydrophobic g‐C3N4.The obtained NPs showed a higher degradation potential for rhodamine B(RhB)dye than those of Fe3O4and g‐C3N4.As compared to photocatalysis,the efficiency of RhB degradation in the Fenton and photo‐Fenton reactions was increased by20%and90%,respectively.Additionally,the horseradish peroxidase(HRP)activity of the prepared nanomaterials was studied with3,3,5,5‐tetramethylbenzidinedihydrochloride(TMB)as a substrate.Dopamine oxidation was also examined.Results indicate that Fe3O4/g‐C3N4nanocomposites offers more efficient degradation of RhB dye in a photo‐Fenton system compared with regular photocatalytic degradation,which requires a long time.Our study also confirmed that Fe3O4/g‐C3N4nanocomposites can be used as a potential material for mimicking HRP owing to its high affinity for TMB.These findings suggest good potential for applications in biosensing and as a catalyst in oxidation reactions.

Effect of Lanthanum Ion on Peroxidase in Plant

The interaction of MP 11 as a model of antioxidatase enzymes with La 3+ was investigated. It was found that La 3+ can increase in the non planarity of heme and the content of α helix and β turn conformations of the MP 11 molecule. The change in the secondary structure of the MP 11 molecule can increase in the exposure extent of heme to the solution. Therefore, the electrochemical reaction of MP 11 is promoted and the electrocatalytic activity to the reduction of H 2O 2 is increased. The results are consistent with that for the interaction of peroxidases(POD), one of the antioxidatase enzymes, obtained in the living plant experiments at low concentration of La 3+ .

Detection mechanism and classification of design principles of peroxidase mimic based colorimetric sensors: A brief overview

Synthesis and characterization of enzyme mimics with characteristic stability and high catalytic efficiency is an interesting field for researchers.Especially,with the development of nanoscience and introducing of Fe3O4 magnetic nanoparticles as peroxidase mimics in 2007,various nanomaterials such as noble metals,metal oxides,and carbon materials were introduced as enzyme mimics(nanozymes).Various nanomaterials exhibit peroxidaselike activity,hence,most of the nanozymes are peroxidase mimetics.Although the nanozyme based sensors were previously classified,the classifications have been focused on the type of nanozyme action.Therefore,the nanozyme based sensors were classified as peroxidase,hydrolase,and urease mimic-based sensors.However,heretofore,these sensors are not classified based on the detection mechanism and principles of system design.The aim of this review is the focus on the peroxidase mimic based colorimetric sensors as the most common nanozyme-based sensors and their classification based on principles of sensor design and review of the detection mechanism of the current mimic peroxidase based sensors.Moreover,some current challenges and future developments in this field are discussed.

Effects of low molecular weight heparin-superoxide dismutase conjugate on serum levels of nitric oxide,glutathione peroxidase,and myeloperoxidase in a gerbil model of cerebral ischemia/reperfusion injury

BACKGROUND： Several studies have demonstrated that low molecular weight heparin-superoxide dismutase （LMWH-SOD） conjugate may exhibit good neuroprotective effects on cerebral ischemia/reperfusion injury though anticoagulation, decreasing blood viscosity, having anti-inflammatory activity, and scavenging oxygen free radicals. OBJECTIVE： To investigate the intervention effects of LMWH-SOD conjugate on serum levels of nitric oxide （NO）, glutathione peroxidase （GSH-Px）, and myeloperoxidase （MPO） following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： A randomized, controlled, and neurobiochemical experiment was performed at the Institute of Biochemical Pharmacy, School of Pharmaceutical Sciences, Shandong University between April and July 2004. MATERIALS： A total of 60 Mongolian gerbils of either gender were included in this study. Total cerebral ischemia/reperfusion injury was induced in 50 gerbils by occluding bilateral common carotid arteries. The remaining 10 gerbils received a sham-operation （sham-operated group）. Kits of SOD, NO, and MPO were sourced from Nanjing Jiancheng Bioengineering Institute, China. LMWH, SOD, and LMWH-SOD conjugates were provided by Institute of Biochemistry and Biotechnique, Shandong University, China. METHODS： Fifty successful gerbil models of total cerebral ischemia/reperfusion injury were evenly randomized to five groups： physiological saline, LMWH-SOD, SOD, LMWH ＋ SOD, and LMWH. At 2 minutes prior to ischemia, 0.5 mL/65 g physiological saline, 20 000 U/kg LMWH-SOD conjugate, 20 000 U/kg SOD, a mixture of SOD （20 000 U/kg） and LMWH （LMWH dose calculated according to weight ratio, LMWH： SOD = 23.6：51）, and LMWH （dose as in the LMWH ＋ SOD group） were administered through the femoral artery in each above-mentioned group, respectively. MAIN OUTCOME MEASURES： Serum levels of NO, MPO, and GSH-Px. RESULTS： Compared with 10 sham-operated gerbils, the cerebral ischemia/reperfusion injury gerbils exhibited decreased serum levels of GSH-Px and increased serum levels of NO and MPO （P 〈 0.01）. The serum level of GSH-Px was significantly upregulated in all groups, in particular in the LMWH-SOD group （P 〈 0.01）, compared with the physiological saline group （P 〈 0.05-0.01）. Following medical treatment, serum levels of NO and MPO were significantly downregulated in all groups, in particular in the LMWH-SOD group （P 〈 0.01）. Serum levels of GSH-Px, NO, and MPO in the LMWH-SOD group were close to those in the sham-operated group （P 〉 0.05）. CONCLUSION： In cerebral ischemia/reperfusion injury, LMWH-SOD conjugate exhibits stronger neuroprotective effects on free radical scavenging, inflammation inhibition, and cytotoxicity inhibition than simple or combined application of LMWH and SOD by downregulating NO and MPO levels and upregulating the GSH-Px level.

Microscopic Characteristics, Chromatographic Profiles and Inhibition of Peroxidase Activity of the Leaves of <i>Manihot esculenta</i>and <i>Manihot glaziovii</i>, Consumed as Traditional Vegetables

Methanolic extracts from the leaves of Manihot esculenta (Two cultivars) and Manihot glaziovii, consumed as traditional vegetables in DR. Congo was chemically characterized by Thin layer Chromatography and High Performance Liquid Chromatography. In vitro biochemical activities of extracts against Radical Oxidative Species (ROS) production were assessed in cellular models, on enzymes, Myeloperoxidase (MPO) and Horseradish Peroxidase (HRP) involved in inflammation. The microscopic analysis of the powder of leaves showed that each species displays specific and discriminating botanical microscopic features. Varieties of M. esculenta had a chemical fingerprint different from M. glaziovii. The majority of compounds were polyphenols, represented mainly by rutin, kaempferol-3-O-rutinoside, amentoflavone, phenolic acids such as gallic acid. All extracts exhibited high cellular antioxidant activity in the range of 0.1 to 10 μg·mL-1 using lucigenin with neutrophils, but a moderate cellular antioxidant activity ranging between 10 and 100 μg·mL-1 with DCFDA on HL60 monocytes. Extracts from Manihot leaves showed a pronounced inhibitory effect on the production of extracellular ROS, on HRP and myeloperoxidase activity. Cellular antioxidant activities, the inhibitory effect on HRP of extracts from M. glaziovii, M. esculenta cultivar Mwambu were significantly higher, but their inhibitory effect on the activity of MPO was lower than those of M. esculenta cultivar TEM 419. The biological activities of Manihot esculenta and Manihot glaziovii were well correlated to their phytochemicals that could justify their traditional use as vegetables, potential functional foods or nutraceutical resources and medicines.

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Studies on Manganese Peroxidase Immobilized in Gelatin-containing Microemulsion-based Gels

The immobilized technique of manganese peroxidase(MnP) in gelatin-containing microemulsion-based gels and the effects of storage time and reuse times on its catalytic activity were studied. The results show that the MnP immobilized together with Mn 2+ and H_2O_2 could effectively oxidize syringaldazine in n-heptane. The immobilized MnP still had a high catalytic activity after one-month storage under a freezing condition. The reuse times have a relation to the amount of the immobilized H_2O_2. When the amount of the immobilized H_2O_2 is sufficient, the microemulsion-based gels containing MnP could be used many times.

Enhancement of Phenylalanine Ammonia Lyase,Polyphenoloxidase,and Peroxidase in Cucumber Seedlings by Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) Infestation

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.

Cloning of catalase and expression patterns of catalase and selenium-dependent glutathione peroxidase from Exopalaemon carinicauda in response to low salinity stress

Catalase （CAT） and selenium-dependent glutathione peroxidase （Se-GPx） play a vital role in protecting organisms against various oxidative stresses by eliminating H202, The objective of this paper is to evaluate the roles of these antioxidant molecules in the ridgetail white prawn Exopalaemon carinicauda in response to low salinity stress. A complementary DNA （cDNA） containing the complete coding sequence of CAT was cloned from the hepatopancreas using reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends. The full-length cDNA of CAT （2 649 bp） contains a 5＇-untranslated region （UTR） of 78 bp, a 3＇- UTR of 1 017 bp, with a poly （A） tail, and an open reading frame of 1 554 bp encoding a 517-amino-acid polypeptide with predicted molecular mass of 58.46 kDa and estimated isoelectric point of 6.64. This CAT sequence contained the proximal active site signature （60FDRERIPERWHAKGAG76）, proximal heme-ligand signature sequence （350RLFSYPDTH358） and three catalytic amino acid residues （His71, Asn144 and Tyr354）. Sequence comparison showed that the CAT deduced amino acid sequence of E. carinicauda shared 68%-92% of identities with those of other species. Quantitative real-time PCR analysis revealed that CAT mRNA was widely expressed in the hepatopancreas （highest）, hemocyte, eyestalk, heart, gill, muscle, ovary and stomach. Under low salinity stress, CAT and GPx mRNA expression levels both in the gill and hepatopancreas increased significantly at the first 48 h and 6 h respectively, indicating a tissue- and time-dependent antioxidant response in E. carinicauda. All these results indicate that E. carinicauda CAT is a member of the CAT family and might be involved in the acute response against low salinity stress.

Imaging of Activity of Horseradish Peroxidase at β-Cyclodextrin Polymer by Scanning Electrochemical Microscopy

The activity of horseradish peroxidase at b-cyclodextrin polymer was imaged by scanning electrochemical microscopy using 3, 3', 5, 5'-tetramethylbenzide and H2O2 as the substrates.

Revealing the Intrinsic Peroxidase-Like Catalytic Mechanism of Heterogeneous Single-Atom Co-MoS2

The single-atom nanozyme is a new concept and has tremendous prospects to become a next-generation nanozyme.However,few studies have been carried out to elucidate the intrinsic mechanisms for both the single atoms and the supports in single-atom nanozymes.Herein,the heterogeneous single-atom Co-MoS2(SA Co-MoS2)is demonstrated to have excellent potential as a high-performance peroxidase mimic.Because of the well-defined structure of SA Co-MoS2,its peroxidase-like mechanism is extensively interpreted through experimental and theoretical studies.Due to the different adsorption energies of substrates on different parts of SA Co-MoS2 in the peroxidase-like reaction,SA Co favors electron transfer mechanisms,while MoS2 relies on Fenton-like reactions.The different catalytic pathways provide an intrinsic understanding of the remarkable performance of SA Co-MoS2.The present study not only develops a new kind of single-atom catalyst(SAC)as an elegant platform for understanding the enzyme-like activities of heterogeneous nanomaterials but also facilitates the novel application of SACs in biocatalysis.

Mechanisms of Salt Tolerance in Transgenic Arabidopsis thaliana Carrying a Peroxisomal Ascorbate Peroxidase Gene from Barley

Ascorbate peroxidases （APX）, localized in the cytosol, peroxisome, mitochondria, and chloroplasts of plant cells, catalyze the reduction of H2O2 to water by using ascorbic acid as the specific electron donor. To determine the role of peroxisomal type ascorbate peroxidase （pAPX）, an antioxidant enzyme, in protection against salt-induced oxidative stress, transgenic Arabidopsis thaliana plant carrying a pAPX gene （HvAPX1） from barley （Hordeum vulgate L.） was analyzed. The transgenic line pAPX3 was found to be more tolerant to salt stress than the wild type. Irrespective of salt stress, there were no significant differences in Na^＋, K^＋, Ca^2＋, and Mg^2＋ contents and the ratio of K^＋ to Na^＋ between pAPX3 and the wild type. Clearly, the salt tolerance in pAPX3 was not due to the maintenance and reestablishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation （measured as the levels of malondialdehyde） accumulation under salt stress was higher in the wild type than in pAPX3. The mechanism of salt tolerance in transgenic pAPX3 can thus be explained by reduction of oxidative stress injury. Under all conditions tested, activities of superoxide, glutathione reductase, and catalase were not significantly different between pAPX3 and the wild type. In contrast, the activity of APX was significantly higher in the transgenic plant than in wild type under salt stress. These results suggested that in higher plants, HvAPX1 played an important role in salt tolerance and was a candidate gene for developing salttolerant crop plants.

Purification and Characterization of a Versatile Peroxidase from Edible Mushroom Pleurotus eryngii

A versatile peroxidase (VP-Peco60-7 ) was generated and purified from the liquid culture of Pleurotus eryngii. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, and gel chromatography. The molecular weight and isoelectric point (pI) of VP-Peco60-7 were determined to be approxi-mately 40 kDa and 4.1, respectively. By N-terminal sequence determination and peptide mapping analysis, VP-Peco60-7 was found to be similar to the versatile peroxidase isoenzyme VPL1, which was previously isolated from liquid cultures of the same species. However, the molecular weight and pI of VP-Peco60-7 were different from those of versatile peroxidases of liquid cultures, implying that the VP-Peco60-7 in this study is of a novel type. With 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate, the maximal enzyme activity was obtained at 50 °C and pH 3.0. The catalysis of ABTS by VP-Peco60-7 was expressed by the Michaelis-Menten equa-tion. At 50 °C and pH 3.0, the maximum velocity (V max ) was 188.68 U·mg-1 and the michaelis constant (K m ) was 203.09 μmol·L-1 .

Overexpression of a Cytosolic Ascorbate Peroxidase Gene, OsAPX2, Increases Salt Tolerance in Transgenic Alfalfa

Alfalfa （Medicago sativa L.） is an important forage crop in the world and it is of great signiifcance for the improvement of its salt tolerance. To improve salt tolerance in alfalfa, a rice ascorbate peroxidase gene （OsAPX2） was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation with marker gene bar. The different T-DNA insertions in T1 transgenic alfalfa were identiifed by Southern hybridization. Three independent T2 transgenic lines were selected for stress analysis and the results showed that all of them were salt tolerant compared with wild-type plants. The transgenic plants had low levels of H2O2, malondialdehyde and relative electrical conductivity under salt and drought stresses. Moreover, the contents of chlorophyll and proline, and APX activity were high in transgenic plants under salt and drought stresses. Taken together, the overexpression of OsAPX2 enhances salt tolerance in alfalfa through scavenging reactive oxygen species.

Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination with horseradish peroxidase

AIM： To explore the mechanisms underlying the apoptosis of human pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid （IAA） in combination with horseradish peroxidase （HRP）. METHODS： BXPC-3 cells derived from human pancreatic cancer were exposed to 40 or 80 μmol/L IAA and 1.2 μg/mL HRP at different times. Then, Mn- assay was used to detect the cell proliferation. Flow cytometry was performed to analyze cell cycle. Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling assay was used to detect apoptosis. 2,7-Dichlorofluorescin diacetate uptake was measured by confocal microscopy to determine free radicals. Level of malondialdehyde （MDA） and activity of superoxide dismutase （SOD） were measured by biochemical methods. RESULTS： IAA/HRP initiated growth inhibition of BXPC-3 cells in a dose- and time-dependent manner. Flow cytometry revealed that the cells treated for 48 h were arrested at G1/G0. After exposure to 80 μmol/L IAA plus 1.2 μg/mL HRP for 72 h, the apoptosis rate increased to 72.5‰, which was nine times that of control. Content of MDA and activity of SOD increased respectively after treatment compared to control. Meanwhile, IAA/HRP stimulated the formation of free radicals. CONCLUSION： The combination of IAA and HRP can inhibit the growth of human pancreatic cancer BXPC-3 cells in vitro by inducing apoptosis.

Expression of lignin peroxidase H2 from Phanerochaete chrysosporium by multi-copy recombinant Pichia strain

The lipH2 gene, encoding the expression of lignin peroxidase, was cloned from Phanerochaete chrysosporium BKM-F-1767 and expressed in Pichia pastoris X-33, a yeast. The cDNA of LiPH2 was generated from total RNA extracted from P chrysosporium by PCR with primers that do not contain a P. chrysosporium lignin peroxidase secretion signal. The gene was then successfully inserted into the expression vector pPICZα, and resulted in the recombinant vector pPICZα-lipH2. The transformation was conducted in two ways. One was using the wild Pichia pastoris as the recipients, which results in the recombinant P. pastoris with single or low lipH2 gene copy. The second was using P. pastoris and single or low lipH2 gene copy as the recipients, which results in the recombinant P. pastoris with multi-copies of lipH2 genes. This study firstly expressed the gene lipH2 in P. pastoris and achieved the successful expression of the lipH2 depending upon the generation of a recombinant strain that contained multiple copies. The lignin peroxidase activity reached a maximum of 15 U/L after 12 h induction.