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Archives of Oral Biology Jul 2015To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase...
OBJECTIVE
To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system.
DESIGN
Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804.
RESULTS
While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system.
CONCLUSIONS
Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities.
Topics: Animals; Candida; Cattle; Chickens; Colony Count, Microbial; Durapatite; Female; Lactoperoxidase; Male; Microbial Viability; Muramidase; Nephelometry and Turbidimetry; Peroxidases; Saliva; Sorbitol; Stem Cells; Surface Properties; Xylitol
PubMed: 25874813
DOI: 10.1016/j.archoralbio.2015.03.011 -
Zebrafish Aug 2016Thrombosis is a leading cause of death and the development of effective and safe therapeutic agents for thrombotic diseases has been proven challenging. In this study,...
Thrombosis is a leading cause of death and the development of effective and safe therapeutic agents for thrombotic diseases has been proven challenging. In this study, taking advantage of the transparency of larval zebrafish, we developed a larval zebrafish thrombosis model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days post fertilization) were treated with phenylhydrazine (PHZ) and a testing drug for 24 h. Tested drugs were administered into the zebrafish either by direct soaking or circulation microinjection. Antithrombotic efficacy was quantitatively evaluated based on our previously patented technology characterized as an image analysis of the heart red blood cells stained with O-dianisidine staining. Zebrafish at 2 dpf treated with PHZ at a concentration of 1.5 μM for a time period of 24 h were determined as the optimum conditions for the zebrafish thrombosis model development. Induced thrombosis in zebrafish was visually confirmed under a dissecting stereomicroscope and quantified by the image assay. All 6 human antithrombotic drugs (aspirin, clopidogrel, diltiazem hydrochloride injection, xuanshuantong injection, salvianolate injection, and astragalus injection) showed significant preventive and therapeutic effects on zebrafish thrombosis (p < 0.05, p < 0.01, & p < 0.001) in this zebrafish thrombosis model. The larval zebrafish thrombosis model developed and validated in this study could be used for in vivo thrombosis studies and for rapid screening and efficacy assessment of antithrombotic drugs.
Topics: Animals; Disease Models, Animal; Fibrinolytic Agents; Humans; Microinjections; Thrombosis; Zebrafish
PubMed: 27333081
DOI: 10.1089/zeb.2016.1263 -
Bioprocess and Biosystems Engineering Apr 2023Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests,...
Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.
Topics: Recombinant Proteins; Isoenzymes; Horseradish Peroxidase; Escherichia coli; Pichia
PubMed: 36527454
DOI: 10.1007/s00449-022-02837-2 -
Heliyon Jan 2021Kinetic and physicochemical properties of peroxidase purified using a novel and cost efficient protocol was investigated with a view to providing information on its...
Kinetic and physicochemical properties of peroxidase purified using a novel and cost efficient protocol was investigated with a view to providing information on its possible biotechnological potentials. peroxidase was purified to homogeneity in two steps, involving ATPS and size exclusion chromatography on Sephadex G-100 with a yield of 84.12 %. In-gel activity staining revealed the presence of one isoform of peroxidase. The purified peroxidase is monomeric with native and subunits molecular weight of 38.9 and 43.5 kDa respectively. Kinetic parameters - , , of the purified enzyme were 2.5 units/mg protein, 0.020 ± 0.04 mM and 1.37 ± 0.18 mM respectively. Its optimum pH and temperature were 5 and 30 °C respectively. The purified enzyme cross-linked BSA into an insoluble matrix with the aid of caffeic acid. The study concluded that the purification scheme adopted is rapid and efficient, the purified enzyme exhibited some physiochemical properties that make it suitable for various biotechnological applications.
PubMed: 33521366
DOI: 10.1016/j.heliyon.2021.e06032 -
Indoor Air Mar 2021With an increasing use of indoor disinfectants such as chlorine (Cl ) and hypochlorous acid, a convenient sampler for estimating exposure to oxidants, such as effective...
With an increasing use of indoor disinfectants such as chlorine (Cl ) and hypochlorous acid, a convenient sampler for estimating exposure to oxidants, such as effective chlorine, is necessary. Here, we developed a personal passive air sampler (PPAS) composed of a redox dye, o-dianisidine, in a polydimethylsiloxane (PDMS) sheet. o-Dianisidine readily reacts with gaseous oxidants generated by bleach usage, and its color changes as the reaction progresses; hence, personal exposure to effective chlorine could be easily detected by the naked eye, while cumulative exposure could be determined by measuring concentrations of o-dianisidine reacting with it. The PPAS was calibrated, and a sampling rate of 0.00253 m /h was obtained using a small test chamber. The PPAS was tested with the help of ten volunteers whose personal exposure to Cl -equivalent gas was estimated after bathrooms were cleaned using spray and liquid-type household disinfection products, and the accumulated exposure-gas concentrations ranged from 69 to 408 ppbv and 148 to 435 ppbv, respectively. These PPAS-derived exposure concentrations were approximately two orders lower than those estimated using ConsExpo, suggesting a significant overestimation by prevailing screening models, possibly due to the ignorance of transformation reactions.
Topics: Air Pollutants; Air Pollution, Indoor; Chlorine; Dimethylpolysiloxanes; Disinfectants; Disinfection; Environmental Monitoring; Humans; Hypochlorous Acid; Inhalation Exposure
PubMed: 32978992
DOI: 10.1111/ina.12747 -
Comparative Biochemistry and... Jun 2021We investigated whether ferulic acid (FA), a nutraceutical could mitigate the arsenic (As) induced cardiotoxicity. Zebrafish larvae (60 and 72 h post-fertilization...
We investigated whether ferulic acid (FA), a nutraceutical could mitigate the arsenic (As) induced cardiotoxicity. Zebrafish larvae (60 and 72 h post-fertilization [hpf]) were used to study the effect of FA on As at different time points (24 and 48 h after exposure). The FA exposure was given as pre-treatment (60 hpf) and simultaneous treatment (72 hpf) to translate the results for As contaminated areas. To accomplish this, the lethality assay was done, and based on the results, the dosage for As (1 mM) and FA (30 μM) was fixed. The FA intervention (30 μM) as 12 h pre-treatment (60 hpf) and simultaneous treatment along with As (72 hpf) decreased the As content in zebrafish larvae as evidenced by inductively coupled plasma-mass spectrometry. As exposure showed congenital deformities especially cardiac malformations in zebrafish larvae after 24 and 48 h. These teratogenic effects induced by As were reduced by FA supplementation in both groups. Also, o-dianisidine staining demonstrated that As treated larvae encountered abnormal cardiac function with reduced blood circulation, while FA supplementation reversed these effects. The acetylcholinesterase activity, a biomarker of As-induced cardiotoxicity was also found to be decreased in As group, which was rescued by FA. The modulation in the expression of the genes involved in cardiogenesis (nkx2.5, bmp2b, gata4, gata5, myh6, myl7, and tnnt2) further confirmed the ameliorative effect of FA on As induced malformations.
Topics: Animals; Arsenic; Cardiotoxicity; Coumaric Acids; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Water Pollutants, Chemical; Zebrafish; Zebrafish Proteins
PubMed: 33631344
DOI: 10.1016/j.cbpc.2021.109021 -
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 -
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 -
Access Microbiology 2019Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (HO) in diluted honey is central to this...
Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (HO) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of HO in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that HO production by these honeys varies greatly, with some samples producing negligible levels of HO. We assessed the effect of colour on the assay by measuring the recovery of spiked HO from light and dark honey and from serially diluted dark corn syrup, and found the amount of HO that could be detected was lower in dark corn syrup and darker honey samples.
PubMed: 32974499
DOI: 10.1099/acmi.0.000065 -
Spectrochimica Acta. Part A, Molecular... Jan 2023In this paper, we report a novel surface-enhanced Raman spectroscopy (SERS) substrate based on hierarchical β-BiO/AuAg microspheres for rapid, sensitive and selective...
In this paper, we report a novel surface-enhanced Raman spectroscopy (SERS) substrate based on hierarchical β-BiO/AuAg microspheres for rapid, sensitive and selective detection of environment pollutants including o-dianisidine (o-diASD) and Hg in environmental samples. The sheet-like β-BiO not only provides large specific surface areas for adsorption of molecules and AuAg, but also emerges as semiconductor matrix with chemical enhancement combined with AuAg with electromagnetic enhancement, making promising SERS activity. Particularly, the β-BiO/AuAg shows high SERS performance for 4-mercaptobenzoic acid and TMB with minimum detectable concentration of 0.1 μg/L with enhancement factor of 3.1 × 10 and 6.3 × 10, respectively. The density functional theory simulations were further adopted to explain the high SERS activity and selectivity for o-diASD and TMB. Finally, the β-BiO/AuAg was applied to direct detection of o-diASD, and indirect detection of Hg by TMB marking in environmental samples. The linearity range of 0.5-200.0 and 0.2-500.0 μg/L with limit of detection of 0.2 and 0.07 μg/L for o-diASD and Hg ions can be achieved, respectively. This method provides a novel strategy in designing and fabricating SERS substrates with high performance for rapid, sensitive and accurate analysis of environmental pollutants.
Topics: Spectrum Analysis, Raman; Metal Nanoparticles; Environmental Pollutants; Microspheres; Mercury
PubMed: 36179562
DOI: 10.1016/j.saa.2022.121907