-
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 -
Neurotoxicology and Teratology 2019Clinically approved iron chelators are effective in decreasing significant transfusional iron accumulation. Starch-Deferoxamine (S-DFO), a novel high molecular weight...
Biocompatibility and toxicity of novel iron chelator Starch-Deferoxamine (S-DFO) compared to zinc oxide nanoparticles to zebrafish embryo: An oxidative stress based apoptosis, physicochemical and neurological study profile.
OBJECTIVES
Clinically approved iron chelators are effective in decreasing significant transfusional iron accumulation. Starch-Deferoxamine (S-DFO), a novel high molecular weight iron chelator, was produced to increase binding capacity to iron and reduce toxicity. Although its efficacy was established in one small cohort clinical trial, its potential adverse effect was not adequately addressed.
METHODS
We utilized zebrafish model to assess S-DFO toxicity using following assays: mortality, teratogenicity, hatching rate, tail flicking, Acridine Orange staining for apoptosis detection, o-dianisidine staining for hemoglobin synthesis, and the level of Hsp70 as a general stress indicator. Embryos were exposed to different concentrations of S-DFO, Zinc Oxide nanoparticle (ZnO) (positive control), along with untreated control (UC).
RESULTS
S-DFO showed no significant mortality nor deformities at all tested concentrations (0.0-1000 μM). Thus, the LC50 is expected to >1000 μM. 100 μM S-DFO treatment did not affect embryo development (as judged by hatching rate); neuromuscular activity (as judged by tail flicking); and hemoglobin synthesis. Neither apoptosis, nor increase in Hsp70 level was noticed upon S-DFO treatment.
CONCLUSION
Our assays demonstrate that S-DFO does not induce cellular or biochemical stress and has no adverse effect on organ development of zebrafish embryos, suggesting its safe use as an iron chelator.
Topics: Animals; Apoptosis; Behavior, Animal; Deferoxamine; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Embryonic Development; Iron Chelating Agents; Materials Testing; Nanoparticles; Oxidative Stress; Zebrafish; Zinc Oxide
PubMed: 30710618
DOI: 10.1016/j.ntt.2019.01.004 -
Bulletin of Experimental Biology and... May 2020A novel express method is developed to determine activity of antitumor enzyme L-lysine-α-oxidase obtained by culturing Trichoderma harzianum Rifai F-180 fungus. The...
A novel express method is developed to determine activity of antitumor enzyme L-lysine-α-oxidase obtained by culturing Trichoderma harzianum Rifai F-180 fungus. The carcinogenic reagent ortho-dianisidine-hydrochloride was replaced in the reaction medium with environmentally friendly reagents of the chromogenic mixture that included tetramethylbenzidine. This method improved precision and sensitivity of ELISA by 10 and 40 times, respectively. In addition, it could detect activity of L-lysine-α-oxidase not only in the producer strains with a pronounced activity of this enzyme, but also in the strains where this activity has not been previously determined.
Topics: Amino Acid Oxidoreductases; Antineoplastic Agents; Colorimetry; Culture Media; Drug Screening Assays, Antitumor; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hypocreales; Temperature; Time Factors
PubMed: 32488773
DOI: 10.1007/s10517-020-04837-2 -
Biotechnology Letters Jan 2018To develop a rapid, dual-parameter, plate-based screening process to improve production and secretion rate of glucose oxidase simultaneously in Aspergillus niger.
OBJECTIVES
To develop a rapid, dual-parameter, plate-based screening process to improve production and secretion rate of glucose oxidase simultaneously in Aspergillus niger.
RESULTS
A morphology engineering based on CaCO was implemented, where the yield of GOD by A. niger was increased by up to 50%. Analysis of extracellular GOD activity was achieved in 96-well plates. There was a close negative correlation between the total GOD activity and its residual glucose of the fermentation broth. Based on this, a rapid, plate-based, qualitative analysis method of the total GOD activity was developed. Compared with the conventional analysis method using o-dianisidine, a correlation coefficient of -0.92 by statistical analysis was obtained.
CONCLUSION
Using this dual-parameter screening method, we acquired a strain with GOD activity of 3126 U l, which was 146% higher than the original strain. Its secretion rate of GOD was 83, 32% higher than the original strain.
Topics: Aspergillus niger; Culture Media; Glucose; Glucose Oxidase; Mass Screening; Microbiological Techniques
PubMed: 28939970
DOI: 10.1007/s10529-017-2442-y -
Journal of Pharmaceutics 2016Two simple methods are described for the determination of ethionamide (ETM) in bulk drug and tablets using cerium (IV) sulphate as the oxidimetric agent. In both...
Two simple methods are described for the determination of ethionamide (ETM) in bulk drug and tablets using cerium (IV) sulphate as the oxidimetric agent. In both methods, the sample solution is treated with a measured excess of cerium (IV) solution in HSO medium, and after a fixed standing time, the residual oxidant is determined either by back titration with standard iron (II) solution to a ferroin end point in titrimetry or by reacting with o-dianisidine followed by measurement of the absorbance of the orange-red coloured product at 470 nm in spectrophotometry. In titrimetry, the reaction proceeded with a stoichiometry of 1 : 2 (ETM : Ce (IV)) and the amount of cerium (IV) consumed by ETM was related to the latter's amount, and the method was applicable over 1.0-8.0 mg of drug. In spectrophotometry, Beer's law was obeyed over the concentration range of 0.5-5.0 g/mL ETM with a molar absorptivity value of 2.66 × 10 L/(mol·cm). The limits of detection (LOD) and quantification (LOQ) calculated according to ICH guidelines were 0.013 and 0.043 g/mL, respectively. The proposed titrimetric and spectrophotometric methods were found to yield reliable results when applied to bulk drug and tablets analysis, and hence they can be applied in quality control laboratories.
PubMed: 27818836
DOI: 10.1155/2016/5410573 -
Aquatic Toxicology (Amsterdam,... Sep 2017Saturated fluorotelomer carboxylic acids (FTCAs) are intermediates in the degradation of fluorotelomer alcohols (FTOHs) to perfluorinated carboxylic acids (PFCAs)....
Saturated fluorotelomer carboxylic acids (FTCAs) are intermediates in the degradation of fluorotelomer alcohols (FTOHs) to perfluorinated carboxylic acids (PFCAs). Recent studies have detected FTCAs in precipitation, surface waters, and wildlife, but few studies have focused on their toxicity. In this study, zebrafish embryos were exposed to different concentrations of 6:2 FTCA (0, 4, 8, and 12mg/L) from 6 to 120h post-fertilization (hpf) to investigate its developmental toxicity. Results showed that 6:2 FTCA exposure decreased the hatching and survival percentages, reduced the heart rate, and increased the malformation of zebrafish embryos. The median lethal concentration of 6:2 FTCA was 7.33mg/L at 120 hpf, which was lower than that of perfluorooctanoic acid (PFOA), thus indicating higher toxicity for zebrafish. The most common developmental malformation was pericardial edema, which appeared in the 8 and 12mg/L 6:2 FTCA-exposed embryos from 60 hpf. Using o-dianisidine staining, we found that the hemoglobin content in embryos was reduced in a concentration-dependent manner after 6:2 FTCA exposure at 72 hpf. Based on quantitative real-time polymerase chain reaction (q-RT-PCR) and whole-mount in situ hybridization, the transcriptional levels of hemoglobin markers (hbae1, hbbe1, and hbae3) were down-regulated at 48 and 72 hpf, even though no observed malformation appeared in zebrafish at 48 hpf. Moreover, 6:2 FTCA exposure decreased the protein level of gata1, a principal early erythrocytic marker, in Tg (gata1:DsRed) transgenic zebrafish at 72 hpf. We analyzed the transcriptional level of other erythrocyte-related genes using q-RT-PCR assay. For heme formation, the transcription of alas2, which encodes the key enzyme for heme biosynthesis, was down-regulated after 6:2 FTCA exposure, whereas the transcription of ho-1, which is related to heme degradation, was up-regulated at 48 and 72 hpf. The transcriptional patterns of gata1 and gata2, which are related to erythroid differentiation, differed. At 48 hpf, the mRNA level of gata2 was significantly increased, whereas that of gata1 exhibited no significant changes in any treatment group. At 72 hpf, the expressions of both were down-regulated in a concentration-dependent manner. Taken together, 6:2 FTCA exposure decreased the erythrocyte number and disrupted erythroid differentiation during zebrafish embryonic development. Our results suggest that 6:2 FTCA can cause developmental toxicity in zebrafish embryos, and that FTCAs exhibit greater toxicity than that of PFCAs.
Topics: Animals; Caprylates; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Embryonic Development; Erythrocytes; Fluorocarbons; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins
PubMed: 28688371
DOI: 10.1016/j.aquatox.2017.06.023 -
Bioelectrochemistry (Amsterdam,... Feb 2015Two modified electrodes with immobilized glucose oxidase were developed. Modification with poly(3,4-ethylenedioxythiophene) (PEDOT) and polyacrylic acid (PAA) doped with...
Two modified electrodes with immobilized glucose oxidase were developed. Modification with poly(3,4-ethylenedioxythiophene) (PEDOT) and polyacrylic acid (PAA) doped with poly(4-lithium styrenesulfonic acid) (PSSLi) in a newly elaborated procedure was used in the first electrode. The second one presents innovative solution and consists of two sublayers; one of them was PEDOT doped with PSSLi and the other was composed of PEDOT and anthranilic acid (AA) doped with poly(4-styrenesulfonic acid) (PSSH). Glucose oxidase was covalently bonded with the carboxyl groups of the polymer through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSC). The activity of immobilized enzyme was confirmed by spectrophotometry using the reaction of the produced hydrogen peroxide with o-dianisidine. The procedure for immobilization was optimized. It was found that the choice of an appropriate doping agent and its concentration were significant and 0.1M PSSLi proved to be the best doping agent. The most efficient immobilization was established for WSC and GOD concentration at the level of 4mg/ml and 5mg/ml respectively. In both cases, it was found that a small deviation from the concentrations determined to cause a sharp decrease in the activity of the enzyme, which was proven by spectrophotometric measurements. Prepared electrodes were active over a month with repeatable measurement results.
Topics: Acrylic Resins; Bridged Bicyclo Compounds, Heterocyclic; Carbodiimides; Electrochemical Techniques; Enzymes, Immobilized; Glucose Oxidase; Methylamines; Polymers; Styrenes; Sulfonic Acids; ortho-Aminobenzoates
PubMed: 25023029
DOI: 10.1016/j.bioelechem.2014.06.009 -
International Journal of Biological... Nov 2015Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate...
Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o-dianisidine). Catalytic activity decreased following the sequence ABTS>guaiacol>ferulic acid>o-dianisidine. Once the intermediate complex (compound III-H2O2) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H2O2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering.
Topics: Cytisus; Enzyme Activation; Hydrogen Peroxide; Kinetics; Least-Squares Analysis; Time Factors
PubMed: 26407901
DOI: 10.1016/j.ijbiomac.2015.09.033 -
Nanotoxicology 2016The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic...
The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic effect and mechanisms of low-dose exposure of silica nanoparticles (SiNPs) on cardiac function in zebrafish embryos via the intravenous microinjection. The dosage of SiNPs was based on the no observed adverse effect level (NOAEL) of malformation assessment in zebrafish embryos. The mainly cardiac toxicity phenotypes induced by SiNPs were pericardial edema and bradycardia but had no effect on atrioventricular block. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased in a dose-dependent manner. Microarray analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway involved in cardiac function. SiNPs induced whole-embryo oxidative stress and neutrophil-mediated cardiac inflammation in Tg(mpo:GFP) zebrafish. Inflammatory cells were observed in atrium of SiNPs-treated zebrafish heart by histopathological examination. In addition, the expression of TNNT2 protein, a cardiac contraction marker in heart tissue had been down-regulated compared to control group using immunohistochemistry. Confirmed by qRT-PCR and western blot assays, results showed that SiNPs inhibited the calcium signaling pathway and cardiac muscle contraction via the down-regulated of related genes, such as ATPase-related genes (atp2a1l, atp1b2b, atp1a3b), calcium channel-related genes (cacna1ab, cacna1da) and the regulatory gene tnnc1a for cardiac troponin C. Moreover, the protein level of TNNT2 was decreased in a dose-dependent manner. For the first time, our results demonstrated that SiNPs induced cardiac dysfunction via the neutrophil-mediated cardiac inflammation and cardiac contraction in zebrafish embryos.
Topics: Animals; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Heart; Heart Function Tests; Inflammation; Myocardial Contraction; Nanoparticles; Neutrophils; Oxidative Stress; Silicon Dioxide; Zebrafish
PubMed: 26551753
DOI: 10.3109/17435390.2015.1102981 -
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