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Fu Wei, Siyi Liu, Li Luo, Nina Gu, Yan Zeng, Xiaoying Chen, Shan Xu, Dan Zhang
OBJECTIVE: Ulinastatin reduces the high permeability of vascular endothelial cells induced by tumor necrosis factor alpha (TNF-α). This study investigated the molecular mechanism behind this effect, with the aim of understanding the action of ulinastatin in sepsis therapy and exploring novel therapeutic strategies for sepsis patients. METHODS: A TNF-α treated human umbilical vein endothelial cell line (EA.hy926) was employed as an inflammation model. Horseradish peroxidase permeability assays and an epithelial voltmeter method were used to measure the permeability of EA...
March 19, 2017: International Immunopharmacology
Yiping Feng, Kun Lu, Shixiang Gao, Liang Mao
In this study, we examined the fate and transformation of tetrabromobisphenol A (TBBPA), mediated by the representative oxidoreductases (laccase and horseradish peroxidase (HRP)) in natural waters. Both enzymes could readily degrade TBBPA at environmentally relevant concentrations (e.g., 10 nmol L(-1)) in natural waters. After 2 hour treatment, 0.5-25% and 35-65% of TBBPA were degraded in municipal wastewater and natural surface waters by a laccase or HRP-catalyzed reaction, respectively. Enzyme kinetics evaluations indicated that the kCAT/KM ratio of HRP (1...
March 22, 2017: Environmental Science. Processes & Impacts
P Hattay, D K Prusator, L Tran, B Greenwood-Van Meerveld
BACKGROUND: Evidence suggests that patients with irritable bowel syndrome (IBS) exhibit increases in gut permeability and alterations in tight junction (TJ) protein expression. Although psychological stress worsens IBS symptoms, the mechanisms by which stress enhances gut permeability and affects TJ protein expression remain to be determined. Here, we test the hypothesis that chronic intermittent psychological stress activates the release of proinflammatory cytokines to alter TJ proteins and promotes increased gut permeability...
March 16, 2017: Neurogastroenterology and Motility: the Official Journal of the European Gastrointestinal Motility Society
Rickdeb Sen, Digvijay Gahtory, Rui Rijo Carvalho, Bauke Albada, Floris L van Delft, Han Zuilhof
Mica is the substrate of choice for microscopic visualization of a wide variety of intricate nanostructures. Unfortunately, the lack of a facile strategy for its modification has prevented the on-mica assembly of nanostructures. Herein, we disclose a convenient catechol-based linker that enables various surface-bound metal-free click reactions, and an easy modification of mica with DNA nanostructures and a horseradish peroxidase mimicking hemin/G-quadruplex DNAzyme.
March 15, 2017: Angewandte Chemie
Jun-Hui Guo, Guo-Lan Xing, Xin-Hui Fang, Hui-Fang Wu, Bo Zhang, Jin-Zhong Yu, Zong-Min Fan, Li-Dong Wang
AIM: To understand the molecular mechanism of esophageal cancer development and provide molecular markers for screening high-risk populations and early diagnosis. METHODS: Two-dimensional electrophoresis combined with mass spectrometry were adopted to screen differentially expressed proteins in nine cases of fetal esophageal epithelium, eight cases of esophageal cancer, and eight cases of tumor-adjacent normal esophageal epithelium collected from fetuses of different gestational age, or esophageal cancer patients from a high-risk area of esophageal cancer in China...
February 28, 2017: World Journal of Gastroenterology: WJG
Thomas Gundinger, Oliver Spadiut
Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E...
March 11, 2017: Journal of Biotechnology
Qian Xiao, Yizhi Zheng, Jing Liu, Suiping Wang, Bo Feng
In this study, we designed a comprehensive strategy for the ultrasensitive detection of alpha fetoprotein (AFP) with high specificity using gold nanoparticle (AuNP)-based enzyme-linked immunosorbent assay (ELISA). A dual-film modified probe was synthesized by coating AuNPs with horseradish peroxidase (HRP) on its surface. Anti-AFP monoclonal antibody (McAb) was immobilized on the surface of the enzyme using glutaraldehyde cross-linking method. AuNPs, employed as support for the immobilization of HRP. HRP was used not only as the enzymatic-amplified tracer but also as a bridge for loading McAb...
March 8, 2017: Biologicals: Journal of the International Association of Biological Standardization
Le Truc Nguyen, Kun-Lin Yang
Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)...
May 2017: Enzyme and Microbial Technology
Negar Alizadeh, Rahman Hallaj, Abdollah Salimi
Here we prepared an electrochemical immunosensor employing Au sheet as working electrode, Fe3O4 magnetic nanoparticles (MNPs) as supporting matrix and hemin/G-quadruplex DNAzyme as signal amplifier for determination of hepatitis B virus surface antigen (HBsAg). First, the primary antibody of HBs (Ab1) was immobilized on the surface of the carboxyl-modified MNPs. Then, the assembly of antibody and alkylthiol/G-quadruplex DNA/hemin on gold nanoparticles was used as bio-bar-coded nanoparticle probe. Protein target was sandwiched between the primary antibody of HBs (Ab1) immobilized on the MNPs and hemin bio-bar-coded AuNPs probe labeled antibody (Ab2)...
February 24, 2017: Biosensors & Bioelectronics
Jennifer L Larson-Casey, A Brent Carter
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (1-5). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5' diacetate [(pHPA)2])...
November 20, 2016: Bio-protocol
Stephanie P Cartwright, Richard A J Darby, Debasmita Sarkar, Nicklas Bonander, Stephane R Gross, Mark P Ashe, Roslyn M Bill
BACKGROUND: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid...
March 9, 2017: Microbial Cell Factories
Paula B Luis, Odaine N Gordon, Fumie Nakashima, Akil I Joseph, Takahiro Shibata, Koji Uchida, Claus Schneider
Conjugation with glucuronic acid is a prevalent metabolic pathway of orally administrated curcumin, the bioactive diphenol of the spice turmeric. The major in vitro degradation reaction of curcumin is autoxidative transformation resulting in oxygenation and cyclization of the heptadienedione chain to form cyclopentadione derivatives. Here we show that curcumin-glucuronide is much more stable than curcumin, degrading about two orders of magnitude slower. Horseradish peroxidase-catalyzed oxidation of curcumin-glucuronide occurred at about 80% of the rate with curcumin, achieving efficient transformation...
March 6, 2017: Biochemical Pharmacology
Wentao Xu, Jingjing Tian, Yunbo Luo, Longjiao Zhu, Kunlun Huang
To solve the requirement of on-site, rapid, and visual detection of copper (II) (Cu(2+)) in aqueous solution, a turn-off sensor for detecting copper (II) ion was developed based on Cu(2+)-dependent DNAzyme as the recognition element and hybridization chain reaction (HCR)-based horseradish peroxidase (HRP) concatemers as the signal amplifier and the signal report element. The detection unit, which was composed of the immobilized Cu(2+)-dependent DNAzyme coupled with HCR-based HRP concatemers via Waston-Crick base pairing, could catalyze hydrogen peroxide (H2O2) via TMB, generating obvious green color and turning yellow after sulfuric acid termination with optical absorption at 450 nm...
March 7, 2017: Scientific Reports
Gary Leung, Melissa Tully, Jonathan Tang, Shengxi Wu, Riyi Shi
BACKGROUND: It is increasingly clear that in addition to myelin disruption, axonal degeneration may also represent a key pathology in multiple sclerosis (MS). Hence, elucidating the mechanisms of axonal degeneration may not only enhance our understanding of the overall MS pathology, but also elucidate additional therapeutic targets. The objective of this study is assess the degree of axonal membrane disruption and its significance in motor deficits in EAE mice. METHODS: Experimental Autoimmune Encephalomyelitis was induced in mice by subcutaneous injection of myelin oligodendrocyte glycoprotein/complete Freud's adjuvant emulsion, followed by two intraperitoneal injections of pertussis toxin...
2017: Translational Neurodegeneration
Jinyang Chen, Xinghu Ji, Zhike He
Based on an interesting experiment phenomenon, a novel and smart composite reagent consist of dsDNA-templated copper nanoparticles (dsDNA-CuNPs) and DNA intercalator (SYBR Green I) was developed, and exploited for the H2O2 detection as well as oxidase-based biosensing. The study found that, within the composite reagent, the small molecule SYBR Green I was easily adsorbed on the surfaces of CuNPs, instead of intercalating into the dsDNA. So the composite reagent only exhibited the red fluorescence generated from dsDNA-CuNPs...
March 7, 2017: Analytical Chemistry
Pavla Martinkova, Miroslav Pohanka
OBJECTIVES: Glucose is an important diagnostic biochemical marker of diabetes but also for organophosphates, carbamates, acetaminophens or salicylates poisoning. Hence, innovation of accurate and fast detection assay is still one of priorities in biomedical research. METHODS: Glucose sensor based on magnetic particles (MPs) with immobilized enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) was developed and the GOx catalyzed reaction was visualized by a smart-phone-integrated camera...
December 18, 2016: Neuro Endocrinology Letters
Jiawang Ding, Enguang Lv, Liyan Zhu, Wei Qin
We report here on an optical ion sensing platform, in which a polymeric membrane ion-selective electrode (ISE) serves as not only a potentiometric transducer for ion activities in the sample solution but also a reference electrode for the potential-modulated release of enzyme from an iron-alginate-horseradish peroxidase (HRP) thin film modified working electrode. The ISE and working electrode are physically separated by a salt bridge. The dissolution of the HRP-embedded thin film can be triggered by the reduction of Fe(3+), which is modulated by the potential response of the ISE to the target ion in the sample...
March 21, 2017: Analytical Chemistry
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
March 3, 2017: Current Protocols in Cell Biology
Jonathan Sabaté Del Río, Ivan Magriñà Lobato, Olena Mayboroda, Ioanis Katakis, Ciara K O'Sullivan
Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers...
March 2, 2017: Analytical and Bioanalytical Chemistry
Rakesh Gupta, Nishi Kashyap, Beena Rai
Transdermal delivery, where the skin acts as the route for local or systemic distribution, presents a lot of advantages over conventional routes such as oral and intravenous and intramuscular injections. However, the delivery of large biomolecules like proteins through the skin is challenging due to their size and structural properties. A molecular level understanding of their transport across the skin barrier is desirable to design successful formulations. We have employed constrained and unconstrained coarse grained molecular dynamics simulation techniques to obtain the molecular mechanism of penetration of the horseradish peroxidase (HRP) protein into the skin, in the presence and absence of gold nanoparticles (AuNPs)...
March 2, 2017: Physical Chemistry Chemical Physics: PCCP
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