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International Journal of Biological... Dec 2011The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with...
The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.
Topics: Arecaceae; Benzothiazoles; Biotechnology; Chromatography; Dianisidine; Electrophoresis, Polyacrylamide Gel; Guaiacol; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Oxidation-Reduction; Peroxidase; Phenylenediamines; Plant Leaves; Plant Proteins; Solutions; Substrate Specificity; Sulfonic Acids
PubMed: 21925205
DOI: 10.1016/j.ijbiomac.2011.09.001 -
Journal of Biomedical Science Sep 2011β-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we...
BACKGROUND
β-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including β-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of β-lapachone on zebrafish embryonic heart development.
METHODS
Embryos were treated with β-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation.
RESULTS
We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in β-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of β-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of β-lapachone-treated embryos. Reduction in wall shear stress was uncovered in β-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by β-lapachone is mediated through an NQO1- and calcium-dependent pathway.
CONCLUSIONS
The novel finding of this study is that β-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.
Topics: Animals; Apoptosis; Dicumarol; Dimethyl Sulfoxide; Embryonic Development; Endocardium; Erythrocyte Count; Erythrocytes; Gene Expression Regulation, Developmental; Heart; Heart Defects, Congenital; Microscopy, Fluorescence; Morphogenesis; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Reactive Oxygen Species; Time-Lapse Imaging; Zebrafish
PubMed: 21936955
DOI: 10.1186/1423-0127-18-70 -
Pharmacological Research Dec 2015The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly...
The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and β-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.
Topics: Amino Acid Sequence; Animals; Biosimilar Pharmaceuticals; Computational Biology; Embryo, Nonmammalian; Epoetin Alfa; Erythropoietin; GATA1 Transcription Factor; Humans; Models, Animal; Molecular Sequence Data; Receptors, Erythropoietin; Recombinant Proteins; Sequence Alignment; Up-Regulation; Zebrafish
PubMed: 26361727
DOI: 10.1016/j.phrs.2015.09.004 -
The FEBS Journal Jul 2006Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we... (Comparative Study)
Comparative Study
Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.
Topics: Amino Acid Motifs; Amino Acid Sequence; Aminobiphenyl Compounds; Biphenyl Compounds; Candida albicans; Conserved Sequence; Dihydroorotate Dehydrogenase; Enzyme Inhibitors; Escherichia coli; Glutathione Transferase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Kinetics; Molecular Sequence Data; Molecular Structure; Mutation; Oxidoreductases Acting on CH-CH Group Donors; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 16774642
DOI: 10.1111/j.1742-4658.2006.05327.x -
Plant Physiology Apr 1987All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as...
All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as H(2)O(2) and free radicals of O(2). H(2)O(2) may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN(3), CO, and C(2)H(2) were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of (1/2) V(max) was 70 micromolar for ascorbate and 3 micromolar for H(2)O(2). The high affinity of ascorbate peroxidase for H(2)O(2) indicates that this enzyme, rather than catalase, is responsible for most H(2)O(2) removal outside of peroxisomes in root nodules.
PubMed: 16665340
DOI: 10.1104/pp.83.4.789 -
Molecules (Basel, Switzerland) Dec 2023Porous covalent organic frameworks (COFs) have been widely used for the efficient removal of iodine from solution due to their abundance of electron-rich sites. In this...
Porous covalent organic frameworks (COFs) have been widely used for the efficient removal of iodine from solution due to their abundance of electron-rich sites. In this study, two kinds of ketoenamine-based COFs, TpBD-(OMe) and TpBD-Me, are successfully synthesized via Schiff base reaction under solvothermal conditions using 1, 3, 5-triformylphoroglucinol as aldehyde monomer, o-tolidine and o-dianisidine as amino monomers. The ability of TpBD-(OMe) and TpBD-Me to adsorb iodine in cyclohexane or aqueous solutions has been quantitatively analyzed and interpreted in terms of adsorption sites. TpBD-Me possesses two adsorption sites, -NH- and -C=O, and exhibits an adsorption capacity of 681.67 mg/g in cyclohexane, with an initial adsorption rate of 0.6 g/mol/min with respect to COF unit cell. The adsorption capacity of TpBD-(OMe) can be as high as 728.77 mg/g, and the initial adsorption rate of TpBD-(OMe) can reach 1.2 g/mol/min in the presence of oxygen atoms between the methyl group and the benzene ring. Compared with TpBD-Me, the higher adsorption capacity and adsorption rate of TpBD-(OMe) towards iodine are not only reflected in organic solvents, but also in aqueous solutions. It is proven through X-ray photoelectron spectroscopy and Raman spectroscopy that iodine exists in the form of I, I, and I within TpBD-(OMe) and TpBD-Me after adsorption. This work not only expands the application of COFs in the field of iodine adsorption, but also provides research ideas and important an experimental basis for the optimization of iodine adsorption sites.
PubMed: 38138639
DOI: 10.3390/molecules28248151 -
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 -
British Medical Journal Aug 1967
Topics: Biochemical Phenomena; Biochemistry; Biphenyl Compounds; Carcinogens; Humans; Occupational Diseases
PubMed: 6038325
DOI: 10.1136/bmj.3.5564.557-d -
The International Journal of... 2010The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However,...
The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.
Topics: Animals; Cardiovascular System; Dianisidine; Embryo, Nonmammalian; Embryonic Development; Female; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Nervous System; Receptor, IGF Type 1; Signal Transduction; Somatomedins; Zebrafish
PubMed: 19757379
DOI: 10.1387/ijdb.092922lh -
The Biochemical Journal Dec 19661. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2....
1. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2. Measurements of the activities of horseradish and tea peroxidase by this and two other methods, involving respectively pyrogallol and o-dianisidine, are compared. 3. It is claimed that the chronometric method is relatively simple, rapid and accurate. 4. The method can be used in the presence of polyphenol oxidases.
PubMed: 16742428
DOI: 10.1042/bj1010582