SeGPX activity of three organoselenium-containing cyclodextrins and their scavenging effects on HO center dot

Three organoselenium-containing derivatives of beta-cyclodextrins (beta-CD), mono-6-benzylseleno-6-deoxy-beta-cyclodextrin (compound 1), 6,6'-trimethylenediseleno bridged beta-cyclodextrin dimer(compound 2) and 6,6'- (o-phenylene)diseleno bridged beta-cyclodextrin dimer (compound 3) functioned as mimics of selenium-containing glutathione peroxidase(SeGPX). Acting on H2O2 and GSH, the SeGPX activities of these compounds were 0.83-, 0.26-, and 1. 23-fold of that of Ebselen (0.99 U/mu mol), respectively. The relationship between the structure and the function of these compounds was studied. The results suggested that the hydrophobicity and rigidity of phenyl group is the main reason that accounted for the higher activity of compounds 3 and 1. Phenyl group not only provided the hydrophobic circumstance which is necessary for the catalytic function of selenium, but also make it possible that the cyclodextrin unit of compounds 1 and 3 combines the substrate with a more effective direction. Fluorometric techniques were utilized to determine the yields of the hydroxyl radical produced by Fenton reactions through the formation of hydroxy benzoic acids from benzoate. Compared with Ebselen which showed a significant inhibition effect on the formation of HO., these organoselenium-containing cyclodextrins showed a little scavenging effect on the formation of HO. throughout the whole process.

Functionalized inorganic-organic composite material derivated by sol-gel for construction of mediated amperometric hydrogen peroxide biosensor

A novel functionalized inorganic-organic hybrid material with cation exchange property was prepared by sol-gel method. The H2O2 biosensor was fabricated by simply dipping the horseradish peroxidase-containing functionalized membrane modified electrode into Meldola's blue (MDB) solution. MDB was adsorbed and firmly immobilized within the membrane. The electrochemical behavior of MDB incorporated in the membrane was more reversible compared with that of the solution species and suitable as mediator for the horseradish peroxidase. The response time was less than 25 s. Linear range is up to 0.6 mM (COH. coeff. 0.9998) with detection Limit of 9 x 10(-7) M. High sensitivity of 75 nA mu M cm(-2) was obtained due to high MDB-loading. The biosensor exhibited a good stability. (C) 1999 Elsevier Science B.V. All rights reserved.

Dimerization of hydroxylated species of m-aminophenol by cytochrome c with hydrogen peroxide

The cytochrome c and hydrogen peroxide-dependent oxidation of m-aminophenol was investigated by electrochemistry and spectrophotometry. The results indicated that the hydroxylated species of m-aminophenol have at least two conjugated substituted groups on the ring system (most possibly, its oxidized form 2-hydroxy-4-iminoquinone), and that the degradation of cytochrome c by hydrogen peroxide can also be prevented in the presence of m-aminophenol. The hydroxyl radical scavengers, mannitol and sodium benzoate, almost completely eliminate the hydroxylation of m-aminophenol. But oxo-heme species scavenger, uric acid, does not inhibit the hydroxylation. Combining the results of mass spectrum, nuclear magnetic resonance and element analysis with that of spectrophotometry, electrochemistry and chemical scavengers, it is suggested that cytochrome c may act as a peroxidase, which facilitates the hydroxylation and subsequent dimerization of m-aminophenol. (C) 1998 Elsevier Science B.V. All rights reserved.

Self-assembled monolayers of thiols on gold electrodes for bioelectrochemistry and biosensors

Monolayers of biological compounds including redox proteins and enzymes, and phospholipids have been immobilized on a gold electrode surface through self-assembling. These proteins and enzymes, such as cytochrome c, cytochrome c oxidase and horseradish peroxidase (HRP), immobilized covalently to the self-assembled monolayers (SAMs) of 3-mercaptopropionic acid on a gold electrode, communicate directly electrons with the electrode surface without mediators and keep their physiological activities. The electron transfer of HRP with the gold electrode can also be mediated by the alkanethiol SAMs with electroactive group viologens on the gold electrode surface. All these direct electrochemistries of proteins and enzymes might offer an opportunity to build a third generation of biosensors without mediators for analytes, such as H2O2, glucose and cholesterol. Monensin and valinomycin have been incorporated into the bilayers on the gold electrode consisting of the SAMs of alkanethiol and a lipid monolayer, which have high selectivity for monovalent ions, and the resulting Na+ or K+ sensor has a wide linear range and high stability. These self-assembly systems provide a good mimetic model for studying the physiological function of a membrane and its associated enzyme. (C) 1997 Elsevier Science S.A.

Organic phase enzyme electrodes based on organohydrogel

A dimethylformamide-polyhydroxyl cellulose organo-hydrogel has been prepared, and its applications for enzyme immobilization in construction of organic phase biosensors have been exploited. With horseradish peroxidase, tyrosinase, and bilirubin oxidase immobilized in the organohydrogel, enzyme electrodes can be operated in various situations, including aqueous buffer, oil/water mixtures, and anhydrous organic solvents, and even in dimethylformamide, to determine analytes of different solubilities, e.g., organic peroxides, phenolic compounds and bilirubin. Biosensing has no restrictions in terms of measuring media and solubilities of analytes.

Influence of intraperitoneal injection of rare earth compounds on the activities of antioxidant enzymes and the level of lipid peroxidation in mice livers

Following intraperitoneal injection of lanthanum and terbium chloride and their complexes of diethyltriaminopentagacetic acid (DTPA) to adult mice with a dose of 0.28 mmol/kg body weight/day for three days. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of lipid end product, malonaldehyde (MDA) in the mice livers have been assayed respectively. The results show that the activity of SOD was increased and the content of MDA was reduced for LaCl3 treated mice and the two targets were not changed for TbCl3, but the activity of GSH-Px was reduced markedly for both LaCl3 and TbCl3 while the above three targets were not changed for La-DTPA and Tb-DTPA complexes.

DIRECT ELECTROCHEMISTRY AND SURFACE CHARACTERIZATION OF GLUCOSE-OXIDASE ADSORBED ON ANODIZED CARBON ELECTRODES

The glassy carbon electrode (gce) and highly oriented pyrolytic graphite (hopg) were electrochemically anodized at a potential of +2.0 V (vs. Ag/AgCl) to create active sites and to improve the adsorption of glucose oxidase (GOD) and flavin adenine dinucle

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.

Examination of Antioxidative System's Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant Reaumuria soongorica (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H2O2), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H2O2 in the early phase of drought and enzymes of ascorbate-glutathione cycle scavenge H2O2 in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H2O2 under drought stress.

cDNA cloning and gene expression pattern following bacterial challenge of peroxinectin in Chinese shrimp Fenneropenaeus chinensis

Peroxinectin, a cell-adhesive hemoperoxidase that binds superoxide dismutase and mediates blood cells adhesion and migration in invertebrate, is believed to play an important role in cellular immune reaction. In this study, we reported a new peroxinectin gene homologue from Chinese shrimp Fenneropenaeus chinensis. Based on expressed sequence tags (ESTs) of haemocyte cDNA library, we cloned a 2,611 bps full-length cDNA of peroxinectin gene homologue encoded 801 amino acids. Motif scanning of the predicted polypeptide revealed a peroxidase domain and an integrin binding motif (Lys-Gly-Asp, KGD). Peroxinectin gene expressed constitutively in haemocyte as determined by quantitative real-time RT-PCR, the expression level varied following bacterial challenge. These findings suggested that peroxinectin expression is susceptible to exterior stimulus and maintains at a high expression level during bacterial infection.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotem nm23, crustacyanin-Cl and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Analysis of the expression and antioxidative property of a peroxiredoxin 6 from Scophthalmus maximus

Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5'- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3'-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81-87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.