-
PloS One 2017We have previously shown that (1) an acute deficiency in blood serum holo-ceruloplasmin (Cp) developed in rats that were fed fodder containing silver ions (Ag-fodder)...
We have previously shown that (1) an acute deficiency in blood serum holo-ceruloplasmin (Cp) developed in rats that were fed fodder containing silver ions (Ag-fodder) for one month and (2) the deficiency in holo-Cp was compensated by non-hepatic holo-Cp synthesis in rats that were chronically fed Ag-fodder for 6 months (Ag-rats). The purpose of the present study is to identify the organ(s) that compensate for the hepatic holo-Cp deficiency in the circulation. This study was performed on rats that were fed Ag-fodder (40 mg Ag·kg-1 body mass daily) for 6 months. The relative expression levels of the genes responsible for copper status were measured by RT-PCR. The in vitro synthesis and secretion of [14C]Cp were analyzed using a metabolic labeling approach. Oxidase activity was determined using a gel assay with o-dianisidine. Copper status and some hematological indexes were measured. Differential centrifugation, immunoblotting, immunoelectrophoresis, and atomic absorption spectrometry were included in the investigation. In the Ag-rats, silver accumulation was tissue-specific. Skeletal muscles and internal (IAT) and subcutaneous (SAT) adipose tissues did not accumulate silver significantly. In SAT, the mRNAs for the soluble and glycosylphosphatidylinositol-anchored ceruloplasmin isoforms were expressed, and their relative levels were increased two-fold in the Ag-rats. In parallel, the levels of the genes responsible for Cp metallation (Ctr1 and Atp7a/b) increased correspondingly. In the SAT of the Ag-rats, Cp oxidase activity was observed in the Golgi complex and plasma membrane. Moreover, full-length [14C]Cp polypeptides were released into the medium by slices of SAT. The possibilities that SAT is part of a system that controls the copper balance in mammals, and it plays a significant role in supporting copper homeostasis throughout the body are discussed.
Topics: Animals; Ceruloplasmin; Copper; Female; Immunoblotting; Immunoprecipitation; Male; Muscle, Skeletal; Rats; Rats, Wistar; Silver; Subcutaneous Fat
PubMed: 28380026
DOI: 10.1371/journal.pone.0175214 -
PloS One 2017Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino... (Comparative Study)
Comparative Study
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos.
Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Base Sequence; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Hematopoietic System; In Situ Hybridization; Membrane Proteins; Morpholinos; Mutation; Phenotype; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Zebrafish; Zebrafish Proteins
PubMed: 28192479
DOI: 10.1371/journal.pone.0172227 -
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 -
Naunyn-Schmiedeberg's Archives of... May 2016Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory...
Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory bowel diseases. The aim of the present study was to characterize the role of melatonin receptors (MT) in the anti-inflammatory effect of melatonin and to assess the anti-inflammatory potential of two novel MT receptor agonists, Neu-P11 and Neu-P67, in the mouse model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Colitis was induced on day 1 by intracolonic (i.c.) administration of TNBS in 30 % ethanol in saline. Melatonin (4 mg/kg, per os (p.o.)), Neu-P11 (20 mg/kg, p.o.; 50 mg/kg, intraperitoneally (i.p.), 50 mg/kg, i.c.), and Neu-P67 (20 mg/kg, p.o.) were given twice daily for 3 days. Luzindole (5 mg/kg, i.p.) was injected 15 min prior to melatonin administration. On day 4, macroscopic and microscopic damage scores were assessed and myeloperoxidase (MPO) activity quantified using O-dianisidine-based assay. Melatonin significantly attenuated colitis in mice, as indicated by the macroscopic score (1.90 ± 0.34 vs. 3.82 ± 0.62 for melatonin- and TNBS-treated mice, respectively), ulcer score (0.87 ± 0.18 vs. 1.31 ± 0.19, respectively), and MPO activity (4.68 ± 0.70 vs.6.26 ± 0.94, respectively). Luzindole, a MT receptor antagonist, did not inhibit the anti-inflammatory effect of melatonin (macroscopic score 1.12 ± 0.22, ulcer score 0.50 ± 0.16); however, luzindole increased MPO activity (7.57 ± 1.05). MT receptor agonists Neu-P11 and Neu-P67 did not improve inflammation induced by TNBS. Melatonin, but not MT receptor agonists, exerts potent anti-inflammatory action in acute TNBS-induced colitis. Our data suggests that melatonin attenuates colitis by additional, MT receptor-independent pathways.
Topics: Animals; Anti-Inflammatory Agents; Colitis; Colon; Indoles; Male; Melatonin; Mice, Inbred BALB C; Peroxidase; Pyrans; Receptors, Melatonin; Trinitrobenzenesulfonic Acid
PubMed: 26899972
DOI: 10.1007/s00210-016-1214-x -
Iranian Journal of Microbiology Feb 2015Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the...
BACKGROUND AND OBJECTIVE
Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the interests of researchers. In this study, molecular, chemical and biological screening of soil actinomycetes was carried out in order to search for peptide-producing actinomycetes.
MATERIALS AND METHODS
60 actinomycetes were isolated from soils of Iran. The isolates were subjected to molecular screening for detection NRPS (non-ribosomal peptide synthetases) gene. Phylogenic identification of NRPS containing isolates was performed. Chemical screening of the crude extracts was performed using chlorine o-dianisidine as peptide detector reagent and bioactivity of peptide producing strains was determined by antimicrobial bioassay. High pressure liquid chromatography- mass spectrometry (HPLC-MS) with UV-visible spectroscopy was performed for detection of the metabolite diversity in selected strain.
RESULTS
Amplified NRPS adenylation gene (700 bp) was detected among 30 strains. Phylogenic identification of these isolates showed presence of rare actinomycetes genera among the isolates and 10 out of 30 strains were subjected to chemical screening. Nocardia sp. UTMC 751 showed antimicrobial activity against bacterial and fungal test pathogens. HPLC-MS and UV-visible spectroscopy results from the crude extract showed that this strain has probably the ability to produce new metabolites.
CONCLUSION
By application of a combined approach, including molecular, chemical and bioactivity analysis, a promising strain of Nocardia sp. UTMC 751 was obtained. This strain had significant activity against Staphylococcus aureus and Pseudomonas aeruginosa. Strain Nocardia sp. UTMC 751 produce five unknown and most probably new metabolites with molecular weights of 274.2, 390.3, 415.3, 598.4 and 772.5. This strain had showed 99% similarity to Nocardia ignorata DSM 44496 T.
PubMed: 26644870
DOI: No ID Found -
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 -
Chemical Science Jul 2015As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel...
As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase bearing a putative metal binding site leads to the emergence of peroxidase activity ( 7.8 × 10 s, 1.1 × 10 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme reveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the -dianisidine substrate.
PubMed: 29218172
DOI: 10.1039/c5sc01065a -
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 -
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 -
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