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Polish Journal of Microbiology 2015We investigated the effect of ciprofloxacin, rifampicine and doxycycline on myeloperoxidase (MPO) activity, glutathione (GSH) and malondialdehyde (MDA) levels in...
We investigated the effect of ciprofloxacin, rifampicine and doxycycline on myeloperoxidase (MPO) activity, glutathione (GSH) and malondialdehyde (MDA) levels in allergic asthma patients and healthy volunteers. Polymorphonuclear leukocytes (PMNs) were isolated with ficoll-hypaque gradient centrifugation method. MPO activity was assayed with modified o-dianisidine, GSH by Ellman's and MDA levels by Beuge's method. PMN functions and MDA levels of patients significantly decreased when compared with healthy volunteers. Ciprofloxacin significantly increased PMN functions, MPO activity and MDA levels of both groups. We have demonstrated that ciprofloxacin has beneficial effects on MPO activity and PMN functions in allergic asthma patients and healthy volunteers.
Topics: Anti-Bacterial Agents; Asthma; Case-Control Studies; Glutathione; Humans; Hypersensitivity; Malondialdehyde; Neutrophils; Peroxidase
PubMed: 26094319
DOI: No ID Found -
Archives of Oral Biology Jul 2015To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase...
OBJECTIVE
To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system.
DESIGN
Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804.
RESULTS
While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system.
CONCLUSIONS
Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities.
Topics: Animals; Candida; Cattle; Chickens; Colony Count, Microbial; Durapatite; Female; Lactoperoxidase; Male; Microbial Viability; Muramidase; Nephelometry and Turbidimetry; Peroxidases; Saliva; Sorbitol; Stem Cells; Surface Properties; Xylitol
PubMed: 25874813
DOI: 10.1016/j.archoralbio.2015.03.011 -
Free Radical Research Jun 2015Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex... (Comparative Study)
Comparative Study
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3',5,5'-tetramethylbenzidine, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.
Topics: Animals; Ceruloplasmin; Enzyme Inhibitors; Eosinophil Peroxidase; Halogenation; Humans; Kinetics; Peroxidase; Protein Binding; Protein Structure, Tertiary
PubMed: 25762223
DOI: 10.3109/10715762.2015.1005615 -
Analytical Chemistry Apr 2015N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology...
N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.
Topics: Amino Acid Isomerases; Colorimetry; High-Throughput Screening Assays; Molecular Structure
PubMed: 25716802
DOI: 10.1021/ac5047328 -
Biochemical and Biophysical Research... Jan 2015Hemoglobin synthesis by erythrocytes continues throughout a vertebrate's lifetime. The mechanism of mammalian heme synthesis has been studied for many years;...
Hemoglobin synthesis by erythrocytes continues throughout a vertebrate's lifetime. The mechanism of mammalian heme synthesis has been studied for many years; aminolevulinate synthase 2 (ALAS2), a heme synthetase, is associated with X-linked dominant protoporphyria in humans. Amphibian and mammalian blood cells differ, but little is known about amphibian embryonic hemoglobin synthesis. We investigated the function of the Xenopus alas2 gene (Xalas2) in primitive amphibian erythrocytes and found that it is first expressed in primitive erythroid cells before hemoglobin alpha 3 subunit (hba3) during primary hematopoiesis and in the posterior ventral blood islands at the tailbud stage. Xalas2 is not expressed during secondary hematopoiesis in the dorsal lateral plate. Hemoglobin was barely detectable by o-dianisidine staining and hba3 transcript levels decreased in Xalas2-knockdown embryos. These results suggest that Xalas2 might be able to synthesize hemoglobin during hematopoiesis and mediate erythrocyte differentiation by regulating hba3 expression in Xenopus laevis.
Topics: 5-Aminolevulinate Synthetase; Amino Acid Motifs; Amino Acid Sequence; Animals; Catalytic Domain; Cell Differentiation; Erythrocytes; Erythropoiesis; Gene Expression Regulation, Developmental; Hemangioblasts; Heme; Hemoglobins; Molecular Sequence Data; Oligonucleotides, Antisense; RNA, Messenger; Sequence Homology, Amino Acid; Stem Cells; Xenopus Proteins; Xenopus laevis
PubMed: 25482442
DOI: 10.1016/j.bbrc.2014.11.110 -
Gene Jan 2015Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro...
Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.
Topics: Amino Acid Sequence; Carica; Chromatography, Affinity; Cloning, Molecular; DNA, Complementary; Dianisidine; Escherichia coli; Guaiacol; Heme; Hydrogen-Ion Concentration; Molecular Docking Simulation; Molecular Sequence Data; Peroxidases; Plant Proteins; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrogallol; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Substrate Specificity; Temperature
PubMed: 25447898
DOI: 10.1016/j.gene.2014.11.013 -
Marine Biotechnology (New York, N.Y.) Apr 2015A 2,158 bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of...
A 2,158 bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of BPOs suggested that P. yezoensis and cyanobacteria were grouped in the same clade and separated from brown algae. Genomic Southern blot analysis suggested that PyBPO1 existed as a single copy per haploid genome. RT-PCR revealed that PyBPO1 was actively expressed in filamentous sporophytes but repressed in leafy gametophytes under normal growth conditions. High expression levels of PyBPO1 in sporophytes were observed when sporophytes were grown under gametophyte conditions, suggesting that preferential expression of PyBPO1 occurs during the sporophyte phase. BPO activity of cell-free extracts from sporophytes and gametophytes was examined by activity staining on native PAGE gel using o-dianisidine. One activity band was detected in sporophyte sample, but not in gametophyte sample. In addition, we found that bromide and iodide were effective substrate, but chloride was not. BPO activity was observed-likely in chloroplasts-when sporophyte cells were incubated with o-dianisidine and hydrogen peroxide. Cellular BPO staining showed the same halogen preference identified by in-gel BPO staining. Based on GS-MS analysis, bromoform was detected in medium containing sporophytes. Bromoform was not detected under dark culture conditions but was detected in the culture exposed to low light intensity (5 μmol m(-2) s(-1)) and increased under a moderate light intensity (30 μmol m(-2) s(-1)).
Topics: Amino Acid Sequence; Base Sequence; Blotting, Southern; Cluster Analysis; DNA Primers; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Developmental; Life Cycle Stages; Molecular Sequence Data; Peroxidases; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Rhodophyta; Sequence Analysis, DNA; Trihalomethanes
PubMed: 25407492
DOI: 10.1007/s10126-014-9608-6 -
TheScientificWorldJournal 2014An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration...
An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and V max for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5-5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.
Topics: Chromatography, Gel; Coloring Agents; Garlic; Hydrogen Peroxide; Industry; Peroxidase; Wastewater
PubMed: 25401128
DOI: 10.1155/2014/183163 -
The Analyst Dec 2014A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose...
A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 μL of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 °C as long as four months.
Topics: Benzothiazoles; Biosensing Techniques; Blood Glucose; Colorimetry; Coloring Agents; Dianisidine; Enzymes, Immobilized; Equipment Design; Glucose Oxidase; Horseradish Peroxidase; Humans; Limit of Detection; Membranes, Artificial; Nanofibers; Reagent Strips; Sulfonic Acids
PubMed: 25343161
DOI: 10.1039/c4an01354a -
PloS One 2014Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and...
Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.
Topics: Anemia; Animals; Animals, Genetically Modified; Cardiovascular System; Embryo, Nonmammalian; Endothelial Cells; Endothelium, Vascular; Environmental Pollutants; Green Fluorescent Proteins; Humans; Hydrocarbons, Fluorinated; Pyrimidines; United States; Zebrafish
PubMed: 25090246
DOI: 10.1371/journal.pone.0104190