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Methods in Molecular Biology (Clifton,... 2024Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an...
Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O and releases HO. The oxidation of o-dianisidine by HO catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.
Topics: Recombinant Proteins; Arabidopsis; Alcohol Oxidoreductases; Phosphoric Monoester Hydrolases; Glycolates; Enzyme Assays; Hydrogen Peroxide; Oxidation-Reduction; Plant Leaves; Arabidopsis Proteins; Spectrophotometry
PubMed: 38861076
DOI: 10.1007/978-1-0716-3802-6_3 -
Life Sciences May 2024Prolyl hydroxylase domain 2 (PHD2), encoded by the Egln1 gene, serves as a pivotal regulator of the hypoxia-inducible factor (HIF) pathway and acts as a cellular oxygen...
AIMS
Prolyl hydroxylase domain 2 (PHD2), encoded by the Egln1 gene, serves as a pivotal regulator of the hypoxia-inducible factor (HIF) pathway and acts as a cellular oxygen sensor. Somatic inactivation of Phd2 in mice results in polycythemia and congestive heart failure. However, due to the embryonic lethality of Phd2 deficiency, its role in development remains elusive. Here, we investigated the function of two egln1 paralogous genes, egln1a and egln1b, in zebrafish.
MAIN METHODS
The egln1 null zebrafish were generated using the CRISPR/Cas9 system. Quantitative real-time PCR assays and Western blot analysis were employed to detect the effect of egln1 deficiency on the hypoxia signaling pathway. The hypoxia response of egln1 mutant zebrafish were assessed by analyzing heart rate, gill agitation frequency, and blood flow velocity. Subsequently, o-dianisidine staining and in situ hybridization were used to investigate the role of egln1 in zebrafish hematopoietic function.
KEY FINDINGS
Our data show that the loss of egln1a or egln1b individually has no visible effects on growth rate. However, the egln1a; egln1b double mutant displayed significant growth retardation and elevated mortality at around 2.5 months old. Both egln1a-null and egln1b-null zebrafish embryo exhibited enhanced tolerance to hypoxia, systemic hypoxic response that include hif pathway activation, increased cardiac activity, and polycythemia.
SIGNIFICANCE
Our research introduces zebrafish egln1 mutants as the first congenital embryonic viable systemic vertebrate animal model for PHD2, providing novel insights into hypoxic signaling and the progression of PHD2- associated disease.
Topics: Animals; Mice; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Polycythemia; Procollagen-Proline Dioxygenase; Zebrafish
PubMed: 38492922
DOI: 10.1016/j.lfs.2024.122564 -
Environmental Pollution (Barking, Essex... Apr 2024PFDMO2OA (C8 HFPO-TA), a novel substitute for perfluorooctanoic acid (PFOA), has been frequently detected in surface waters. However, information on its toxicity remains...
PFDMO2OA (C8 HFPO-TA), a novel substitute for perfluorooctanoic acid (PFOA), has been frequently detected in surface waters. However, information on its toxicity remains scarce. In the present study, zebrafish embryos were exposed to varying concentrations of PFDMO2OA, ranging from 80 to 800 mg/L, until 120 h post-fertilization (hpf) to explore its potential developmental toxicities. The LC50 value for mortality was 505.9 mg/L, comparable to that of PFOA (over 500 mg/L), suggesting a lack of safety of PFDMO2OA compared to PFOA. At 120 hpf, PFDMO2OA exposure led to various malformations in embryos, including uninflated swim bladder, yolk sac oedema, spinal deformation, and pigmentation changes, with pericardial oedema being prominent. Analysis using O-dianisidine stain indicated a decline in erythrocytes over time. Transcriptome analysis further revealed the cardiovascular toxicity caused by PFDMO2OA at the molecular level. Time-course differential analysis pointed to the apoptosis dependent on disrupted mitochondrial function as a significant contributor to erythrocyte disappearance, as confirmed by the TUNEL stain. Therefore, the present findings suggest that PFDMO2OA induces developmental malformations and cardiovascular toxicities in zebrafish embryos, demonstrating a toxic potency comparable to that of PFOA. The results further highlight the significance of evaluating the health risks associated with PFDMO2OA.
Topics: Animals; Zebrafish; Embryo, Nonmammalian; Gene Expression Profiling; Edema; Fluorocarbons; Propionates
PubMed: 38462201
DOI: 10.1016/j.envpol.2024.123729 -
Methods in Molecular Biology (Clifton,... 2024Angiogenesis is the process of new blood vessel formation from preexisting vasculature. It is an integral component in normal embryonic development and tissue repair....
Angiogenesis is the process of new blood vessel formation from preexisting vasculature. It is an integral component in normal embryonic development and tissue repair. Dysregulation of angiogenesis might lead to tissue ischemia (resulting from reduced blood vessel formation) or major diseases such as cancer (abnormal vascular growth). This makes angiogenesis an excellent area of research for cancer therapeutics, and various animal models including zebrafish are used to study blood vessel development. As most of the techniques used to study angiogenesis are complex and cumbersome, in this chapter, we provide two simple assays to study angiogenesis with live and fixed zebrafish embryos/larvae.
Topics: Animals; Female; Angiogenesis; Zebrafish; Embryonic Development; Larva; Perciformes; Neoplasms
PubMed: 38285352
DOI: 10.1007/978-1-0716-3625-1_21 -
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 -
Chemistry & Biodiversity Dec 2023The aim of this study was to examine a collection of 79 honeys derived from plants endemic to several Western Australian unique bioregions for bioactivity and...
The aim of this study was to examine a collection of 79 honeys derived from plants endemic to several Western Australian unique bioregions for bioactivity and physicochemical characteristics. For physicochemical analyses, total phenolic content, high performance thin layer chromatography (HPTLC) fingerprints, pH, Brix, colour and hydrogen peroxide generation were examined. Brix (82.6±1.3) and pH (4.34±0.24) values were within expected ranges, whereas hydrogen peroxide levels determined using an o-dianisidine/horseradish peroxidase assay were relatively low, ranging from 0-244 μM. Antibacterial activity determined by the broth microdilution assay showed that Moort (Eucalyptus platypus) and Yate (Eucalyptus occidentalis) honeys had the highest overall activity with mean minimum inhibitory concentrations of 24.8 % and 25.1 % (w/v) honey, respectively. Yate honey also had the highest overall antioxidant activity (4.38±0.58 mmol Fe /kg of honey), followed by Mallee honeys from various eucalypts, as determined by FRAP (Ferric reducing antioxidant power) and DPPH⋅ (2,2-Diphenyl-1-picrylhydrazyl) assays. This study identified new sources of honeys with potentially useful therapeutic properties from bioregions within Western Australia.
Topics: Western Australia; Honey; Hydrogen Peroxide; Australia; Phenols; Antioxidants; Eucalyptus
PubMed: 37968896
DOI: 10.1002/cbdv.202301678 -
The Journal of Biological Chemistry Dec 2023Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases. PHD3 catalyzes proline...
Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases. PHD3 catalyzes proline hydroxylation of hypoxia-inducible factor α (HIF-α) and promotes HIF-α proteasomal degradation through coordination with the pVHL complex under normoxic conditions. However, the relationship between PHD3 and the hypoxic response is not well understood. In this study, we used quantitative real-time PCR assay and O-dianisidine staining to characterize the hypoxic response in zebrafish deficient in phd3. We found that the hypoxia-responsive genes are upregulated and the number of erythrocytes was increased in phd3-null zebrafish compared with their wild-type siblings. On the other hand, we show overexpression of phd3 suppresses HIF-transcriptional activation. In addition, we demonstrate phd3 promotes polyubiquitination of zebrafish hif-1/2α proteins, leading to their proteasomal degradation. Finally, we found that compared with wild-type zebrafish, phd3-null zebrafish are more resistant to hypoxia treatment. Therefore, we conclude phd3 has a role in hypoxia tolerance. These results highlight the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.
Topics: Animals; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Procollagen-Proline Dioxygenase; Proline; Zebrafish; Zebrafish Proteins; Gene Deletion; Oxygen
PubMed: 37923141
DOI: 10.1016/j.jbc.2023.105420 -
Veterinary Sciences Oct 2023Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as -phenylenediamine (-P), -dianisidine...
Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as -phenylenediamine (-P), -dianisidine (-D) and, most recently, ammonium iron(II) sulfate (AIS). Once developed in humans, these assays are often used in veterinary medicine without appropriately optimizing in the animal species of interest. In this study, two assays using AIS and -D as substrates have been compared and validated for Cp oxidase activity assessment in horse's plasma. The optimization of the assays was performed mainly by varying the buffer pH as well as the buffer and the substrate molar concentration. Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent Cp inhibitor. In the -D assay, 500 µM sodium azide treatment completely inhibits the enzymatic activity of Cp, whereas, using the AIS assay, a residual analytical signal was still present even at the highest (2000 µM) sodium azide concentration. Even though the analytical values obtained from these methods are well correlated, the enzymatic activity values significantly differ when expressed in Units L. A disagreement between these assays has also been detected with the Bland-Altman plot, showing a progressive discrepancy between methods with increasing analytical values.
PubMed: 37888575
DOI: 10.3390/vetsci10100623 -
Journal of Hazardous Materials Oct 2023In this study, we developed a colorimetric ozone passive sampler (OPS) incorporating o-dianisidine, a redox dye, into a polydimethylsiloxane sheet. The reaction between...
In this study, we developed a colorimetric ozone passive sampler (OPS) incorporating o-dianisidine, a redox dye, into a polydimethylsiloxane sheet. The reaction between ozone (O) and o-dianisidine result in a visible yellowish color change. Unlike previous passive methods that rely on nitrate extraction or the color disappearance of indigotrisulfonate, the OPS offered improved recognition of average O exposure. To optimize OPS based on time-weighted average (TWA), we extracted and quantified the amount of reacted o-dianisidine after exposing OPS to O by varying concentrations (0-200 ppb) within 8 h. Colorimetric changes of OPS were further analyzed by capturing images, and the effective absorbance of blue scale showed the best fit (EA, R =0.997). OPS validation on visual detection assessed by six parameters: limit of detection, limit of quantification, reproducibility, sampling rate, selectivity to interfering gases, and sensitivity to environmental factors. To enhance visibility, the OPS was assembled with coloration exposure guidelines, and a smartphone app was developed to quantify average O exposures. We further conducted field tests that showed the significant disparity between O concentrations and personal O exposures, which is considered more crucial for assessing health risks. The OPS was optimized to monitor O exposure levels and raise awareness among workers and occupants regarding invisible indoor hazards.
Topics: Humans; Colorimetry; Dianisidine; Reproducibility of Results; Levonorgestrel; Ozone
PubMed: 37703734
DOI: 10.1016/j.jhazmat.2023.132510 -
Journal of Applied Toxicology : JAT Feb 2024Angiogenesis and hemodynamic instability created by the irregular blood vessels causes hypoperfusion and angiogenesis-mediated diseases. Therefore, therapies focusing on...
Angiogenesis and hemodynamic instability created by the irregular blood vessels causes hypoperfusion and angiogenesis-mediated diseases. Therefore, therapies focusing on controlling angiogenesis will be a valuable approach to treat a broad spectrum of diseases. In this study, we explored the anti-angiogenic potential of berberine (BBR) and also analyzed blood flow hemodynamics using zebrafish embryos. Zebrafish embryos treated with BBR (0.01-0.75 mM) at various doses at 1 hour post-fertilization (hpf) developed a variety of phenotypic variations including aberrant blood vessels, tail bending, edema, and hemorrhage. Survival rates were much lower at higher dosages, and hatching rates were almost 99%, whereas control group appeared normal. Heart rate is an essential measure that has a strong association with hemodynamics. We used ImageJ software to study the heart rate of embryos treated with BBR, preceded by video processing. The resultant graph shows a significant decrease in heart rate of embryos treated with BBR in dose-dependent manner. Also, RBC staining using o-Dianisidine confirms the anti-angiogenic potential of BBR by indicating the decrease in the intersegmental vessels at 0.5 and 0.75 mM treated embryos. Further, the gene expression study determined that the transcripts (vegf, vegfr2, nrp1a, hif-1α, nos2a, nos2b, cox-2a, and cox-2b) measured were found to be downregulated by BBR at 0.5 mM concentration, from which we conclude that enos/vegf signaling could play an important role in modulating angiogenesis. Our data imply that BBR may be an effective compound for suppressing angiogenesis in vivo, which might be helpful in the treatment of vascular disorders like cancer and diabetic retinopathy in future.
Topics: Animals; Zebrafish; Berberine; Vascular Endothelial Growth Factor A; Angiogenesis; Hemodynamics
PubMed: 37615217
DOI: 10.1002/jat.4529