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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 -
3 Biotech Feb 2014A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was...
A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200-700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest K value being for ortho-dianisidine and highest K and K/K for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.
PubMed: 28324461
DOI: 10.1007/s13205-013-0129-1 -
International Journal of Analytical... 2013One titrimetric and two spectrophotometric methods are described for the determination of ketotifen fumarate (KTF) in bulk drug and in tablets using cerium(IV) as the...
One titrimetric and two spectrophotometric methods are described for the determination of ketotifen fumarate (KTF) in bulk drug and in tablets using cerium(IV) as the oxidimetric agent. In titrimetry (method A), the drug was treated with a measured excess of cerium(IV) in H2SO4 medium and after a standing time of 10 min, the surplus oxidant was determined by back titration with iron(II). The spectrophotometric procedures involve addition of a known excess of cerium(IV) to KTF in acid medium followed by the determination of unreacted oxidant by reacting with either p-dimethyl amino benzaldehyde and measuring the resulting colour at 460 nm (method B) or o-dianisidine and subsequent measurement of the absorbance of coloured product at 470 nm (method C). Titrimetric assay is based on a 1 : 2 reaction stoichiometry between KTF and cerium(IV) and the method is applicable over 2-18 mg range. In spectrophotometry, regression analysis of Beer's law plots showed a good correlation in 0.4-8.0 and 0.4-10.0 g mL(-1) KTF ranges for method B and method C, respectively, and the corresponding molar absorptivity coefficients are calculated to be 4.0 × 10(4) and 3.7 × 10(4) L mol(-1) cm(-1).
PubMed: 24324496
DOI: 10.1155/2013/697651 -
International Journal of Molecular... 2012Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was...
Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N(2) adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H(2)O(2) as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme.
Topics: Anti-Bacterial Agents; Bioreactors; Dianisidine; Glutaral; Hydrogen Peroxide; Micrococcus; Muramidase; Myoglobin; Peroxidases; Silicon Dioxide; Spectroscopy, Fourier Transform Infrared
PubMed: 23109864
DOI: 10.3390/ijms130911443 -
Molecules (Basel, Switzerland) Jul 2012The room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis-(trifluorometanesulfonyl)imide BMIm[NTf₂] was used as a novel medium for improvement of...
The room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis-(trifluorometanesulfonyl)imide BMIm[NTf₂] was used as a novel medium for improvement of separation and quantization of 16 aromatic amines typically present as contaminants in consumer products and detected by HPLC coupled to an electrochemical detector. The aromatic amines, namely 4,4'-diaminodiphenylmethane, 4-chloroaniline, 2-methoxy-5-methyl-aniline, 3,3'-dimethylbenzidine, 2,4-diaminotoluidine, 2-chloro-4-nitroaniline, 4,4'-oxydianiline, aniline, 3,3'-ichlorobenzidine, benzidine, 4-aminobiphenyl, o-dianisidine, o-anisidine, o-toluidine, 4,4'-methylene-bis-2-chloroaniline and 2-naphthylamine are oxidized in methanol/BMIm[NTf₂] at a potential around +0.68V to +0.93V vs. Ag/AgCl at a glassy carbon electrode, which is the base for their determination by HPLC/ED. Using the optimized conditions of methanol/BMIm[NTf₂] 70:30 (v/v) as mobile phase, flow-rate of 0.8 mL·min⁻¹, column CLC-ODS, Eap = +1.0 V and T = 40 °C analytical curves were constructed for each of the tested amines. Good linearity was obtained in the concentration range of 1.09 mg·L⁻¹ to 217 mg·L⁻¹, with excellent correlation coefficients. The limits of detection reached 0.021 mg·L⁻¹ to 0.246 mg·L⁻¹ and good relative standard deviations (RSD, n = 3) were obtained from the measurements. Satisfactory recovery for each aromatic amine was achieved, ranging from 95 to 103%. The developed method was successfully applied to determine six aromatic amines present as contaminants in commercial hair dye samples.
Topics: Amines; Chromatography, High Pressure Liquid; Drug Contamination; Electrochemical Techniques; Electrodes; Hair Dyes; Hydrocarbons, Aromatic; Hydrodynamics; Imidazoles; Ionic Liquids; Oxidation-Reduction; Reference Standards; Solutions; Sulfonamides
PubMed: 22751261
DOI: 10.3390/molecules17077961 -
Molecules (Basel, Switzerland) Jan 2012The phenolic Schiff bases I-VI were synthesized by condensation reactions between various diamines, namely o-dianisidine, o-tolidine and ethylenediamine with vanillin or...
The phenolic Schiff bases I-VI were synthesized by condensation reactions between various diamines, namely o-dianisidine, o-tolidine and ethylenediamine with vanillin or p-hydroxybenzaldehyde and subsequent reactions between these phenolic Schiff bases and epichlorohydrin to produce new diglycidyl ethers Ia-VIa. The structures of these compounds were confirmed by CHN, FT-IR, (1)H-NMR, and (13)C-NMR spectroscopy. Their thermotropic liquid crystalline behavior was studied using differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). All the diglycidyl ethers prepared exhibit nematic mesophases, except for Va and VIa, which did not show any transition mesophases, but simply flow to liquids.
Topics: Calorimetry, Differential Scanning; Diglycerides; Ethers; Liquid Crystals; Microscopy, Polarization; Molecular Structure; Phenols; Schiff Bases; Spectroscopy, Fourier Transform Infrared
PubMed: 22233565
DOI: 10.3390/molecules17010645 -
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 -
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 -
Report on Carcinogens : Carcinogen... 2011
Topics: Aniline Compounds; Animals; Anisoles; Biphenyl Compounds; Carcinogens; Coloring Agents; Dianisidine; Environmental Exposure; Government Regulation; Humans; United States
PubMed: 21852827
DOI: No ID Found -
Report on Carcinogens : Carcinogen... 2011
Topics: Aniline Compounds; Animals; Anisoles; Biphenyl Compounds; Carcinogens; Dianisidine; Environmental Exposure; Government Regulation; Humans; United States
PubMed: 21852826
DOI: No ID Found