-
ACS Applied Bio Materials Jun 2019Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes...
Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes is their low reactivity because of the permeability resistance of lipid bilayers to substrates. In the present work, the polyunsaturated bilayers of 1,2-dilinolenoyl--glycero-3-phosphocholine (18:3-PC) were elucidated to be highly permeable to 5(6)-carboxyfluorescein at high temperatures up to 60 °C and applied to fabricate vesicle-based reactive enzyme reactors. d-Amino acid oxidase (DAO) from porcine kidney was encapsulated in 18:3-PC vesicles with each aqueous volume of 3.4 × 10 m (=3.4 aL). The DAO-containing vesicles were highly reactive at 40 °C toward d-alanine being added to bulk solution at pH 9.0 and stably catalyzed following two types of reactions. One is the DAO-catalyzed continuous production of HO in the vesicles for 30 min being detected by the free peroxidase-catalyzed oxidation of -dianisidine in bulk solution. The other is the cascade reaction in the vesicles coencapsulating DAO and catalase being followed for 5 h on the basis of the concentration of unreacted d-alanine. In the latter reaction, the intermediate product HO was decomposed by catalase producing oxygen allowing its cyclical use for the DAO-catalyzed oxidation. Furthermore, thanks to the highly temperature-dependent permeability of 18:3-PC bilayers, on/off-like switching in the activity could be induced with respect to the vesicle-confined enzyme by shifting the reaction temperature between 20 and 40 °C. The above reactive vesicles can offer the opportunity of the HO-based reliable detection of d-amino acids and the continuous optical resolution of racemic mixtures of amino acids.
PubMed: 35030702
DOI: 10.1021/acsabm.9b00165 -
Biochemical and Biophysical Research... Jul 2019ABCD4, a member of the ATP-binding cassette transporter superfamily, is associated with the transport of vitamin B which is crucial for the development of red blood...
ABCD4, a member of the ATP-binding cassette transporter superfamily, is associated with the transport of vitamin B which is crucial for the development of red blood cells (RBCs) and may also be involved in its metabolism. However, the molecular function of ABCD4 during RBC development in zebrafish is mostly unknown. Using a morpholino-based knockdown approach, we found that abcd4-knockdown resulted in abnormal RBCs of irregular shapes and various sizes. o-Dianisidine staining, as an indicator of hemoglobin in RBCs, further confirmed that abcd4 morphants possessed fewer hemoglobinized cells and impaired blood circulation. Multiple protein sequence alignment revealed that the amino acid sequence for residues 13-292, which is the domain of vitamin B transport, of the zebrafish Abcd4 was highly conserved compared to that of other species. Accordingly, the abcd4 morphants can be rescued with human ABCD4, demonstrating a conserved role of ABCD4 in vertebrates. Notably, the vitamin B-deficient phenotype in abcd4 morphants, which causes anemia, was recapitulated in the newly-established abcd4 mutant, indicating the possibility that the abcd4 mutant could be used as a disease model of vitamin B-deficiency anemia. Our study provides an insight that the analysis of the newly-established abcd4 mutant may contribute to understanding its roles in ABCD4-related vitamin B-deficiency anemia and the associated pathogeneses in humans.
Topics: ATP-Binding Cassette Transporters; Anemia; Animals; Mutation; Vitamin B 12 Deficiency; Zebrafish
PubMed: 31113616
DOI: 10.1016/j.bbrc.2019.05.099 -
Journal of Environmental Management May 2019Studies on the oxidation products of organic pollutants and their toxicity in textile dyeing sludge after the sludge was treated by the advance oxidation processes were...
Studies on the oxidation products of organic pollutants and their toxicity in textile dyeing sludge after the sludge was treated by the advance oxidation processes were limited, since textile dyeing sludge was a complicated mixture. For the first time, simulated sludge was used to study the degradation mechanism of 3,3'-dimethoxybenzidine (DMB) during the combined ultrasound-Mn(VII) treatment. The toxicity of DMB and its products was also evaluated. The results indicated that the compositions and microstructures of polyaluminium chloride (PAC)- and polyferric sulphate (PFS)-based simulated sludge were similar to those of real textile dyeing sludge. The optimum conditions of ultrasound-Mn(VII) treatment were: a KMnO dosage of 40 μM, an ultrasound power density of 0.36 W cm, and a reaction time of 20 min. 98.24% of DMB and 63.04% of total organic carbon (TOC) in the simulated sludge were removed. Six products, that is, 2-nitroanisole, 3-methoxy-4-nitrophenol, vanillylmandelic acid, vanillyl alcohol, m-anisic acid, and benzoic acid, were identified by GC-MS and LC-MS-MS. It was noted that all of these identified products were also detected in the real textile dyeing sludge after the ultrasound-Mn(VII) treatment. All of them were less toxic than DMB. Moreover, 53.30% and 54.80% of toxicity toward the alga Desmodesmus subspicatus and the bacterium Vibrio fischeri were removed in simulated sludge, respectively. Therefore, simulated sludge was helpful for studying a pollutant's degradation mechanism in the complex sludge mixtures. The results would also provide some useful suggestions for the sludge disposal after the sludge was treated by the advance oxidation processes.
Topics: Dianisidine; Oxidation-Reduction; Sewage; Waste Disposal, Fluid; Water Pollutants, Chemical
PubMed: 30849594
DOI: 10.1016/j.jenvman.2018.11.135 -
Chemosphere May 2019Triclosan (TCS), one of the important bactericides, is widely used in personal care products, and its chronic exposure leads to severe toxic effects on the growth and...
Triclosan (TCS), one of the important bactericides, is widely used in personal care products, and its chronic exposure leads to severe toxic effects on the growth and development of blood vessels in zebrafish (Danio rerio). Herein, we screened out three differentially expressed miRNAs (miR-181a-5p, miR-132-3p and miR-128-3p) by sequencing and qRT-PCR analyses of 4-96-hpf TCS-exposed zebrafish, among which miR-181a-5p was found to regulate many signaling pathways involved in fatty acid biosynthesis and phosphatidylimositol signaling systems. By O-dianisidine staining, TCS-exposure resulted in decreased distribution of red blood cells and induced blood hypercoagulable state and thrombotic effects. Defective subintestinal veins (SIVs), and decreased branching and curvature of blood vessels were observed with increasing TCS-exposure concentrations. After microinjection of miR-181a-5p mimic and inhibitor, zebrafish malformation type and percentage were prominently increased such as distorted SIV vessels along with reduced venation and abnormal branches by ALP staining. Overexpressed miR-181a-5p had a greater effect on development and branching patterns of arteries and veins than its knockdown. By laser confocal microscopy observation, the 72-hpf Tg (flk1: mCherry) zebrafish obviously displayed vascular proliferation and ablation in the miR-181a-5p mimic group. Microinjection of miR-181a-5p mimics and inhibitors led to abnormal expressions (20-50%) of two key target genes (pax2a and vash2) by WISH, and increased malformation percentages (18-45%) by IOD analysis. Overexpression of vash2 led to the inhibitory or promoting effects on the expression of PI3K signaling pathway-related genes, proving that the effect of vash2 on development of blood vessels could be realized by inhibiting PI3K signaling pathway. These observations lay theoretical foundation for deep insight into the molecular mechanisms on TCS-induced cardiovascular diseases.
Topics: Animals; Anti-Infective Agents, Local; MicroRNAs; Triclosan; Zebrafish
PubMed: 30784759
DOI: 10.1016/j.chemosphere.2019.02.038 -
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 -
Acta Crystallographica. Section E,... Mar 2018The title compound, (systematic name: ,'-dibenzyl-3,3'-dimeth-oxy-1,1'-biphenyl-4,4'-di-amine), CHNO, was synthesized by the reduction of a Schiff base prepared a...
The title compound, (systematic name: ,'-dibenzyl-3,3'-dimeth-oxy-1,1'-biphenyl-4,4'-di-amine), CHNO, was synthesized by the reduction of a Schiff base prepared a condensation reaction between -dianisidine and benzaldehyde under acidic conditions. The mol-ecule lies on a crystallographic inversion centre so that the asymmetric unit contains one half-mol-ecule. The biphenyl moiety compound is essentially planar. Two intra-molecular N-H⋯O hydrogen bonds occur. The dihedral angle between the terminal phenyl and phenyl-ene rings of a benzidine unit is 48.68 (6)°. The methyl-ene C atom of the benzyl group is disordered over two sets of sites, with occupancy ratio 0.779 (18):0.221 (18). In the crystal, mol-ecules are connected by hydrogen bonding between -dianisidine O atoms and H atoms of the terminal benzyl groups, forming a one-dimensional ladder-like structure. In the data from DFT calculations, the central biphenyl showed a twisted conformation.
PubMed: 29765704
DOI: 10.1107/S2056989018001688 -
Biochemical and Biophysical Research... Jun 2018Chionodraco hamatus is a teleost within the suborder Notothenioidei, the members of which are known to lack functional erythrocytes with modified hematopoiesis....
Chionodraco hamatus is a teleost within the suborder Notothenioidei, the members of which are known to lack functional erythrocytes with modified hematopoiesis. Hematopoiesis is an essential process during the development of animals, where it is tightly regulated by many different transcription factors, signaling proteins, chromatin modifications, and microRNAs (miRNAs). The miRNAs are known to regulate the expression of their target genes at the post-transcriptional level. However, little is known about the miRNA-mediated regulation of hematopoiesis. In this study, we confirmed that miR-152 plays a crucial role in hematopoiesis during the development of C. hamatus. The overexpression of miR-152 reduced hematopoiesis according to the decreased expression of GATA1 and reduced o-dianisidine staining of hemoglobin. Mechanistically, reduced hematopoiesis was regulated by the miR-152-mediated down-regulated expression of GATA1. Bioinformatics analysis was used to predict the target gene of miR-152. Western blotting as well as dual luciferase and EGFP reporter assays were employed to investigate the expression of GATA1 mediated by miR-152. Finally, verification experiments in the zebrafish autologous model strongly supported the effect of miR-152 on hematopoiesis. In conclusion, we suggest that miR-152 is a novel molecular factor that regulates hematopoiesis during the development of C. hamatus by down-regulating the expression of GATA1.
Topics: Animals; Erythropoiesis; Fish Proteins; GATA1 Transcription Factor; Gene Expression Regulation; MicroRNAs; Perciformes
PubMed: 29753742
DOI: 10.1016/j.bbrc.2018.05.053 -
ACS Chemical Biology May 2018X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding...
X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding and homohexameric protein organization. Three dimers interwoven in contact regions and tightened by hydrogen-bond-clamped guanidinium stacks along with regularly aligned water molecules form the basic structure of the enyzme. Intra- and intermolecular disulfide bridges further stabilize the enzyme preventing altogether the protein from denaturing up to a temperature of 90 °C, as evident from dynamic light scattering and the on-gel ortho-dianisidine assay. Every monomer binds one equivalent of orthovanadate in a cavity formed from side chains of three histidines, two arginines, one lysine, serine, and tryptophan. Protein binding occurs primarily through hydrogen bridges and superimposed by Coulomb attraction according to thermochemical model on density functional level of theory (B3LYP/6-311++G**). The strongest attractor is the arginine side chain mimic N-methylguanidinium, enhancing in positive cooperative manner hydrogen bridges toward weaker acceptors, such as residues from lysine and serine. Activating hydrogen peroxide occurs in the thermochemical model by side-on binding in orthovanadium peroxoic acid, oxidizing bromide with virtually no activation energy to hydrogen bonded hypobromous acid.
Topics: Binding Sites; Bromine; Density Functional Theory; Oxidation-Reduction; Peroxidases; Vanadates; X-Ray Diffraction
PubMed: 29665335
DOI: 10.1021/acschembio.8b00041 -
Nanotoxicology Jun 2018Nowadays, nanotechnology environmental health and safety (nanoEHS) is gaining attention. We previously found that silica nanoparticles (SiNPs) could induce vascular...
Nowadays, nanotechnology environmental health and safety (nanoEHS) is gaining attention. We previously found that silica nanoparticles (SiNPs) could induce vascular endothelial damage. However, the subsequent toxicologic response to SiNPs-induced endothelial damage was still largely unknown. In this study, we explored the inflammation-coagulation response and thrombotic effects of SiNPs in endothelial cells and zebrafish embryos. For in vitro study, swollen mitochondria and autophagosome were observed in ultrastructural analysis. The cytoskeleton organization was disrupted by SiNPs in vascular endothelial cells. The release of proinflammatory and procoagulant cytokines including IL-6, IL-8, MCP-1, PECAM-1, TF and vWF, were markedly elevated in a dose-dependent manner. For in vivo study, based on the NOAEL for dosimetry selection, and using two transgenic zebrafish, Tg(mpo:GFP) and Tg(fli-1:EGFP), SiNPs-induced neutrophil-mediated inflammation and impaired vascular endothelial cells. With the dosage higher than NOAEL, SiNPs significantly decreased blood flow and velocity, exhibiting a blood hypercoagulable state in zebrafish embryos. The thrombotic effect was assessed by o-dianisidine staining, showed that an increasing of erythrocyte aggregation occurred in SiNPs-treated zebrafish. Microarray analysis was used to screen the possible genes for inflammation-coagulation response to SiNPs in zebrafish, and the JAK1/TF signaling pathway was further verified by qRT-PCR and Western blot assays. For in-deepth study, il6st was knocked down with specific morpholinos. The whole-mount in situ hybridization and qRT-PCR analysis showed that the expression jak1 and f3b were attenuated in il6st knockdown groups. In summary, our data demonstrated that SiNPs could induce inflammation-coagulation response and thrombotic effects via JAK1/TF signaling pathway.
Topics: Animals; Blood Coagulation; Cells, Cultured; Embryo, Nonmammalian; Endothelium, Vascular; Humans; Inflammation; Janus Kinase 1; Nanoparticles; Signal Transduction; Silicon Dioxide; Thrombosis; Zebrafish
PubMed: 29658397
DOI: 10.1080/17435390.2018.1461267 -
Biochimica Et Biophysica Acta. General... Jul 2018A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based... (Comparative Study)
Comparative Study
BACKGROUND
A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated.
METHODS
Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of carbidopa, an anti-Parkinsonian drug, and other catechols, including l-dopa, were monitored spectrophotometrically and by measuring hydrogen peroxide consumption.
RESULTS
Chromophore formation in all three enzyme/substrate systems was blocked in the presence of carbidopa and other catechols. However, the rates of hydrogen peroxide consumption were not much affected. Irreversible enzyme inhibition was also insignificant.
CONCLUSIONS
Tested compounds reduced the oxidation products or intermediates of model substrates thus preventing chromophore formation. This interference may affect interpretation of results of diagnostic tests in samples from patients with Parkinson's disease treated with carbidopa and l-dopa.
GENERAL SIGNIFICANCE
This mechanism allows prediction of interference in peroxidase-based diagnostic tests for other compounds, including drugs and natural products.
Topics: Animals; Carbidopa; Catalysis; Catechols; Cattle; Chromogenic Compounds; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Lactoperoxidase; Molecular Docking Simulation; Molecular Structure; Monophenol Monooxygenase; Oxidation-Reduction; Peroxidase; Peroxidases
PubMed: 29649511
DOI: 10.1016/j.bbagen.2018.04.007