Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

PubMed Central

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-01-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

PubMed

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1985-01-01

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

Novel enzymatic method for assaying Lp-PLA2 in serum.

PubMed

Yamaura, Saki; Sakasegawa, Shin-Ichi; Koguma, Emisa; Ueda, Shigeru; Kayamori, Yuzo; Sugimori, Daisuke; Karasawa, Ken

2018-06-01

Measurement of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA 2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA 2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C 16 PAF) was developed. The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA 2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C 16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. Regression analysis of Lp-PLA 2 activity measured by the enzymatic Lp-PLA 2 activity assay vs. two chemical Lp-PLA 2 activity assays, i.e. LpPLA 2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). Advantages of this enzymatic Lp-PLA 2 activity assay compared with chemical Lp-PLA 2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. Copyright © 2018 Elsevier B.V. All rights reserved.

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

PubMed Central

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format.

PubMed

Berkowitz, Oliver; Jost, Ricarda; Pearse, Stuart J; Lambers, Hans; Finnegan, Patrick M; Hardy, Giles E St J; O'Brien, Philip A

2011-05-01

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD(+) as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application. Copyright © 2011 Elsevier Inc. All rights reserved.

Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana.

PubMed

Udompaisarn, Somsiri; Arthan, Dumrongkiet; Somana, Jamorn

2017-04-19

An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r 2 = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

PubMed

Wang, Xiao-Peng; Zhao, Xin-Huai

2017-06-01

Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Evaluation of a next generation direct whole blood enzymatic assay for hemoglobin A1c on the ARCHITECT c8000 chemistry system.

PubMed

Teodoro-Morrison, Tracy; Janssen, Marcel J W; Mols, Jasper; Hendrickx, Ben H E; Velmans, Mathieu H; Lotz, Johannes; Lackner, Karl; Lennartz, Lieselotte; Armbruster, David; Maine, Gregory; Yip, Paul M

2015-01-01

The utility of HbA1c for the diagnosis of type 2 diabetes requires an accurate, precise and robust test measurement system. Currently, immunoassay and HPLC are the most popular methods for HbA1c quantification, noting however the limitations associated with some platforms, such as imprecision or interference from common hemoglobin variants. Abbott Diagnostics has introduced a fully automated direct enzymatic method for the quantification of HbA1c from whole blood on the ARCHITECT chemistry system. Here we completed a method evaluation of the ARCHITECT HbA1c enzymatic assay for imprecision, accuracy, method comparison, interference from hemoglobin variants and specimen stability. This was completed at three independent clinical laboratories in North America and Europe. The total imprecision ranged from 0.5% to 2.2% CV with low and high level control materials. Around the diagnostic cut-off of 48 mmol/mol, the total imprecision was 0.6% CV. Mean bias using reference samples from IFCC and CAP ranged from -1.1 to 1.0 mmol/mol. The enzymatic assay also showed excellent agreement with HPLC methods, with slopes of 1.01 and correlation coefficients ranging from 0.984 to 0.996 compared to Menarini Adams HA-8160, Bio-Rad Variant II and Variant II Turbo instruments. Finally, no significant effect was observed for erythrocyte sedimentation or interference from common hemoglobin variants in patient samples containing heterozygous HbS, HbC, HbD, HbE, and up to 10% HbF. The ARCHITECT enzymatic assay for HbA1c is a robust and fully automated method that meets the performance requirements to support the diagnosis of type 2 diabetes.

An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

PubMed

Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

2017-02-01

Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles. Copyright © 2016 Elsevier Inc. All rights reserved.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

A microfluorometric assay for cholinesterases, suitable for multiple kinetic determinations of picomoles of released thiocholine.

PubMed

Parvari, R; Pecht, I; Soreq, H

1983-09-01

A highly sensitive microfluorometric assay for cholinesterases has been developed. Enzymatic activity is measured by monitoring the thiocholine produced by specific hydrolysis of acetylthiocholine. This is carried out by reacting the thiocholine formed with the fluorogenic compound N-(4(7 diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide to yield an intensely fluorescent product. The assay is linear over a range extending from a few picomoles to nanomoles of thiocholine. The specificity and accuracy of this microfluorometric assay were examined using microgram quantities of rat brain tissue as a source for cholinesterases. The specific activities and the Km values determined by this new method for both cholinesterase activities present in the brain (acetylcholine hydrolase, EC 3.1.1.7, and "nonspecific" cholinesterase-acylcholine acylhydrolase, EC 3.1.1.8) were identical to those reported earlier using the less sensitive spectrophotometric and radiometric methods. The background emission caused by nonenzymatic hydrolysis of the substrate is relatively low, and does not exceed background values encountered in other methods. The assay may be used for monitoring the kinetics of enzymatic activities in microscale reaction mixtures, providing a linear determination of the thiocholine produced over a period of at least 30 h at room temperature. The method can also be adapted for use in other enzymatic assays where reagents containing thiol groups can be produced or consumed.

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Development of a thyroperoxidase inhibition assay for high-throughput screening

EPA Science Inventory

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

NASA Astrophysics Data System (ADS)

Stasyuk, Nataliya; Gayda, Galina; Zakalskiy, Andriy; Zakalska, Oksana; Errachid, Abdelhamid; Gonchar, Mykhailo

2018-03-01

A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2 +-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2 + or Co2 + with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2 + assay is from 4 pM to 1.10 nM with a limit of detection of 1 pM Mn2 +, whereas the linearity range of the present method in the case of Co2 + assay is from 8 pM to 45 nM with a limit of detection of 2.5 pM Co2 +. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2 + and Co2 + content in clinical laboratories, food industry and environmental control service.

Multi-wavelength dye concentration determination for enzymatic assays: evaluation of chromogenic para-nitrophenol over a wide pH range.

PubMed

Max, Jean-Joseph; Meddeb-Mouelhi, Fatma; Beauregard, Marc; Chapados, Camille

2012-12-01

Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of para-nitrophenol (pNP) in basic and acidic solutions. First, we confirmed by factor analysis that pNP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates pNP after hydrolysis catalyzed by a lipase from the fungus Yarrowia lipolytica was used. Over the pH range of 3-11, accurate amounts of acidic and basic pNP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect pNP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

PubMed Central

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

Determination of optimal biomass pretreatment strategies for biofuel production: investigation of relationships between surface-exposed polysaccharides and their enzymatic conversion using carbohydrate-binding modules.

PubMed

Khatri, Vinay; Meddeb-Mouelhi, Fatma; Adjallé, Kokou; Barnabé, Simon; Beauregard, Marc

2018-01-01

Pretreatment of lignocellulosic biomass (LCB) is a key step for its efficient bioconversion into ethanol. Determining the best pretreatment and its parameters requires monitoring its impacts on the biomass material. Here, we used fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay to study the relationship between surface-exposed polysaccharides and enzymatic hydrolysis of LCB. Our results indicated that alkali extrusion pretreatment led to the highest hydrolysis rates for alfalfa stover, cattail stems and flax shives, despite its lower lignin removal efficiency compared to alkali pretreatment. Corn crop residues were more sensitive to alkali pretreatments, leading to higher hydrolysis rates. A clear relationship was consistently observed between total surface-exposed cellulose detected by the FTCM-depletion assay and biomass enzymatic hydrolysis. Comparison of bioconversion yield and total composition analysis (by NREL/TP-510-42618) of LCB prior to or after pretreatments did not show any close relationship. Lignin removal efficiency and total cellulose content (by NREL/TP-510-42618) led to an unreliable prediction of enzymatic polysaccharide hydrolysis. Fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay provided direct evidence that cellulose exposure is the key determinant of hydrolysis yield. The clear and robust relationships that were observed between the cellulose accessibility by FTCM probes and enzymatic hydrolysis rates change could be evolved into a powerful prediction tool that might help develop optimal biomass pretreatment strategies for biofuel production.

Combined subcritical water and enzymatic hydrolysis for reducing sugar production from coconut husk

NASA Astrophysics Data System (ADS)

Muharja, Maktum; Junianti, Fitri; Nurtono, Tantular; Widjaja, Arief

2017-05-01

Coconut husk wastes are abundantly available in Indonesia. It has a potential to be used into alternative renewable energy sources such as hydrogen using enzymatic hydrolysis followed by a fermentation process. Unfortunately, enzymatic hydrolysis is hampered by the complex structure of lignocellulose, so the cellulose component is hard to degrade. In this study, Combined Subcritical Water (SCW) and enzymatic hydrolysis are applied to enhance fermentable, thereby reducing production of sugar from coconut husk. There were two steps in this study, the first step was coconut husk pretreated by SCW in batch reactor at 80 bar and 150-200°C for 60 minutes reaction time. Secondly, solid fraction from the results of SCW was hydrolyzed using the mixture of pure cellulose and xylanase enzymes. Analysis was conducted on untreated and SCW-treated by gravimetric assay, liquid fraction after SCW and solid fraction after enzymatic hydrolysis using DNS assay. The maximum yield of reducing sugar (including xylose, arabinose glucose, galactose, mannose) was 1.254 gr per 6 gr raw material, representing 53.95% of total sugar in coconut husk biomass which was obtained at 150°C 80 bar for 60 minutes reaction time of SCW-treated and 6 hour of enzymatic hydrolysis using mixture of pure cellulose and xylanase enzymes (18.6 U /gram of coconut husk).

[Enzymatic analysis of the quality of foodstuffs].

PubMed

Kolesnov, A Iu

1997-01-01

Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

Use of Fructosyl Peptide Oxidase for HbA1c Assay

PubMed Central

Yonehara, Satoshi; Inamura, Norio; Fukuda, Miho; Sugiyama, Koji

2015-01-01

ARKRAY, Inc developed the world’s first automatic glycohemoglobin analyzer based on HPLC (1981). After that, ARKRAY developed enzymatic HbA1c assay “CinQ HbA1c” with the spread and diversification of HbA1c measurement (2007). CinQ HbA1c is the kit of Clinical Chemistry Analyzer, which uses fructosyl peptide oxidase (FPOX) for a measurement reaction. This report mainly indicates the developmental background, measurement principle, and future of the enzymatic method HbA1c reagent. PMID:25633966

A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.

PubMed

Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

2016-01-01

The α/β-hydrolase domain-containing 6 (ABHD6) enzyme is a newly found serine hydrolase whose substrate profile resembles that of monoacylglycerol lipase (MAGL), the major 2-arachidonoyl glycerol (2-AG) hydrolase in the brain. Here, we describe a sensitive fluorescent assay of ABHD6 activity in a 96-well-plate format that allows parallel testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD6 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred arachidonoyl glycerol isomer. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. The approach has major benefits compared to laborious traditional mass spectrometric methods and liquid scintillation-based assays, or approaches using unnatural substrates.

Quantification of diphtheria toxin mediated ADP-ribosylation in a solid-phase assay.

PubMed

Bachran, Christopher; Sutherland, Mark; Bachran, Diana; Fuchs, Hendrik

2007-09-01

Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT. Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

Analytical performances of a new enzymatic assay for hemoglobin A1c.

PubMed

Jaisson, Stéphane; Desmons, Aurore; Renard, Benoît; Chevelle, Benjamin; Leroy, Nathalie; Gillery, Philippe

2014-07-01

HbA1c is considered the gold standard for the follow-up of diabetic patients and a new diagnostic tool for diabetes mellitus, which implies the availability of reliable assay methods. We have evaluated a new assay developed by Abbott Laboratories, based on the enzymatic quantification of HbA1c by a fructosyl dipeptide oxidase using Architect analyzers. Precision, linearity, correlation with a HPLC method, accuracy and potential impact interferences on HbA1c measurement have been evaluated. Intra-day and between-day CVs were lower than 1.2% and linearity was excellent from 19 mmol/mol (3.9%) to 163 mmol/mol (17.1%). The results were well correlated with those obtained by the HPLC (Variant II device, kit NU - BioRad): HbA1c [Architect, mmol/mol]=0.986×HbA1c [Variant II, mmol/mol]+0.713 (r=0.998, n=109). This method provided consistent results with IFCC titrated quality control samples. Classical interferences in HbA1c assays (i.e. labile HbA1c, carbamylated hemoglobin, triglycerides or bilirubin) did not have an impact on HbA1c quantification by this method. This new enzymatic assay proved to be a robust and reliable method for HbA1c measurement suitable for routine practice in clinical chemistry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS[S

PubMed Central

Fang, Nianbai; Yu, Shanggong; Adams, Sean H.; Ronis, Martin J. J.; Badger, Thomas M.

2016-01-01

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets. PMID:27538824

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Colorimetric micro-assay for accelerated screening of mould inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2013-01-01

Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

A novel procedure to assess the non-enzymatic hydrogen-peroxide antioxidant capacity of metabolites with high UV absorption.

PubMed

Csepregi, Kristóf; Hideg, Éva

2016-12-01

Assays assessing non-enzymatic hydrogen peroxide antioxidant capacities are often hampered by the high UV absorption of the sample itself. This is a typical problem in studies using plant extracts with high polyphenol content. Our assay is based on comparing the 405 nm absorption of the product of potassium iodine and hydrogen peroxide in the presence and absence of a putative hydrogen peroxide reactive antioxidant. This method is free of interference with either hydrogen peroxide or antioxidant self-absorption and it is also suitable for high-throughput plate reader applications.

Comparison of the fluorescent polarization (TDx) and the enzymatic competitive (EMIT 2000) immune assays for the measurement of cyclosporin A blood concentration.

PubMed

David-Neto, E; Ballarati, C A; Freitas, O J; Lemos, F C; Nahas, W C; Arap, S; Kalil, J

2000-01-01

Evaluation of Cyclosporin A (CyA) blood concentration is imperative in solid organ transplantation in order to achieve maximal immunosuppression with the least side effects. We compared the results of whole blood concentrations of CyA in 50 blood samples simultaneously evaluated by the fluorescent polarization immune assay (TDx) and the enzymatic competitive immune assay (EMIT 2000). There was a strong correlation between both kits for any range of CyA blood concentration (R=0.99, p<0.001). The within-run and between-days coefficient of variation were less than 4% for both assays. The cost for each CyA measurement was 50% lower for the EMIT assay when compared to the TDx assay. We concluded that the EMIT is as accurate as the TDx in measuring CyA blood concentration and has the advantage of a lower cost, as well as the possibility of widespread access to the EMIT methodology in contrast to the TDx equipment, allowing the laboratory to perform several routines within a working day.

Galactomannan Enzymatic Immunoassay Cross-Reactivity Caused by Prototheca Species

PubMed Central

Van den Bossche, D.; Hendrickx, M.; De Becker, A.; Jacobs, R.; Naessens, A.; Piérard, D.

2012-01-01

We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis. PMID:22837317

Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

PubMed

Cozma, Claudia; Eichler, Sabrina; Wittmann, Gyula; Flores Bonet, Alba; Kramp, Guido Johannes; Giese, Anne-Katrin; Rolfs, Arndt

2015-01-01

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance. We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone. The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays. The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

PubMed

Krappmann, Michael; de Boer, Arjen R; Kool, Daniël R W; Irth, Hubertus; Letzel, Thomas

2016-04-30

Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated. Copyright © 2016 John Wiley & Sons, Ltd.

Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

2017-04-01

Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems for ship-borne measurements of physico-chemical parameters, such as the FLAMe (Crawford et al., 2015), paves new ground for data interpretation and process understanding. Cabral, J.P.S., 2010. Water Microbiology. Bacterial Pathogens and Water. Int. J. Environ. Res. Public. Health 7, 3657-3703. doi:10.3390/ijerph7103657 Crawford, J.T., Loken, L.C., Casson, N.J., Smith, C., Stone, A.G., Winslow, L.A., 2015. High-speed limnology: using advanced sensors to investigate spatial variability in biogeochemistry and hydrology. Environ. Sci. Technol. 49, 442-450. doi:10.1021/es504773x Farnleitner, A. h., Hocke, L., Beiwl, C., Kavka, G. c., Zechmeister, T., Kirschner, A. k. t., Mach, R. l., 2001. Rapid enzymatic detection of Escherichia coli contamination in polluted river water. Lett. Appl. Microbiol. 33, 246-250. doi:10.1046/j.1472-765x.2001.00990.x Farnleitner, A.H., Hocke, L., Beiwl, C., Kavka, G.G., Mach, R.L., 2002. Hydrolysis of 4-methylumbelliferyl-β-d-glucuronide in differing sample fractions of river waters and its implication for the detection of fecal pollution. Water Res. 36, 975-981. doi:10.1016/S0043-1354(01)00288-3 Ryzinska-Paier, G., Lendenfeld, T., Correa, K., Stadler, P., Blaschke, A.P., Mach, R.L., Stadler, H., Kirschner, A.K.T., Farnleitner, A.H., 2014. A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 69, 1349-1358. doi:10.2166/wst.2014.032 Stadler, P., Blöschl, G., Vogl, W., Koschelnik, J., Epp, M., Lackner, M., Oismüller, M., Kumpan, M., Nemeth, L., Strauss, P., Sommer, R., Ryzinska-Paier, G., Farnleitner, A.H., Zessner, M., 2016. Real-time monitoring of beta-d-glucuronidase activity in sediment laden streams: A comparison of prototypes. Water Res. 101, 252-261. doi:10.1016/j.watres.2016.05.072 Stadler, P., Farnleitner, A.H., Zessner, M., 2017. Development and evaluation of a self-cleaning custom-built auto sampler controlled by a low-cost RaspberryPi microcomputer for online enzymatic activity measurements. Talanta 162, 390-397. doi:10.1016/j.talanta.2016.10.031

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS.

PubMed

Fang, Nianbai; Yu, Shanggong; Adams, Sean H; Ronis, Martin J J; Badger, Thomas M

2016-10-01

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

In vitro stemness characterization of radio-resistant clones isolated from a medulloblastoma cell line ONS-76

PubMed Central

Sun, Lue; Moritake, Takashi; Zheng, Yun-Wen; Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Zenkoh, Junko; Taniguchi, Hideki; Tsuboi, Koji

2013-01-01

One-third of patients with medulloblastoma die due to recurrence after various treatments including radiotherapy. Although it has been postulated that cancer stem-like cells are radio-resistant and play an important role in tumor recurrence, the “stemness” of medulloblastoma cells surviving irradiation has not yet been elucidated. Using a medulloblastoma cell line ONS-76, cells that survived gamma irradiation were investigated on their “stemness” in vitro. From 10 500 cells, 20 radio-resistant clones were selected after gamma ray irradiation (5 Gy × two fractions) using the replica micro-well technique. These 20 resistant clones were screened for CD133 positivity by flow cytometry followed by side population assay, tumor sphere formation assay and clonogenic survival assay. Results revealed CD133 fractions were significantly elevated in three clones, which also exhibited significantly increased levels of tumor sphere formation ability and side population fraction. Clonogenic survival assay demonstrated that their radio-resistance was significantly higher than the parental ONS-76. This may support the hypothesis that a small number of cancer stem-like cells (CSCs) are the main culprits in local recurrence after radiotherapy, and disruption of the resistance mechanism of these CSCs is a critical future issue in improving the outcome of patients with medulloblastoma. PMID:22951319

Phylogenetic analysis of platelet-derived growth factor by radio- receptor assay

PubMed Central

1982-01-01

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo. PMID:7142300

Correlation between radio-induced lymphocyte apoptosis measurements obtained from two French centres.

PubMed

Mirjolet, C; Merlin, J L; Dalban, C; Maingon, P; Azria, D

2016-07-01

In the era of modern treatment delivery, increasing the dose delivered to the target to improve local control might be modulated by the patient's intrinsic radio-sensitivity. A predictive assay based on radio-induced lymphocyte apoptosis quantification highlighted the significant correlation between CD4 and CD8 T-lymphocyte apoptosis and grade 2 or 3 radiation-induced late toxicities. By conducting this assay at several technical platforms, the aim of this study was to demonstrate that radio-induced lymphocyte apoptosis values obtained from two different platforms were comparable. For 25 patients included in the PARATOXOR trial running in Dijon the radio-induced lymphocyte apoptosis results obtained from the laboratory of Montpellier (IRCM, Inserm U1194, France), considered as the reference (referred to as Lab 1), were compared with those from the laboratory located at the Institut de cancérologie de Lorraine (ICL, France), referred to as Lab 2. Different statistical methods were used to measure the agreement between the radio-induced lymphocyte apoptosis data from the two laboratories (quantitative data). The Bland-Altman plot was used to identify potential bias. All statistical tests demonstrated good agreement between radio-induced lymphocyte apoptosis values obtained from both sites and no major bias was identified. Since radio-induced lymphocyte apoptosis values, which predict tolerance to radiotherapy, could be assessed by two laboratories and showed a high level of robustness and consistency, we can suggest that this assay be extended to any laboratories that use the same technique. Copyright © 2016 Société française de radiothérapie oncologique (SFRO). Published by Elsevier SAS. All rights reserved.

Genetic analysis--a diagnostic tool for primary hyperoxaluria type I.

PubMed

Milosevic, Danko; Rinat, Choni; Batinic, Danica; Frishberg, Yaacov

2002-11-01

Primary hyperoxaluria type I is an autosomal recessive metabolic disease in which excessive oxalates are formed by the liver and excreted by the kidneys, causing a wide spectrum of disease, ranging from renal failure in infancy to mere renal stones in late adulthood. The diagnosis may be suspected when clinical signs and increased urinary oxalate and glycolate excretion present, and is confirmed by the measurement of decreased alanine:glyoxylate aminotransferase activity in a liver sample. The enzymatic assay is not readily available to pediatric nephrologists in many parts of the world. We describe three families from Croatia in whom the diagnosis of primary hyperoxaluria was solely based on clinical findings that included nephrolithiasis and nephrocalcinosis accompanied by increased urinary oxalates and glycolate excretion, as enzymatic assays of liver samples could not be performed. Mutation analysis of the AGXT gene encoding the defective enzyme confirmed the diagnosis, revealing three alleles carrying the C156ins mutation and two the G630A mutation. Screening first-degree relatives for the relevant mutation disclosed an asymptomatic affected sibling. Mutation analysis of the AGXT gene is a non-invasive and accurate tool for the diagnosis of type I primary hyperoxaluria that may replace enzymatic assays of liver biopsies.

A simple and rapid microplate assay for glycoprotein-processing glycosidases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, M.S.; Zwolshen, J.H.; Harry, B.S.

1989-08-15

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates (( 3H)glucose for glucosidases and (3H)mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported. These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. Thismore » procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.« less

PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

PubMed Central

2015-01-01

Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter. PMID:25634295

Chronic fatigue syndrome: exercise performance related to immune dysfunction.

PubMed

Nijs, Jo; Meeus, Mira; McGregor, Neil R; Meeusen, Romain; de Schutter, Guy; van Hoof, Elke; de Meirleir, Kenny

2005-10-01

To date, the exact cause of abnormal exercise response in chronic fatigue syndrome (CFS) remains to be revealed, but evidence addressing intracellular immune deregulation in CFS is growing. Therefore, the aim of this cross-sectional study was to examine the interactions between several intracellular immune variables and exercise performance in CFS patients. After venous blood sampling, subjects (16 CFS patients) performed a maximal exercise stress test on a bicycle ergometer with continuous monitoring of cardiorespiratory variables. The following immune variables were assessed: the ratio of 37 kDa Ribonuclease (RNase) L to the 83 kDa native RNase L (using a radiolabeled ligand/receptor assay), RNase L enzymatic activity (enzymatic assay), protein kinase R activity assay (comparison Western blot), elastase activity (enzymatic-colorimetric assay), the percent of monocytes, and nitric oxide determination (for monocytes and lymphocytes; flow cytometry, live cell assay). Forward stepwise multiple regression analysis revealed 1) that elastase activity was the only factor related to the reduction in oxygen uptake at a respiratory exchange ratio (RER) of 1.0 (regression model: R = 0.53, F (1,14) = 15.5, P < 0.002; elastase activity P < 0.002); 2) that the protein kinase R activity was the principle factor related to the reduction in workload at RER = 1.0; and 3) that elastase activity was the principle factor related to the reduction in percent of target heart rate achieved. These data provide evidence for an association between intracellular immune deregulation and exercise performance in patients with CFS. To establish a causal relationship, further study of these interactions using a prospective longitudinal design is required.

Enzymatic Formulation Capable of Degrading Scrapie Prion under Mild Digestion Conditions

PubMed Central

Okoroma, Emeka A.; Purchase, Diane; Garelick, Hemda; Morris, Roger; Neale, Michael H.; Windl, Otto; Abiola, Oduola O.

2013-01-01

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrPSc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrPSc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrPSc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrPSc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions. PMID:23874511

Fingerprint Analysis: Moving Toward Multiattribute Determination via Individual Markers.

PubMed

Brunelle, Erica; Huynh, Crystal; Alin, Eden; Eldridge, Morgan; Le, Anh Minh; Halámková, Lenka; Halámek, Jan

2018-01-02

Forensic science will be forever revolutionized if law enforcement can identify personal attributes of a person of interest solely from a fingerprint. For the past 2 years, the goal of our group has been to establish a way to identify originator attributes, specifically biological sex, from a single analyte. To date, an enzymatic assay and two chemical assays have been developed for the analysis of multiple analytes. In this manuscript, two additional assays have been developed. This time, however, the assays utilize only one amino acid each. The enzymatic assay targets alanine and employs alanine transaminase (ALT), pyruvate oxidase (POx), and horseradish peroxidase (HRP). The other, a chemical assay, is known as the Sakaguchi test and targets arginine. It is important to note that alanine has a significantly higher concentration than arginine in the fingerprint content of both males and females. Both assays proved to be capable of accurately differentiating between male and female fingerprints, regardless of their respective average concentration. The ability to target a single analyte will transform forensic science as each originator attribute can be correlated to a different analyte. This would then lead to the possibility of identifying multiple attributes from a single fingerprint sample. Ultimately, this would allow for a profile of a person of interest to be established without the need for time-consuming lab processes.

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

PubMed

Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

1993-08-01

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Rapid Analysis of Nonstructural Carbohydrate Components in Grass Forage Using Microplate Enzymatic Assays

USDA-ARS?s Scientific Manuscript database

Measurements of nonstructural carbohydrates (NSC) in plant tissues are important to estimate plant organ resources available for plant growth and stress tolerance or for feed value to grazing animals. A popular commercially available assay kit used to detect glucose with a light sensitive dye reacti...

Epoxide hydrolases: structure, function, mechanism, and assay.

PubMed

Arand, Michael; Cronin, Annette; Adamska, Magdalena; Oesch, Franz

2005-01-01

Epoxide hydrolases are a class of enzymes important in the detoxification of genotoxic compounds, as well as in the control of physiological signaling molecules. This chapter gives an overview on the function, structure, and enzymatic mechanism of structurally characterized epoxide hydrolases and describes selected assays for the quantification of epoxide hydrolase activity.

RAPID TETRAZOLIUM DYE REDUCTION ASSAY TO ASSESS THE BACTERICIDAL ACTIVITY OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES AGAINST VIBRIO PARAHAEMOLYTICUS

EPA Science Inventory

An assay was developed to assess the ability of oyster, Crassostrea virginica, hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethyl...

Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase - Degradation of compounds does not always eliminate toxicity.

PubMed

Becker, Dennis; Varela Della Giustina, Saulo; Rodriguez-Mozaz, Sara; Schoevaart, Rob; Barceló, Damià; de Cazes, Matthias; Belleville, Marie-Pierre; Sanchez-Marcano, José; de Gunzburg, Jean; Couillerot, Olivier; Völker, Johannes; Oehlmann, Jörg; Wagner, Martin

2016-11-01

In this study, the performance of immobilised laccase (Trametes versicolor) was investigated in combination with the mediator syringaldehyde (SYR) in removing a mixture of 38 antibiotics in an enzymatic membrane reactor (EMR). Antibiotics were spiked in osmosed water at concentrations of 10μg·L(-1) each. Laccase without mediator did not reduce the load of antibiotics significantly. The addition of SYR enhanced the removal: out of the 38 antibiotics, 32 were degraded by >50% after 24h. In addition to chemical analysis, the samples' toxicity was evaluated in two bioassays (a growth inhibition assay and the Microtox assay). Here, the addition of SYR resulted in a time-dependent increase of toxicity in both bioassays. In cooperation with SYR, laccase effectively removes a broad range of antibiotics. However, this enhanced degradation induces unspecific toxicity. If this issue is resolved, enzymatic treatment may be a valuable addition to existing water treatment technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic analysis of α-ketoglutaramate—A biomarker for hyperammonemia

PubMed Central

Halámková, Lenka; Mailloux, Shay; Halámek, Jan; Cooper, Arthur J.L.; Katz, Evgeny

2012-01-01

Two enzymatic assays were developed for the analysis of α-ketoglutaramate (KGM)—an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to α-ketoglutarate (KTG) via a reaction catalyzed by ω-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (λ=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids. PMID:23141304

Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s bearing functionalizable carbonate building blocks: II. Enzymatic biodegradation and in vitro biocompatibility assay.

PubMed

Yang, Jing; Tian, Weisheng; Li, Qiaobo; Li, Yang; Cao, Amin

2004-01-01

In a previous study, we have reported chemical synthesis of novel aliphatic poly(butylene succinate-co-cyclic carbonate) P(BS-co-CC)s bearing various functionalizable carbonate building blocks, and this work will continue to present our new studies on their enzymatic degradation and in vitro cell biocompatibility assay. First, enzymatic degradation of the novel P(BS-co-CC) film samples was investigated with two enzymes of lipase B Candida Antartic (Novozyme 435) and lipase Porcine Pancreas PPL, and it was revealed that copolymerizing linear poly(butylene succinate) PBS with a functionalizable carbonate building block could remarkably accelerate the enzymatic degradation of a synthesized product P(BS-co-CC), and its biodegradation behavior was found to strongly depend on the overall impacts of several important factors as the cyclic carbonate (CC) comonomer structure and molar content, molar mass, thermal characteristics, morphology, the enzyme-substrate specificity, and so forth. Further, the biodegraded residual film samples and water-soluble enzymatic degradation products were allowed to be analyzed by means of proton nuclear magnetic resonance (1H NMR), gel permeation chromatograph (GPC), differential scanning calorimeter (DSC), attenuated total reflection FTIR (ATR-FTIR), scanning electron microscope (SEM), and liquid chromatograph-mass spectrometry (LC-MS). On the experimental evidences, an exo-type mechanism of enzymatic chain hydrolysis preferentially occurring in the noncrystalline domains was suggested for the synthesized new P(BS-co-CC) film samples. With regard to their cell biocompatibilities, an assay with NIH 3T3 mouse fibroblast cell was conducted using the novel synthesized P(BS-co-CC) films as substrates with respect to the cell adhesion and proliferation, and these new biodegradable P(BS-co-CC) samples were found to exhibit as low cell toxicity as the PLLA control, particularly the two samples of poly(butylene succinate-co-18.7 mol % dimethyl trimethylene carbonate) P(BS-co-18.7 mol % DMTMC) and poly(butylene succinate-co-21.9 mol % 5-benzyloxy trimethylene carbonate) P(BS-co-21.9 mol % BTMC) were interestingly found to show much better cell biocompatibilities than the PLLA reference.

Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda

PubMed Central

2013-01-01

Background The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. Methods The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. Results The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. Conclusions The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation. PMID:23782846

Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda.

PubMed

De Niz, Mariana; Eziefula, Alice C; Othieno, Lucas; Mbabazi, Edith; Nabukeera, Damalie; Ssemmondo, Emmanuel; Gonahasa, Samuel; Tumwebaze, Patrick; Diliberto, Deborah; Maiteki-Sebuguzi, Catherine; Staedke, Sarah G; Drakeley, Chris

2013-06-19

The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

Determination of dietary starch in animal feeds and pet food by an enzymatic-colorimetric method: collaborative study.

PubMed

Hall, Mary Beth

2015-01-01

Starch, glycogen, maltooligosaccharides, and other α-1,4- and α-1,6-linked glucose carbohydrates, exclusive of resistant starch, are collectively termed "dietary starch". This nutritionally important fraction is increasingly measured for use in diet formulation for animals as it can have positive or negative effects on animal performance and health by affecting energy supply, glycemic index, and formation of fermentation products by gut microbes. AOAC Method 920.40 that was used for measuring dietary starch in animal feeds was invalidated due to discontinued production of a required enzyme. As a replacement, an enzymatic-colorimetric starch assay developed in 1997 that had advantages in ease of sample handling and accuracy compared to other methods was considered. The assay was further modified to improve utilization of laboratory resources and reduce time required for the assay. The assay is quasi-empirical: glucose is the analyte detected, but its release is determined by run conditions and specification of enzymes. The modified assay was tested in an AOAC collaborative study to evaluate its accuracy and reliability for determination of dietary starch in animal feedstuffs and pet foods. In the assay, samples are incubated in screw cap tubes with thermostable α-amylase in pH 5.0 sodium acetate buffer for 1 h at 100°C with periodic mixing to gelatinize and partially hydrolyze α-glucan. Amyloglucosidase is added, and the reaction mixture is incubated at 50°C for 2 h and mixed once. After subsequent addition of water, mixing, clarification, and dilution as needed, free + enzymatically released glucose are measured. Values from a separate determination of free glucose are subtracted to give values for enzymatically released glucose. Dietary starch equals enzymatically released glucose multiplied by 162/180 (or 0.9) divided by the weight of the as received sample. Fifteen laboratories that represented feed company, regulatory, research, and commercial feed testing laboratories analyzed 10 homogenous test materials representing animal feedstuffs and pet foods in duplicate using the dietary starch assay. The test samples ranged from 1 to 70% in dietary starch content and included moist canned dog food, alfalfa pellets, distillers grains, ground corn grain, poultry feed, low starch horse feed, dry dog kibbles, complete dairy cattle feed, soybean meal, and corn silage. The average within-laboratory repeatability SD (sr) for percentage dietary starch in the test samples was 0.49 with a range of 0.03 to 1.56, and among-laboratory repeatability SDs (sR) averaged 0.96 with a range of 0.09 to 2.69. The HorRat averaged 2.0 for all test samples and 1.9 for test samples containing greater than 2% dietary starch. The HorRat results are comparable to those found for AOAC Method 996.11, which measures starch in cereal products. It is recommended that the dietary starch method be accepted for Official First Action status.

Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

PubMed

Skandrani, Ines; Bouhlel, Ines; Limem, Ilef; Boubaker, Jihed; Bhouri, Wissem; Neffati, Aicha; Ben Sghaier, Mohamed; Kilani, Soumaya; Ghedira, Kamel; Ghedira-Chekir, Leila

2009-02-01

The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction.

Commutability of NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum with selected total homocysteine immunoassays and enzymatic assays.

PubMed

Nelson, Bryant C; Pfeiffer, Christine M; Zhang, Ming; Duewer, David L; Sharpless, Katherine E; Lippa, Katrice A

2008-09-01

The National Institute of Standards and Technology (NIST) has recently developed Standard Reference Material (SRM) 1955 Homocysteine and Folate in Frozen Human Serum with certified values for total homocysteine (tHcy) and 5-methyl-tetrahydrofolic acid. NIST has performed an international, interlaboratory assessment of SRM 1955 commutability; results are reported for tHcy only. Total Hcy was measured in 20 patient sera and in 3 levels of SRM 1955 using 14 immunoassays and/or enzymatic assays. Liquid chromatography/tandem mass spectrometry was utilized as the reference assay. An "errors-in-variables" statistical model was utilized to assess the commutability of SRM 1955. Normalized residuals ranged from -2.65 to 2.19 for SRM 1955. The median interlaboratory/interassay imprecision (CV) was approximately 4% for patient specimens and ranged from approximately 3% to approximately 7% for SRM 1955. The median intra-assay imprecision ranged from approximately 1% to approximately 13%. Orthogonal residuals, as a descriptor of assay accuracy, ranged from 0.29 to 7.71 and from 0.20 to 2.22 for patient specimens and SRM 1955 samples, respectively. The current study suggests that SRM 1955 is commutable with the investigated tHcy assays; however, a broader specimen set needs to be evaluated to completely substantiate this conclusion.

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

PubMed Central

Salas-Sarduy, Emir; Guerra, Yasel; Covaleda Cortés, Giovanni; Avilés, Francesc Xavier; Chávez Planes, María A.

2017-01-01

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented. PMID:28430158

Application of chemical arrays in screening elastase inhibitors.

PubMed

Gao, Feng; Du, Guan-Hua

2006-06-01

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

PubMed

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de Los Angeles; Kirchmayr, Manuel R; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim; Rodríguez, Jorge A

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl- sn -glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

PubMed Central

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de los Angeles; Kirchmayr, Manuel R.; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology. PMID:28695068

[Assays of HbA1c and Amadori products in human biology].

PubMed

Gillery, P

2014-09-01

Different Amadori products, formed during the early steps of the non-enzymatic glycation of proteins, may be assayed in current practice in human biology. The most important marker is HbA1c, resulting from the binding of glucose to the N-terminal extremity of HbA beta chains. HbA1c may be evaluated by various techniques (ion exchange or affinity high performance liquid chromatography, capillary electrophoresis, immunoassay, enzymatic technique) and is considered the best marker of diabetic patient survey. Due to its irreversible and cumulative formation, it provides a retrospective information on the glycemic balance over the four to eight weeks preceding blood collection. It benefits from an international standardization, based on a reference method using liquid chromatography coupled to capillary electrophoresis or mass spectrometry, maintained by an international network of reference laboratories. When HbA1c assay cannot be used (anemia, hemolysis, hemoglobinopathy) or when a shorter period of glycemic equilibrium must be evaluated (child and adolescent, pregnancy, therapeutic changes), other Amadori products may be assayed, like plasma fructosamine (all plasma glycated proteins) or glycated albumin. Nevertheless, these assays are less used in practice, because their semiological value has been less evidenced. Besides, fructosamine assay lacks specificity, and glycated albumin assay has been described recently. An expanding use of HbA1c assay is expected, especially for the diagnosis of diabetes mellitus and the evaluation of other risks, especially cardiovascular ones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Radio-methyl vorozole and methods for making and using the same

DOEpatents

Kim, Sung Won; Biegon, Anat; Fowler, Joanna S.

2014-08-12

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

Radio-methyl vorozole and methods for making and using the same

DOEpatents

Kim, Sung Won; Biegon, Anat; Fowler, Joanna S.

2014-08-05

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

NASA Astrophysics Data System (ADS)

Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

2001-12-01

Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans.

PubMed

Georgieva, Mariyana; Rashydov, Namik M; Hajduch, Martin

2017-02-01

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzyme catalysis-electrophoresis titration for multiplex enzymatic assay via moving reaction boundary chip.

PubMed

Zhong, Ran; Xie, Haiyang; Kong, Fanzhi; Zhang, Qiang; Jahan, Sharmin; Xiao, Hua; Fan, Liuyin; Cao, Chengxi

2016-09-21

In this work, we developed the concept of enzyme catalysis-electrophoresis titration (EC-ET) under ideal conditions, the theory of EC-ET for multiplex enzymatic assay (MEA), and a related method based on a moving reaction boundary (MRB) chip with a collateral channel and cell phone imaging. As a proof of principle, the model enzymes horseradish peroxidase (HRP), laccase and myeloperoxidase (MPO) were chosen for the tests of the EC-ET model. The experiments revealed that the EC-ET model could be achieved via coupling EC with ET within a MRB chip; particularly the MEA analyses of catalysis rate, maximum rate, activity, Km and Kcat could be conducted via a single run of the EC-ET chip, systemically demonstrating the validity of the EC-ET theory. Moreover, the developed method had these merits: (i) two orders of magnitude higher sensitivity than a fluorescence microplate reader, (ii) simplicity and low cost, and (iii) fairly rapid (30 min incubation, 20 s imaging) analysis, fair stability (<5.0% RSD) and accuracy, thus validating the EC-ET method. Finally, the developed EC-ET method was used for the clinical assay of MPO activity in blood samples; the values of MPO activity detected via the EC-ET chip were in agreement with those obtained by a traditional fluorescence microplate reader, indicating the applicability of the EC-ET method. The work opens a window for the development of enzymatic research, enzyme assay, immunoassay, and point-of-care testing as well as titration, one of the oldest methods of analysis, based on a simple chip.

Radio-protective effect of cinnamic acid, a phenolic phytochemical, on genomic instability induced by X-rays in human blood lymphocytes in vitro.

PubMed

Cinkilic, Nilufer; Tüzün, Ece; Çetintaş, Sibel Kahraman; Vatan, Özgür; Yılmaz, Dilek; Çavaş, Tolga; Tunç, Sema; Özkan, Lütfi; Bilaloğlu, Rahmi

2014-08-01

The present study was designed to determine the protective activity of cinnamic acid against induction by X-rays of genomic instability in normal human blood lymphocytes. This radio-protective activity was assessed by use of the cytokinesis-block micronucleus test and the alkaline comet assay, with human blood lymphocytes isolated from two healthy donors. A Siemens Mevatron MD2 (Siemens AG, USA, 1994) linear accelerator was used for the irradiation with 1 or 2 Gy. Treatment of the lymphocytes with cinnamic acid prior to irradiation reduced the number of micronuclei when compared with that in control samples. Treatment with cinnamic acid without irradiation did not increase the number of micronuclei and did not show a cytostatic effect in the lymphocytes. The results of the alkaline comet assay revealed that cinnamic acid reduces the DNA damage induced by X-rays, showing a significant radio-protective effect. Cinnamic acid decreased the frequency of irradiation-induced micronuclei by 16-55% and reduced DNA breakage by 17-50%, as determined by the alkaline comet assay. Cinnamic acid may thus act as a radio-protective compound, and future studies may focus on elucidating the mechanism by which cinnamic acid offers radioprotection. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of mobile phone radiation (900 MHz radiofrequency) on structure and functions of rat brain.

PubMed

Saikhedkar, Nidhi; Bhatnagar, Maheep; Jain, Ayushi; Sukhwal, Pooja; Sharma, Chhavi; Jaiswal, Neha

2014-12-01

The goals of this study were: (1) to obtain basic information about the effects of long-term use of mobile phones on cytological makeup of the hippocampus in rat brains (2) to evaluate the effects on antioxidant status, and (3) to evaluate the effects on cognitive behavior particularly on learning and memory. Rats (age 30 days, 120 ± 5 g) were exposed to 900 MHz radio waves by means of a mobile hand set for 4 hours per day for 15 days. Effects on anxiety, spatial learning, and memory were studied using the open field test, the elevated plus maze, the Morris water maze (MWM), and the classic maze test. Effects on brain antioxidant status were also studied. Cresyl violet staining was done to assess the neuronal damage. A significant change in behavior, i.e., more anxiety and poor learning was shown by test animals as compared to controls and sham group. A significant change in the level of antioxidant enzymes and non-enzymatic antioxidants, and an increase in lipid peroxidation were observed in the test rats. Histological examination showed neurodegenerative cells in hippocampal sub regions and the cerebral cortex. Thus our findings indicate extensive neurodegeneration on exposure to radio waves. Increased production of reactive oxygen species due to exhaustion of enzymatic and non-enzymatic antioxidants and increased lipid peroxidation indicate extensive neurodegeneration in selective areas of CA1, CA3, DG, and the cerebral cortex. This extensive neuronal damage results in alterations in behavior related to memory and learning.

A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.

PubMed

Gräber, Martin; Andersson, Mats; Rundbäck, Fabian; Pozzo, Tania; Karlsson, Eva Nordberg; Adlercreutz, Patrick

2010-01-15

The present work describes the development of a novel direct screening method, assayed in 96-well format, for evaluation of enzymatic alkyl glycoside production in a hexanol-water two-phase system. Alkyl glycosides are surfactants with a range of applications and with good biodegradability and low toxicity. Enzymatic synthesis makes it possible to prepare beta-d-glucopyranosides with high purity. In the developed screening assay, hexyl-beta-d-glucopyranoside was chosen as a model product to be synthesised by reversed hydrolysis in a water-hexanol two-phase system. In a first step the model product is produced by glucosidases expressed in E. coli cells in 96-deep-well plates. After phase separation, the hexyl-beta-d-glucopyranoside in the organic phase is degraded enzymatically and the released glucose detected spectrophotometrically at 405nm utilizing peroxidase/glucose oxidase, and the reagent 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The aqueous phase is used to monitor hydrolysis of p-NPG at 405nm, allowing use of a ratio of the two assays to compensate for expression differences. The complete method was used for comparison of two different beta-glucosidases, classified under glycoside hydrolase family 1 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-beta-d-glucopyranoside by reversed hydrolysis.

"Reagent-free" L-asparaginase activity assay based on CD spectroscopy and conductometry.

PubMed

Kudryashova, Elena V; Sukhoverkov, Kirill V

2016-02-01

A new method to determine the catalytic parameters of L-asparaginase using circular dichroism spectroscopy (CD spectroscopy) has been developed. The assay is based on the difference in CD signal between the substrate (L-asparagine) and the product (L-aspartic acid) of enzymatic reaction. CD spectroscopy, being a direct method, enables continuous measurement, and thus differentiates from multistage and laborious approach based on Nessler's method, and overcomes limitations of conjugated enzymatic reaction methods. In this work, we show robust measurements of L-asparaginase activity in conjugates with PEG-chitosan copolymers, which otherwise would not have been possible. The main limitation associated with the CD method is that the analysis should be performed at substrate saturation conditions (V max regime). For K M measurement, the conductometry method is suggested, which can serve as a complimentary method to CD spectroscopy. The activity assay based on CD spectroscopy and conductometry was successfully implicated to examine the catalytic parameters of L-asparaginase conjugates with chitosan and its derivatives, and for optimization of the molecular architecture and composition of such conjugates for improving biocatalytic properties of the enzyme in the physiological conditions. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not enable direct measurement with absorption or fluorescence spectroscopy. This may include a number of amino acid or glycoside-transforming enzymes.

Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culman, J.; Torda, T.; Weise, V.K.

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments ofmore » other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.« less

Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

PubMed

Yakubu, S I; Yakasai, I A; Musa, A

2011-06-01

The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

Profiling 976 ToxCast Chemicals across 331 Enzymatic and Receptor Signaling Assays

PubMed Central

2013-01-01

Understanding potential health risks is a significant challenge due to the large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes 976 chemicals (including failed pharmaceuticals, alternative plasticizers, food additives, and pesticides) in Phases I and II of the U.S. EPA’s ToxCast project across 331 cell-free enzymatic and ligand-binding high-throughput screening (HTS) assays. Half-maximal activity concentrations (AC50) were identified for 729 chemicals in 256 assays (7,135 chemical–assay pairs). Some of the most commonly affected assays were CYPs (CYP2C9 and CYP2C19), transporters (mitochondrial TSPO, norepinephrine, and dopaminergic), and GPCRs (aminergic). Heavy metals, surfactants, and dithiocarbamate fungicides showed promiscuous but distinctly different patterns of activity, whereas many of the pharmaceutical compounds showed promiscuous activity across GPCRs. Literature analysis confirmed >50% of the activities for the most potent chemical–assay pairs (54) but also revealed 10 missed interactions. Twenty-two chemicals with known estrogenic activity were correctly identified for the majority (77%), missing only the weaker interactions. In many cases, novel findings for previously unreported chemical–target combinations clustered with known chemical–target interactions. Results from this large inventory of chemical–biological interactions can inform read-across methods as well as link potential targets to molecular initiating events in adverse outcome pathways for diverse toxicities. PMID:23611293

The Effect of Phytase on the Oxygen Isotope Composition of Phosphate

NASA Astrophysics Data System (ADS)

von Sperber, C.; Tamburini, F.; Bernasconi, S. M.; Frossard, E.

2013-12-01

Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi) (1-2). These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. The enzymatic hydrolysis leads, via a nucleophilic attack, to the incorporation of one oxygen atom from the water into the newly formed Pi molecule. During the incorporation, an isotopic fractionation occurs, which might be used to identify the origin of Pi in the environment (3-6). While the effect of phosphomonoesterases and phosphodiesterases on the oxygen isotope composition of phosphate has been examined, there are, so far, no studies dealing with the effect of phytases (4-6). Phytases catalyze the hydrolysis of myo-inositol-hexakis-phosphate (IP6), which is an important component of organic P in many ecosystems (7). Enzymatic assays with phytase from wheat germ and Aspergillus niger were prepared under sterile and temperature controlled conditions in order to determine the effect of phytases on the oxygen isotope composition of phosphate, which has been liberated from IP6 via enzymatic hydrolysis. Assays with phytase from wheat germ lead to a turnover of the substrate close to 100%, while assays with phytase from Aspergillus niger lead to a turnover of the substrate close to 80%. In the case of the assays with phytase from wheat germ, our results indicate that one sixth of the total 24 oxygen which are associated to the phosphates in IP6 are exchanged with oxygen from water. From this we conclude that the incorporation of one oxygen atom from water occurs only at four phosphate molecules of IP6, while two phosphate molecules do not experience an incorporation of oxygen. This suggests that during the enzymatic hydrolysis, four P-O bonds and two C-O bonds are broken. Provided that, the isotopic fractionation can be calculated with an isotopic mass balance resulting in -8.4‰ (×3.6 SD). This is a value very similar to those reported for acid phosphatases (6). In contrast, the results from assays with phytase from Aspergillus niger indicate that the exchange of oxygen occurs at more than one third of the total 24 oxygen which are associated to the phosphates in IP6. In addition, we observe a change in the oxygen isotope composition of Pi when using myo-inositol and potassium-dihydrogen-phosphate as sole substrates in the enzymatic assays with phytase from Aspergillus niger. These observations suggest that the reformation of IP6 from the two products of the reaction (myo-inositol and Pi) is taking place at a rate, which is within the time scale of the experiment. In this case, the isotopic fractionation caused by phytase from Aspergillus niger will be determined by the equilibrium of the reaction. Further experiments are in process to verify these findings.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

PubMed

Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

2018-01-01

Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

PubMed

Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

2015-10-01

Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

PubMed

Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

2015-06-01

An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains

DTIC Science & Technology

2012-06-01

trifluoroacetic acid; Tm: melting temperature; TMB, 3,3′,5,5′-tetramethylbenzidine; UPLC , ultra performance liquid chromatography; VAMP, vesicle...activity determination by UPLC . Alternately, in large-scale preparations, phosphoryla- tion reaction was stopped by removing the Src with sepharose beads...peptides. ENZYMATIC ACTIVITY ASSAYS Activity assays were based on UPLC separation and measurement of the cleaved products from a 17-residue SNAP-25

Drug Development and Conservation of Biodiversity in West and Central Africa: Performance of Neurochemical and Radio Receptor Assays of Plant Extracts Drug Discovery for the Central Nervous System

DTIC Science & Technology

2004-09-01

7) Hui, D.; Sao-Xing, C. J. Nat. Prod. 1998, 61, 142-144. (8) Aldrich Libray of 13C and 1H FT NMR spectra 1992, 2, 326A. (9) Kadota, S .; Hui, D...Biodiversity in West and Central Africa: Performance of Neurochemical and Radio Receptor Assays of Plant Extracts Drug Discovery for the Central... s ) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation

Effects of different radio-opacifying agents on physicochemical and biological properties of a novel root-end filling material

PubMed Central

Lü, Xiao-Ying; Liu, Gen-Di

2018-01-01

Background/Purpose Radio-opacity is an essential attribute of ideal root-end filling materials because it is important for clinicians to observe root canal filling and to facilitate the follow-up instructions. The novel root-end filling material (NRFM) has good cytocompatibility and physicochemical properties but low intrinsic radio-opacity value. To improve its radio-opacity value, three novel radio-opaque root-end filling materials (NRRFMs) were developed by adding barium sulphate (NRFM-Ba), bismuth trioxide (NRFM-Bi) and zirconium dioxide (NRFM-Zr) to NRFM, respectively. The purpose of this study was to identify the suitable radio-opacifier for NRFM through evaluating their physicochemical and biological properties, in comparison with NRFM and glass ionomer cement (GIC). Methods NRRFMs were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrophotometry (FTIR). Physicochemical properties including setting time, compressive strength, porosity, pH variation, solubility, washout resistance, contact angle and radiopacity were investigated. Cytocompatibility of both freshly mixed and set NRRFMs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Alkaline phosphatase (ALP) activity assay and alizarin red staining were used to investigate the osteogenic differentiation potential of NRFM-Zr. Data were analyzed using two-way ANOVA (pH variation, solubility and ALP activity) and one-way ANOVA (for the other variables). Results (1) NRRFMs were primarily composed of hydroxyapatite, calcium carboxylate salt and the corresponding radio-opacity agents (barium sulphate, bismuth trioxide or zirconium dioxide). (2) Besides similar physicochemical properties in terms of setting time, pH variation, solubility, washout resistance and contact angle to NRFM, NRFM-Bi and NRFM-Zr exhibited lower porosity and greater compressive strength after being set for 7 days and their radio-opacity were greater than the 3 mm aluminium thickness specified in ISO 6876 (2001). (3) MTT assay revealed that freshly mixed and set NRFM-Zr presented better cell viability than NRFM-Ba and NRFM-Bi at 24 hours and 48 hours (P<0.05). (4) NRFM-Zr significantly enhanced ALP activity and calcium formation of human osteoblast-like Saos-2 cells when compared with negative group and GIC (P<0.05). Conclusion NRFM-Zr presents desirable physicochemical and biological properties, thus zirconium dioxide may be a suitable radio-opacifier for NRFM. PMID:29420559

Effects of different radio-opacifying agents on physicochemical and biological properties of a novel root-end filling material.

PubMed

Chen, Yao-Zhong; Lü, Xiao-Ying; Liu, Gen-Di

2018-01-01

Radio-opacity is an essential attribute of ideal root-end filling materials because it is important for clinicians to observe root canal filling and to facilitate the follow-up instructions. The novel root-end filling material (NRFM) has good cytocompatibility and physicochemical properties but low intrinsic radio-opacity value. To improve its radio-opacity value, three novel radio-opaque root-end filling materials (NRRFMs) were developed by adding barium sulphate (NRFM-Ba), bismuth trioxide (NRFM-Bi) and zirconium dioxide (NRFM-Zr) to NRFM, respectively. The purpose of this study was to identify the suitable radio-opacifier for NRFM through evaluating their physicochemical and biological properties, in comparison with NRFM and glass ionomer cement (GIC). NRRFMs were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrophotometry (FTIR). Physicochemical properties including setting time, compressive strength, porosity, pH variation, solubility, washout resistance, contact angle and radiopacity were investigated. Cytocompatibility of both freshly mixed and set NRRFMs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Alkaline phosphatase (ALP) activity assay and alizarin red staining were used to investigate the osteogenic differentiation potential of NRFM-Zr. Data were analyzed using two-way ANOVA (pH variation, solubility and ALP activity) and one-way ANOVA (for the other variables). (1) NRRFMs were primarily composed of hydroxyapatite, calcium carboxylate salt and the corresponding radio-opacity agents (barium sulphate, bismuth trioxide or zirconium dioxide). (2) Besides similar physicochemical properties in terms of setting time, pH variation, solubility, washout resistance and contact angle to NRFM, NRFM-Bi and NRFM-Zr exhibited lower porosity and greater compressive strength after being set for 7 days and their radio-opacity were greater than the 3 mm aluminium thickness specified in ISO 6876 (2001). (3) MTT assay revealed that freshly mixed and set NRFM-Zr presented better cell viability than NRFM-Ba and NRFM-Bi at 24 hours and 48 hours (P<0.05). (4) NRFM-Zr significantly enhanced ALP activity and calcium formation of human osteoblast-like Saos-2 cells when compared with negative group and GIC (P<0.05). NRFM-Zr presents desirable physicochemical and biological properties, thus zirconium dioxide may be a suitable radio-opacifier for NRFM.

Same ammo, different weapons: enzymatic extracts from two apple genotypes with contrasted susceptibilities to fire blight (Erwinia amylovora) differentially convert phloridzin and phloretin in vitro.

PubMed

Gaucher, Matthieu; Dugé de Bernonville, Thomas; Guyot, Sylvain; Dat, James F; Brisset, Marie-Noëlle

2013-11-01

The necrogenic bacterium Erwinia amylovora responsible for the fire blight disease causes cell death in apple tissues to enrich intercellular spaces with nutrients. Apple leaves contain large amounts of dihydrochalcones (DHCs), including phloridzin and its aglycone phloretin. Previous work showed an important decrease in the constitutive DHCs stock in infected leaves, probably caused by transformation reactions during the infection process. At least two flavonoid transformation pathways have been described so far: deglucosylation and oxidation. The aim of the present study was to determine whether DHCs are differentially converted in two apple genotypes displaying contrasted susceptibilities to the disease. Different analyses were performed: i) enzymatic activity assays in infected leaves, ii) identification/quantification of end-products obtained after in vitro enzymatic reactions with DHCs, iii) evaluation of the bactericidal activity of end-products. The results of the enzymatic assays showed that deglucosylation was dominant over oxidation in the susceptible genotype MM106 while the opposite was observed in the resistant genotype Evereste. These data were confirmed by LC-UV/Vis-MS analysis of in vitro reaction mixtures, especially because higher levels of o-quinoid oxidation products of phloretin were measured by using the enzymatic extracts of Evereste infected leaves. Their presence correlated well with a strong bactericidal activity of the reaction mixtures. Thus, our results suggest that a differential transformation of DHCs occur in apple genotypes with a potential involvement in the establishment of the susceptibility or the resistance to fire blight, through the release of glucose or of highly bactericidal compounds respectively. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Fluorometric enzymatic assay of L-arginine

NASA Astrophysics Data System (ADS)

Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

2017-01-01

The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

Microwave pretreatment of paramylon enhances the enzymatic production of soluble β-1,3-glucans with immunostimulatory activity.

PubMed

Gissibl, Alexander; Care, Andrew; Parker, Lindsay M; Iqbal, Sameera; Hobba, Graham; Nevalainen, Helena; Sunna, Anwar

2018-09-15

A hydrothermal microwave pretreatment was established to facilitate the enzymatic production of soluble bioactive β-1,3-glucans from the recalcitrant substrate paramylon. The efficacy of this pretreatment was monitored with a newly developed direct Congo Red dye-based assay over a range of temperatures. Microwave pretreatment at 170 °C for 2 min resulted in a significantly enhanced enzymatic hydrolysis of paramylon. The action of endo-β-1,3- and exo- β-1,3-glucanases on the microwave-pretreated paramylon produced soluble β-1,3-glucans with degrees of polymerisation (DP) ranging from 2-59 and 2-7, respectively. In comparison, acid-mediated hydrolysis of untreated paramylon resulted in β-1,3-glucans with a DP range of 2-38. The hydrolysates were assayed on their immunostimulatory effect on murine macrophages by measuring the production of the inflammation-linked marker tumour necrosis factor alpha (TNFα) using immunofluorescence. All of the tested hydrolysis products were shown to induce TNFα production, with the most significant immunostimulatory effect observed with the hydrolysate from the exo-β-1,3-glucanase treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enzymatic cycling method using creatine kinase to measure creatine by real-time detection.

PubMed

Ueda, Shigeru; Sakasegawa, Shin-Ichi

2016-08-01

We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP-GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay. Copyright © 2016 Elsevier Inc. All rights reserved.

New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andres, H.H.; Klem, A.J.; Szabo, S.M.

1985-03-01

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of (/sup 3/H)acetyl-CoA in the assaymore » using (/sup 3/H)acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-(/sup 3/H)acetylarylamine after separation from (/sup 3/H)acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.« less

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Newly Designed Quinolinol Inhibitors Mitigate the Effects of Botulinum Neurotoxin A in Enzymatic, Cell-Based, and ex Vivo Assays.

PubMed

Bremer, Paul T; Adler, Michael; Phung, Cecilia H; Singh, Ajay K; Janda, Kim D

2017-01-12

Botulinum neurotoxin A (BoNT/A) is one of the most deadly toxins and is the etiological agent of the potentially fatal condition, botulism. Herein, we investigated 8-hydroxyquinoline (quinolin-8-ol) as a potential inhibitor scaffold for preventing the deadly neurochemical effects of the toxin. Quinolinols are known chelators that can disrupt the BoNT/A metalloprotease zinc-containing active site, thus impeding its proteolysis of the endogenous protein substrate, synaptosomal-associated protein 25 (SNAP-25). By use of this information, the structure-activity relationship (SAR) of the quinolinol-5-sulfonamide scaffold was explored through preparation of a crude sulfonamide library and evaluation of the library in a BoNT/A LC enzymatic assay. Potency optimization of the sulfonamide hit compounds was undertaken as informed by docking studies, granting a lead compound with a submicromolar K i . These quinolinol analogues demonstrated inhibitory activity in a cell-based model for SNAP-25 cleavage and an ex vivo assay for BoNT/A-mediated muscle paralysis.

In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

PubMed

Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

2016-07-09

In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

Probing collagen-enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep

PubMed Central

Zareian, Ramin; Church, Kelli P.; Saeidi, Nima; Flynn, Brendan P.; Beale, John W.; Ruberti, Jeffrey W.

2012-01-01

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially-loaded strips of bovine corneal tissue (0.1, 0.25 or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at ~5% strain) reduces the maximum cutting-rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective “cutoff” occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is non-linear (initially-steep between 4.5 and 6.5% then flattens out from 6.5–9.5%). The high sensitivity to strain at low strain implies that even lightly-loaded collagenous tissue may exhibit significant strain-protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types. PMID:20429513

Self-Powered Wireless Carbohydrate/Oxygen Sensitive Biodevice Based on Radio Signal Transmission

PubMed Central

Falk, Magnus; Alcalde, Miguel; Bartlett, Philip N.; De Lacey, Antonio L.; Gorton, Lo; Gutierrez-Sanchez, Cristina; Haddad, Raoudha; Kilburn, Jeremy; Leech, Dónal; Ludwig, Roland; Magner, Edmond; Mate, Diana M.; Conghaile, Peter Ó.; Ortiz, Roberto; Pita, Marcos; Pöller, Sascha; Ruzgas, Tautgirdas; Salaj-Kosla, Urszula; Schuhmann, Wolfgang; Sebelius, Fredrik; Shao, Minling; Stoica, Leonard; Sygmund, Cristoph; Tilly, Jonas; Toscano, Miguel D.; Vivekananthan, Jeevanthi; Wright, Emma; Shleev, Sergey

2014-01-01

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply. PMID:25310190

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission.

PubMed

Falk, Magnus; Alcalde, Miguel; Bartlett, Philip N; De Lacey, Antonio L; Gorton, Lo; Gutierrez-Sanchez, Cristina; Haddad, Raoudha; Kilburn, Jeremy; Leech, Dónal; Ludwig, Roland; Magner, Edmond; Mate, Diana M; Conghaile, Peter Ó; Ortiz, Roberto; Pita, Marcos; Pöller, Sascha; Ruzgas, Tautgirdas; Salaj-Kosla, Urszula; Schuhmann, Wolfgang; Sebelius, Fredrik; Shao, Minling; Stoica, Leonard; Sygmund, Cristoph; Tilly, Jonas; Toscano, Miguel D; Vivekananthan, Jeevanthi; Wright, Emma; Shleev, Sergey

2014-01-01

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

A novel clot lysis assay for recombinant plasminogen activator.

PubMed

Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

2015-03-01

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

Universal fieldable assay with unassisted visual detection

NASA Technical Reports Server (NTRS)

Chelyapov, Nicolas (Inventor)

2012-01-01

A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

A laboratory-scale pretreatment and hydrolysis assay for determination of reactivity in cellulosic biomass feedstocks.

PubMed

Wolfrum, Edward J; Ness, Ryan M; Nagle, Nicholas J; Peterson, Darren J; Scarlata, Christopher J

2013-11-14

The rapid determination of the release of structural sugars from biomass feedstocks is an important enabling technology for the development of cellulosic biofuels. An assay that is used to determine sugar release for large numbers of samples must be robust, rapid, and easy to perform, and must use modest amounts of the samples to be tested.In this work we present a laboratory-scale combined pretreatment and saccharification assay that can be used as a biomass feedstock screening tool. The assay uses a commercially available automated solvent extraction system for pretreatment followed by a small-scale enzymatic hydrolysis step. The assay allows multiple samples to be screened simultaneously, and uses only ~3 g of biomass per sample. If the composition of the biomass sample is known, the results of the assay can be expressed as reactivity (fraction of structural carbohydrate present in the biomass sample released as monomeric sugars). We first present pretreatment and enzymatic hydrolysis experiments on a set of representative biomass feedstock samples (corn stover, poplar, sorghum, switchgrass) in order to put the assay in context, and then show the results of the assay applied to approximately 150 different feedstock samples covering 5 different materials. From the compositional analysis data we identify a positive correlation between lignin and structural carbohydrates, and from the reactivity data we identify a negative correlation between both carbohydrate and lignin content and total reactivity. The negative correlation between lignin content and total reactivity suggests that lignin may interfere with sugar release, or that more mature samples (with higher structural sugars) may have more recalcitrant lignin. The assay presented in this work provides a robust and straightforward method to measure the sugar release after pretreatment and saccharification that can be used as a biomass feedstock screening tool. We demonstrated the utility of the assay by identifying correlations between feedstock composition and reactivity in a population of 150 samples.

Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

PubMed Central

Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

2015-01-01

BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods.

PubMed

Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao; Yang, Jianming; Bae, Hyeun-Jong

2016-01-01

The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content.

Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods

PubMed Central

Wang, Sumeng; Li, Ruichao; Yi, Xiaohua; Fang, Tigao

2016-01-01

The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content. PMID:27847814

False positive acetaminophen concentrations in patients with liver injury.

PubMed

Polson, Julie; Wians, Frank H; Orsulak, Paul; Fuller, Dwain; Murray, Natalie G; Koff, Jonathan M; Khan, Adil I; Balko, Jody A; Hynan, Linda S; Lee, William M

2008-05-01

Acetaminophen toxicity is the most common form of acute liver failure in the U.S. After acetaminophen overdoses, quantitation of plasma acetaminophen can aid in predicting severity of injury. However, recent case reports have suggested that acetaminophen concentrations may be falsely increased in the presence of hyperbilirubinemia. We tested sera obtained from 43 patients with acute liver failure, mostly unrelated to acetaminophen, utilizing 6 different acetaminophen quantitation systems to determine the significance of this effect. In 36 of the 43 samples with bilirubin concentrations ranging from 1.0-61.5 mg/dl no acetaminophen was detectable by gas chromatography-mass spectroscopy. These 36 samples were then utilized to test the performance characteristics of 2 immunoassay and 4 enzymatic-colorimetric methods. Three of four colorimetric methods demonstrated 'detectable' values for acetaminophen in from 4 to 27 of the 36 negative samples, low concentration positive values being observed when serum bilirubin concentrations exceeded 10 mg/dl. By contrast, the 2 immunoassay methods (EMIT, FPIA) were virtually unaffected. The false positive values obtained were, in general, proportional to the quantity of bilirubin in the sample. However, prepared samples of normal human serum with added bilirubin showed a dose-response curve for only one of the 4 colorimetric assays. False positive acetaminophen tests may result when enzymatic-colorimetric assays are used, most commonly with bilirubin concentrations >10 mg/dl, leading to potential clinical errors in this setting. Bilirubin (or possibly other substances in acute liver failure sera) appears to affect the reliable measurement of acetaminophen, particularly with enzymatic-colorimetric assays.

New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74.

PubMed

Pearson, Mark S; Jariwala, Amar R; Abbenante, Giovanni; Plieskatt, Jordan; Wilson, David; Bottazzi, Maria Elena; Hotez, Peter J; Keegan, Brian; Bethony, Jeffrey M; Loukas, Alex

2015-01-01

Na-APR-1(M74) is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1(M74) and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1 wt in vitro, thereby providing proof of concept of Na-APR-1(M74) as a vaccine antigen. The mutated version, Na-APR-1(M74), was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1(M74) IgG. As little as 0.99 μg of recombinant Na-APR-1(M74) could induce anti Na-APR-1(M74) IgG in mice that were capable of inhibiting Na-APR-1w t-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1(M74) as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1 wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1(M74) which was assessed by fluorescence polarization, but with an ∼ 50-fold reduction in the dissociation constant. Taken together, these assays comprise a "toolbox" that could be useful for the analyses of Na-APR-1(M74) as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline.

New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74

PubMed Central

Pearson, Mark S; Jariwala, Amar R; Abbenante, Giovanni; Plieskatt, Jordan; Wilson, David; Bottazzi, Maria Elena; Hotez, Peter J; Keegan, Brian; Bethony, Jeffrey M; Loukas, Alex

2015-01-01

Na-APR-1M74 is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1M74 and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1wt in vitro, thereby providing proof of concept of Na-APR-1M74 as a vaccine antigen. The mutated version, Na-APR-1M74, was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1M74 IgG. As little as 0.99 μg of recombinant Na-APR-1M74 could induce anti Na-APR-1M74 IgG in mice that were capable of inhibiting Na-APR-1wt-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1M74 as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1M74 which was assessed by fluorescence polarization, but with an ∼50-fold reduction in the dissociation constant. Taken together, these assays comprise a “toolbox” that could be useful for the analyses of Na-APR-1M74 as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline. PMID:26018444

Effects of enzymatic hydrolysis and ultrasounds pretreatments on corn cob and vine trimming shoots for biogas production.

PubMed

Pérez-Rodríguez, N; García-Bernet, D; Domínguez, J M

2016-12-01

Due to their lignocellulosic nature, corn cob and vine trimming shoots (VTS) could be valorized by anaerobic digestion for biogas production. To enhance the digestibility of substrates, pretreatments of lignocellulosic materials are recommended. The effect of enzymatic hydrolysis, ultrasounds pretreatments (US) and the combination of both was assayed in lignocellulosic composition, methane, and biogas yields. The pretreatments leaded to a reduction in lignin and an increase in neutral detergent soluble compounds making corn cob and VTS more amendable for biogas conversion. The US were negative for biogas production from both substrates and in particular strongly detrimental for VTS. On the opposite side, the enzymatic hydrolysis was certainly beneficial increasing 59.8% and 14.6% the methane production from VTS and corn cob, respectively. The prior application of US did not potentiate (or not sufficiently) the improvement in the methane production reflected by the enzymatic hydrolysis pretreatment of VTS and corn cob. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of analytical methods for the determination of histamine in reference canned fish samples

NASA Astrophysics Data System (ADS)

Jakšić, S.; Baloš, M. Ž.; Mihaljev, Ž.; Prodanov Radulović, J.; Nešić, K.

2017-09-01

Two screening methods for histamine in canned fish, an enzymatic test and a competitive direct enzyme-linked immunosorbent assay (CD-ELISA), were compared with the reversed-phase liquid chromatography (RP-HPLC) standard method. For enzymatic and CD-ELISA methods, determination was conducted according to producers’ manuals. For RP-HPLC, histamine was derivatized with dansyl-chloride, followed by RP-HPLC and diode array detection. Results of analysis of canned fish, supplied as reference samples for proficiency testing, showed good agreement when histamine was present at higher concentrations (above 100 mg kg-1). At a lower level (16.95 mg kg-1), the enzymatic test produced some higher results. Generally, analysis of four reference samples according to CD-ELISA and RP-HPLC showed good agreement for histamine determination (r=0.977 in concentration range 16.95-216 mg kg-1) The results show that the applied enzymatic test and CD-ELISA appeared to be suitable screening methods for the determination of histamine in canned fish.

Lipase pre-hydrolysis enhance anaerobic biodigestion of soap stock from an oil refining industry.

PubMed

Cherif, Slim; Aloui, Fathi; Carrière, Frédéric; Sayadi, Sami

2014-01-01

A novel alcalophilic Staphylococcus haemolyticus strain with the lipolytic activity was used to perform enzymatic hydrolysis pretreatment of soap stock: a lipid rich solid waste from an oil refining industry. The culture liquid of the selected bacteria and an enzymatic preparation obtained by precipitation with ammonium sulphate from a filtrate of the same culture liquid were used for enzymatic pretreatment. The hydrolysis was carried with different incubation concentrations (10, 20 and 30%) of soap stock and the pretreatment efficiency was verified by running comparative biodegradability tests (crude and treated lipid waste). All pretreated assays showed higher reaction rate compared to crude lipid waste, which was confirmed by the increased levels of biogas production. The pretreatment of solutions containing 10% emulsified soap stock was optimized for 24 h hydrolysis time, enabling high-biogaz formation (800 ml). The use of enzymatic pre-treatment seemed to be a very promising alternative for treating soap stock having high fat contents.

A sensitive immunosensor via in situ enzymatically generating efficient quencher for electrochemiluminescence of iridium complexes doped SiO2 nanoparticles.

PubMed

Liang, Wenbin; Zhuo, Ying; Xiong, Chengyi; Zheng, Yingning; Chai, Yaqin; Yuan, Ruo

2017-08-15

A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy) 3 ] doped silica nanoparticles (SiO 2 @Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H 2 O 2 for efficiently quenching the ECL emission of SiO 2 @Ir. Typically, the SiO 2 @Ir not only increased the loading amount of Ir(ppy) 3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H 2 O 2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO 2 @Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells. Copyright © 2017 Elsevier B.V. All rights reserved.

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Chemical functionalization of surfaces for building three-dimensional engineered biosensors

NASA Astrophysics Data System (ADS)

Marques, Marco E.; Mansur, Alexandra A. P.; Mansur, Herman S.

2013-06-01

This study presents a new approach for developing biosensors based on enzymatic systems with designed three-dimensional structures. Silica glass slides were chemically functionalized at surfaces by reacting with organosilanes, 3-mercaptopropyltriethoxysilane (MPTES), and 3-aminopropyltriethoxysilane (APTES), using sol-gel process at room temperature. The functionalization of the supports was characterized by contact angle measurements and FTIR spectroscopy. The first enzyme layer was covalently immobilized to the support by a bi-functional linker (glutaraldehyde). The second enzyme layer was deposited using the protein conjugation method based on the high affinity "avidin-biotin" interactions. Each enzyme was biotinylated before being added to the nanostructured system and avidin was used as the binder between consecutive enzyme layers. The biochemical response was assayed at all stages to certify that the enzymatic bioactivity was retained throughout the entire layer-by-layer (LBL) process. The model of building 3D-enzymatic systems was evaluated using the enzymatic structure with glucose oxidase (GOx) and horseradish peroxidase (HRP). It was verified that the amino-modified support presented the highest bioactivity response compared to the other chemical functionalities. Moreover, the bienzyme nanostructure demonstrated relevant biochemical activity upon injecting the glucose substrate into the system. Finally, as a proof of concept, the bienzyme systems were assayed using real samples of regular and sugar-free soft drinks where they effectively behaved as structured biosensor for glucose with the built-in 3D hybrid architecture. Based on the results, it can be foreseen the development of promising new nanomaterials for several analytical applications such as monitoring the quality of food and beverages for nutrition purposes.

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

PubMed

Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

2016-08-01

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

Cellulase activities in biomass conversion: measurement methods and comparison.

PubMed

Dashtban, Mehdi; Maki, Miranda; Leung, Kam Tin; Mao, Canquan; Qin, Wensheng

2010-12-01

Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

Quantification of protein concentration using UV absorbance and Coomassie dyes.

PubMed

Noble, James E

2014-01-01

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used. © 2014 Elsevier Inc. All rights reserved.

Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays

EPA Science Inventory

Understanding potential health risks is a significant challenge for large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes chemical-target activity profiles of 976 chemicals (including failed pharmaceuticals...

Regulation of the oxidative balance with coenzyme Q10 sensitizes human glioblastoma cells to radiation and temozolomide.

PubMed

Frontiñán-Rubio, Javier; Santiago-Mora, Raquel María; Nieva-Velasco, Consuelo María; Ferrín, Gustavo; Martínez-González, Alicia; Gómez, María Victoria; Moreno, María; Ariza, Julia; Lozano, Eva; Arjona-Gutiérrez, Jacinto; Gil-Agudo, Antonio; De la Mata, Manuel; Pesic, Milica; Peinado, Juan Ramón; Villalba, José M; Pérez-Romasanta, Luis; Pérez-García, Víctor M; Alcaín, Francisco J; Durán-Prado, Mario

2018-05-18

To investigate how the modulation of the oxidative balance affects cytotoxic therapies in glioblastoma, in vitro. Human glioblastoma U251 and T98 cells and normal astrocytes C8D1A were loaded with coenzyme Q10 (CoQ). Mitochondrial superoxide ion (O 2 - ) and H 2 O 2 were measured by fluorescence microscopy. OXPHOS performance was assessed in U251 cells with an oxytherm Clark-type electrode. Radio- and chemotherapy cytotoxicity was assessed by immunostaining of γH2AX (24 h), annexin V and nuclei morphology, at short (72 h) and long (15 d) time. Hif-1α, SOD1, SOD2 and NQO1 were determined by immunolabeling. Catalase activity was measured by classic enzymatic assay. Glutathione levels and total antioxidant capacity were quantified using commercial kits. CoQ did not affect oxygen consumption but reduced the level of O 2 - and H 2 O 2 while shifted to a pro-oxidant cell status mainly due to a decrease in catalase activity and SOD2 level. Hif-1α was dampened, echoed by a decrease lactate and several key metabolites involved in glutathione synthesis. CoQ-treated cells were twofold more sensitive than control to radiation-induced DNA damage and apoptosis in short and long-term clonogenic assays, potentiating TMZ-induced cytotoxicity, without affecting non-transformed astrocytes. CoQ acts as sensitizer for cytotoxic therapies, disarming GBM cells, but not normal astrocytes, against further pro-oxidant injuries, being potentially useful in clinical practice for this fatal pathology. Copyright © 2018 Elsevier B.V. All rights reserved.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD12.

PubMed

Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

2016-01-01

Despite great progress in identifying and deorphanizing members of the human metabolic serine hydrolase (mSH) family, the fundamental role of numerous enzymes in this large protein class has remained unclear. One recently found mSH is α/β-hydrolase domain containing 12 (ABHD12) enzyme, whose natural substrate in vivo appears to be the lysophospholipid lysophosphatidylserine (LPS). In vitro, ABHD12 together with monoacylglycerol lipase (MAGL) and ABHD6 hydrolyzes also monoacylglycerols (MAGs) such as the primary endocannabinoid 2-arachidonoyl glycerol (2-AG). Traditional approaches for determining 2-AG hydrolase activity are rather laborious, and often utilize unnatural substrates. Here, we describe a sensitive fluorescent assay of ABHD12 activity in a 96-well-plate format that allows simultaneous testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD12 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred MAG substrate of this enzyme. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. This methodology has helped to identify the first class of inhibitors showing selectivity for ABHD12 over the other mSHs.

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics.

PubMed

Mitchell, Gabriel J; Nelson, Daniel C; Weitz, Joshua S

2010-10-04

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

PubMed

Schmidt, F N; Zimmermann, E A; Campbell, G M; Sroga, G E; Püschel, K; Amling, M; Tang, S Y; Vashishth, D; Busse, B

2017-04-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm -1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

PubMed Central

Schmidt, F.N.; Zimmermann, E.A.; Campbell, G.M.; Sroga, G.E.; Püschel, K.; Amling, M.; Tang, S. Y.; Vashishth, D.; Busse, B.

2017-01-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. PMID:28109917

Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boswell, G.S.; Dimitrijevich, S.D.; Gracy, R.W.

1989-10-01

A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developedmore » an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ((3H)thymidine, (14C)leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.« less

Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

PubMed Central

2015-01-01

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy. PMID:25420087

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

PubMed

Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

2015-07-01

To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. 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. 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. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

MiR-20a-5p promotes radio-resistance by targeting Rab27B in nasopharyngeal cancer cells.

PubMed

Huang, Dabing; Bian, Geng; Pan, Yueyin; Han, Xinghua; Sun, Yubei; Wang, Yong; Shen, Guodong; Cheng, Min; Fang, Xiang; Hu, Shilian

2017-01-01

MicroRNAs (miRNAs) was reported to be involved in cancer radio-resistance, which remains a major obstacle for effective cancer therapy. The differently expressed miRNAs were detected by RNA-seq experiment in nasopharyngeal cancer (NPC) cells. MiR-20a-5p was selected as our target, which was subject to finding its target gene Rab27B via bioinformatics analysis. The qRT-PCR, western blot and the luciferase reporter assays were performed to confirm Rab27B as the target of miR-20a-5p. In addition, the roles of miR-20a-5p in NPC radio-resistance were detected by transfection of either miR-20a-5p-mimic or miR-20a-5p-antagomiR. The involvement of Rab27B with NPC radio-resistance was also detected by the experiments with siRNA-mediated repression of Rab27B or over-expression of GFP-Rab27B. Wound healing and invasion assays were performed to detect the roles of both miR-20a-5p and Rab27B. MiR-20a-5p promotes NPC radio-resistance. We identified that its target gene Rab27B negatively correlates with miR-20a-5p-mediated NPC radio-resistance by systematic studies of a radio-sensitive (CNE-2) and resistant (CNE-1) NPC cell lines. Repression of Rab27B by siRNA suppresses cell apoptosis and passivates CNE-2 cells, whereas over-expression of Rab27B triggered cell apoptosis and sensitizes CNE-1 cells. MiR-20a-5p and its target gene Rab27B might be involved in the NPC radio-resistance. Thus the key players and regulators involved in this pathway might be the potential targets for developing effective therapeutic strategies against NPC.

Non-enzymatic hydrolysis of RNA in workers of the ant Nylanderia pubens

USDA-ARS?s Scientific Manuscript database

During preparation of total RNA from Nylanderia pubens (Forel) workers for use in expression library construction, severe RNA degradation consistently occurred that was masked by spectrophotometric analysis but clearly evident by microfluidic-based assay. Although not specifically identified, the ...

Octopamine: Presence in Single Neurons of Aplysia Suggests Neurotransmitter Function

DTIC Science & Technology

1974-10-01

responsive to octopamine than 2 any other phenylethylamine . All of these observations indicate that octopamine may function as a neurotransmitter...B., Landsberg, L. and Axelrod, J. An enzymatic assay for octop- amine and other g-hydroxylated phenylethylamines . J. Pharmacol. Exptl. Ther

Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays (Communities of Practice)

EPA Science Inventory

Understanding potential health risks is a significant challenge for large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes chemical-target activity profiles of 976 chemicals (including failed pharmaceuticals...

Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors

PubMed Central

Agah, Sayeh; Mendelson, Anna J.; Eletu, Oluwafunmilayo T.; Barkey-Bircann, Peter; Gesualdi, James

2018-01-01

Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors. PMID:29672528

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

[Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

PubMed

Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

2017-03-01

Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

PubMed

Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

Investigation of the degradation-retarding effect caused by the low swelling capacity of a novel hyaluronic Acid filler developed by solid-phase crosslinking technology.

PubMed

Park, Sunyoung; Park, Kui Young; Yeo, In Kwon; Cho, Sung Yeon; Ah, Young Chang; Koh, Hyun Ju; Park, Won Seok; Kim, Beom Joon

2014-06-01

A variety of hyaluronic acid (HA) fillers demonstrate unique physical characteristics, which affect the quality of the HA filler products. The critical factors that affect the degradation of HA gels have not yet been determined. Our objective was to determine the characteristics of HA gels that affect their resistance to the degradation caused by radicals and enzymes. Three types of HA fillers for repairing deep wrinkles, Juvederm Ultra Plus (J-U), Restylane Perlane (Perlane), and Cleviel, were tested in this study. The resistance of these HA fillers to enzymatic degradation was measured by carbazole and displacement assays using hyaluronidase as the enzyme. The resistance of these fillers to radical degradation was measured by the displacement assay using H2O2. Different tests for evaluating the degradation resistance of HA gels can yield different results. The filler most susceptible to enzymatic degradation was J-U, followed by Perlane and Cleviel. The HA filler showing the highest degree of degradation caused by H2O2 treatment was Perlane, followed by J-U, and then Cleviel. Cleviel showed higher enzymatic and radical resistances than J-U and Perlane did. Furthermore, it exhibited the highest resistance to heat and the lowest swelling ratio among all the fillers that were examined. The main factor determining the degradation of HA particles is the gel swelling ratio, which is related to the particle structure of the gel. Our in vitro assays suggest that the decrease in the swelling ratio will lead to a retarding effect on the degradation of HA fillers.

Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method.

PubMed

Keevil, B G; Owen, L; Thornton, S; Kavanagh, J

2005-09-01

Measurement of urine citrate is used to assess the risk of further urinary stone formation and to assess the benefit of treatment in affected individuals. We wanted to develop a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of urinary citrate and to compare it with our current enzymatic assay. For the LC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of urine and 20 microL of internal standard to 400 microL of water. After mixing, 3 microL of the diluted sample was injected into the LC-MS/MS system. An LC system was used to isocratically elute a C18 column (50 x 2.1 mm) with 0.4 mL/min water containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. A step gradient of 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid was used to wash the column. The retention times were 1.4 min for citrate and 1.4 min for d4-citrate. Cycle time was 4.0 min, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions, citrate m/z 191.0>111.0 and d4-citrate m/z 195.0>113.0. Within and between-batch coefficients of variation were <3% over the range 480-3800 micromol/L. The lower limit of quantification was 24.0 micromol/L. Regression analysis showed LC-MS/MS = 0.8781 (enzymatic assay) + 102.5, r = 0.964, n = 73. We have developed a simple LC-MS/MS method for urinary citrate measurement that shows acceptable performance.

Interferences of homogentisic acid (HGA) on routine clinical chemistry assays in serum and urine and the implications for biochemical monitoring of patients with alkaptonuria.

PubMed

Curtis, S L; Roberts, N B; Ranganath, L R

2014-05-01

We have assessed the effect of elevated concentrations of homogentisic acid (HGA) as in alkaptonuria (AKU), on a range of routine chemistry tests in serum and urine. HGA was added to pooled serum and a range of assays was analysed with Roche Modular chemistries. Effects on urine were assessed by diluting normal urine with urine from a patient with AKU, adding HGA to urine and after lowering output of urinary HGA with nitisinone treatment. Serum enzymatic creatinine showed 30% negative interference with 100μmol/L HGA and >50% at 400μmol/L. Serum urate 100 to 480μmol/L was reduced up to 20% at 100 and to 50% with 400μmol/L HGA. Serum cholesterol between 3 and 11mmol/L was reduced by 0.5mmol/L with 400μmol/L HGA. Urine enzymatic creatinine and urate with >2mmol/L HGA showed concentration dependent negative interference up to 80%. A positive interference in urine total protein by benzethonium turbidometric assay was observed, with 10mmol/L HGA equivalent to 1g/L protein. Jaffe creatinine, Na, K, Cl, Mg, Ca, phosphate, ALT, GGT, ALP activities and urea in serum and or urine were not affected by increases in HGA. To avoid interferences by HGA in alkaptonuria concentration of HGA should be established before samples are assayed with peroxidase assays and benzethonium urine protein. Copyright © 2013 The Canadian Society of Clinical Chemists. All rights reserved.

Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core-satellite nanoprobes.

PubMed

Zheng, Tingting; Zhang, Rui; Zhang, Qingfeng; Tan, Tingting; Zhang, Kui; Zhu, Jun-Jie; Wang, Hui

2013-09-18

We have developed a robust enzymatic peptide cleavage-based assay for the ultrasensitive dual-channel detection of matrix metalloproteinase-2 (MMP-2) in human serum using gold-quantum dot (Au-QD) core-satellite nanoprobes.

Lipidomics of oxidized polyunsaturated fatty acids

PubMed Central

Massey, Karen A.; Nicolaou, Anna

2013-01-01

Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions. PMID:22940496

Analysis of Resistant Starches in Rat Cecal Contents Using Fourier Transform Infrared Photoacoustic Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Timothy J.; Ai, Yongfeng; Jones, Roger W.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) qualitatively and quantitatively measured resistant starch (RS) in rat cecal contents. Fisher 344 rats were fed diets of 55% (w/w, dry basis) starch for 8 weeks. Cecal contents were collected from sacrificed rats. A corn starch control was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. To calibrate the FTIR-PAS analysis, samples from each diet were analyzed using an enzymatic assay. A partial least-squares cross-validation plot generated from the enzymatic assay and FTIR-PAS spectral results for starch fitmore » the ideal curve with a R2 of 0.997. A principal component analysis plot of components 1 and 2 showed that spectra from diets clustered significantly from each other. This study clearly showed that FTIR-PAS can accurately quantify starch content and identify the form of starch in complex matrices.« less

Effects of grain species and cultivar, thermal processing, and enzymatic hydrolysis on gluten quantitation.

PubMed

Pahlavan, Autusa; Sharma, Girdhari M; Pereira, Marion; Williams, Kristina M

2016-10-01

Gluten from wheat, rye, and barley can trigger IgE-mediated allergy or Celiac disease in sensitive individuals. Gluten-free labeled foods are available as a safe alternative. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are commonly used to quantify gluten in foods. However, various non-assay related factors can affect gluten quantitation. The effect of gluten-containing grain cultivars, thermal processing, and enzymatic hydrolysis on gluten quantitation by various ELISA kits was evaluated. The ELISA kits exhibited variations in gluten quantitation depending on the gluten-containing grain and their cultivars. Acceptable gluten recoveries were obtained in 200mg/kg wheat, rye, and barley-spiked corn flour thermally processed at various conditions. However, depending on the enzyme, gluten grain source, and ELISA kit used, measured gluten content was significantly reduced in corn flour spiked with 200mg/kg hydrolyzed wheat, rye, and barley flour. Thus, the gluten grain source and processing conditions should be considered for accurate gluten analysis. Published by Elsevier Ltd.

Malolactic bioconversion using a Oenococcus oeni strain for cider production: effect of yeast extract supplementation.

PubMed

Herrero, Mónica; García, Luis A; Díaz, Mario

2003-12-01

Yeast extract addition to reconstituted apple juice had a positive impact on the development of the malolactic starter culture used to ensure malolactic fermentation in cider, using active but non-proliferating cells. In this work, the reuse of fermentation lees from cider is proposed as an alternative to the use of commercial yeast extract products. Malolactic enzymatic assays, both in whole cells and cell-free extracts, were carried out to determine the best time to harvest cells for use as an inoculum in cider. Cells harvested at the late exponential phase, the physiological stage of growth corresponding to the maximum values of specific malolactic activity, achieved a good rate of malic acid degradation in controlled cider fermentation. Under the laboratory conditions used, malic acid degradation rates in the fermentation media turned out to be near 2.0 and 2.5 times lower, compared with the rates obtained in whole-cell enzymatic assays, as useful data applicable to industrial cider production.

Enzyme-Cascade Analysis of the Rio Tinto Subsurface Environment: A Biosensor Experiment

NASA Technical Reports Server (NTRS)

McKay, David S.; Lynch, Kennda; Wainwright, Norman; Child, Alice; Williams, Kendra; McKay, David; Amils, Ricardo; Gonzalez, Elena; Stoker, Carol

2004-01-01

The Portable Test System (PTS), designed & developed by Charles Rivers Laboratories, Inc. (Charleston, SC) is a portable instrument that was designed to perform analysis of enzymatic assays related to rapid assessment of microbial contamination (Wainwright, 2003). The enzymatic cascade of Limulus Amebocyte Lysate (LAL) is known to be one of the most sensitive techniques available for microbial detection, enabling the PTS to be evaluated as a potential life detection instrument for in situ Astrobiology missions. In the summer of 2003 the system was tested as a part of the Mars Astrobiology Research and Technology Experiment (MARTE) ground truth science campaign in the Rio Tinto Analogue environment near Nerva, Spain. The preliminary results show that the PTS analysis correlates well with the contamination control tests and the more traditional lab-based biological assays performed during the MARTE field mission. Further work will be conducted on this research during a second field campaign in 2004 and a technology demonstration of a prototype instrument that includes autonomous sample preparation will occur in 2005.

Novel epoxy-silica nanoparticles to develop non-enzymatic colorimetric probe for analytical immuno/bioassays.

PubMed

Dixit, Chandra K; Bhakta, Snehasis; Macharia, John; Furtado, Jared; Suib, Steven L; Rusling, James F

2018-10-22

We have developed a novel method to develop epoxy silica nanoparticles (EfSiNP) in a single pot. High surface coverage of epoxy functional groups between 150 and 57000 molecules per particles (∼10 13 -10 16 molecules/mL of 200 nm EfSiNPs) was achieved for different preparation conditions. We then created a red colored probe by conjugating Fuchsin dye to the epoxy functionalities of EfSINPs. Anti-mouse IgG was co-immobilized with Fuchsin and their ratios were optimized for achieving optimum ratios by testing those in functional assays. Dye to antibody ratios were in good negative correlation with a coefficient of -1.00 measured at a confidence level of over 99%. We employed the developed non-enzymatic colorimetric immunonanoprobe for detecting mouse IgG in a direct immunoassay format. We achieved a sensitivity of 427 pg/mL with the assay. Copyright © 2018 Elsevier B.V. All rights reserved.

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

PubMed

Koller, E; Wolfbeis, O S

1984-11-15

A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging

PubMed Central

Ponomarev, Vladimir; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Balatoni, Julius; Blasberg, Ronald; Tjuvajev, Juri Gelovani

2003-01-01

Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improvedmetabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and a γ-camera. PMID:12869307

Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

PubMed

Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

2013-09-01

Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.

PubMed

Hitomi, Kiyotaka; Kitamura, Miyako; Alea, Mileidys Perez; Ceylan, Ismail; Thomas, Vincent; El Alaoui, Saïd

2009-11-15

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

False HDAC Inhibition by Aurone Compound.

PubMed

Itoh, Yukihiro; Suzuki, Miki; Matsui, Taiji; Ota, Yosuke; Hui, Zi; Tsubaki, Kazunori; Suzuki, Takayoshi

2016-01-01

Fluorescence assays are useful tools for estimating enzymatic activity. Their simplicity and manageability make them suitable for screening enzyme inhibitors in drug discovery studies. However, researchers need to pay attention to compounds that show auto-fluorescence and quench fluorescence, because such compounds lower the accuracy of the fluorescence assay systems by producing false-positive or negative results. In this study, we found that aurone compound 7, which has been reported as a histone deacetylase (HDAC) inhibitor, gave false-positive results. Although compound 7 was identified by an in vitro HDAC fluorescence assay, it did not show HDAC inhibitory activity in a cell-based assay, leading us to suspect its in vitro HDAC inhibitory activity. As a result of verification experiments, we found that compound 7 interferes with the HDAC fluorescence assay by quenching the HDAC fluorescence signal. Our findings underscore the faults of fluorescence assays and call attention to careless interpretation.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

NASA Astrophysics Data System (ADS)

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-07-01

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT.

PubMed

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-07-16

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

Detection of ingested nitromethane and reliable creatinine assessment using multiple common analytical methods.

PubMed

Murphy, Christine M; Devlin, John J; Beuhler, Michael C; Cheifetz, Paul; Maynard, Susan; Schwartz, Michael D; Kacinko, Sherri

2018-04-01

Nitromethane, found in fuels used for short distance racing, model cars, and model airplanes, produces a falsely elevated serum creatinine with standard creatinine analysis via the Jaffé method. Erroneous creatinine elevation often triggers extensive testing, leads to inaccurate diagnoses, and delayed or inappropriate medical interventions. Multiple reports in the literature identify "enzymatic assays" as an alternative method to detect the true value of creatinine, but this ambiguity does not help providers translate what type of enzymatic assay testing can be done in real time to determine if there is indeed false elevation. We report seven cases of ingested nitromethane where creatinine was determined via Beckman Coulter ® analyser using the Jaffé method, Vitros ® analyser, or i-Stat ® point-of-care testing. Nitromethane was detected and semi-quantified using a common clinical toxic alcohol analysis method, and quantified by headspace-gas chromatography-mass spectrometry. When creatinine was determined using i-Stat ® point-of-care testing or a Vitros ® analyser, levels were within the normal range. Comparatively, all initial creatinine levels obtained via the Jaffé method were elevated. Nitromethane concentrations ranged from 42 to 310 μg/mL. These cases demonstrate reliable assessment of creatinine through other enzymatic methods using a Vitros ® analyser or i-STAT ® . Additionally, nitromethane is detectable and quantifiable using routine alcohols gas chromatography analysis and by headspace-gas chromatography-mass spectrometry.

In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

PubMed

Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

2010-11-15

In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

PubMed Central

SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

PubMed Central

Senevirathne, Mahinda; Kim, Soo-Hyun

2010-01-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro. PMID:20607062

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

PubMed

Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

2010-06-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

Thermal and enzymatic recovering of proteins from untanned leather waste.

PubMed

Bajza, Z; Vrucek, V

2001-01-01

The laboratory trials of a process to treat untanned leather waste to isolate valuable protein products are presented. In this comparative study, both thermal and enzymatic treatments of leather waste were performed. The enzymatic method utilizes commercially available alkaline protease at moderate temperatures and for short periods of time. The concentration of the enzyme was 500 units per gram of leather waste which makes the method cost-effective. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis of untanned leather waste is determined. Chemical and physical properties of protein powder products from untanned leather waste were evaluated by spectrophotometric and chromatographic methods and by use of electron microscope. The results of microbiological assays confirm that these products agree to food safety standards. This relatively simple treatment of untanned leather waste may provide a practical and economical solution to the disposal of potentially dangerous waste.

Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

PubMed

Zhang, Xin; Wang, Zhuang; Wang, Se; Fang, Hao; Zhang, Fan; Wang, De-Gao

2017-01-02

Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 μm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

PubMed

Tanaka, T; Inui, O; Dohi, N; Okada, N; Okada, H; Kikuchi, Y

2001-07-01

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-enzymatic Properties against Urease by an ESI-MS Based Assay.

PubMed

Hassan, Sherif T S; Švajdlenka, Emil; Berchová-Bímová, Kateřina

2017-04-30

For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC 50 values of 0.92 and 1.43 µg∙mL -1 , respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC 50 value of 82.4 µg∙mL -1 . This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

Measurement of the distribution of non-structural carbohydrate composition in onion populations by a high-throughput microplate enzymatic assay.

PubMed

Revanna, Roopashree; Turnbull, Matthew H; Shaw, Martin L; Wright, Kathryn M; Butler, Ruth C; Jameson, Paula E; McCallum, John A

2013-08-15

Non-structural carbohydrate (NSC; glucose, fructose, sucrose and fructan) composition of onions (Allium cepa L.) varies widely and is a key determinant of market usage. To analyse the physiology and genetics of onion carbohydrate metabolism and to enable selective breeding, an inexpensive, reliable and practicable sugar assay is required to phenotype large numbers of samples. A rapid, reliable and cost-effective microplate-based assay was developed for NSC analysis in onions and used to characterise variation in tissue hexose, sucrose and fructan content in open-pollinated breeding populations and in mapping populations developed from a wide onion cross. Sucrose measured in microplates employing maltase as a hydrolytic enzyme was in agreement with HPLC-PAD results. The method revealed significant variation in bulb fructan content within open-pollinated 'Pukekohe Longkeeper' breeding populations over a threefold range. Very wide segregation from 80 to 600 g kg(-1) in fructan content was observed in bulbs of F2 genetic mapping populations from the wide onion cross 'Nasik Red × CUDH2150'. The microplate enzymatic assay is a reliable and practicable method for onion sugar analysis for genetics, breeding and food technology. Open-pollinated onion populations may harbour extensive within-population variability in carbohydrate content, which may be quantified and exploited using this method. The phenotypic data obtained from genetic mapping populations show that the method is well suited to detailed genetic and physiological analysis. © 2013 Society of Chemical Industry.

Using enzyme bioassays as a rapid screen for metal toxicity

USGS Publications Warehouse

Choate, LaDonna M.; Ross, P.E.; Blumenstein, E. P.; Ranville, James F.

2005-01-01

Mine tailings piles and abandoned mine soils are often contaminated by a suite of toxic metals, which were released in the mining process. Traditionally, toxicity of such areas has been determined by numerous chemical methods including the Toxicity Characteristic Leachate Procedure (TCLP) and traditional toxicity tests using organisms such as the cladoceran Ceriodaphnia dubia. Such tests can be expensive and time-consuming. Enzymatic bioassays may provide an easier, less costly, and more time-effective toxicity screening procedure for mine tailings and abandoned mine soil leachates. This study evaluated the commercially available MetPLATE™ enzymatic toxicity assay test kit. The MetPLATE™ assay uses a modified strain of Escherichia coli bacteria as the test organism. Toxicity is defined by the activity of β-galactosidase enzyme which is monitored colorometrically with a 96-well spectrophotometer. The study used water samples collected from North Fork Clear Creek, a mining influenced water (MIW) located in Colorado. A great benefit to using the MetPLATE™ assay over the TCLP is that it shows actual toxicity of a sample by taking into account the bioavailability of the toxicants rather than simply measuring the metal concentration present. Benefits of the MetPLATE™ assay over the use of C. dubia include greatly reduced time for the testing process (∼2 hours), a more continuous variable due to a greater number of organisms present in each sample (100,000+), and the elimination of need to maintain a culture of organisms at all times.

A Compton Suppressed Gamma Ray Counter For Radio Assay of Materials

NASA Astrophysics Data System (ADS)

Godfrey, Benjamin

2016-03-01

Rare event searches, such as direct dark matter experiments, require materials with ultra-low levels of natural radioactivity. We present a neutron activation analysis (NAA) technique for assaying metals, specifically titanium used for cryostat construction. Earlier attempts at NAA encountered limitations due to bulk activation via (n, p) reactions, which contributed to large continuum backgrounds due to Compton tails. Our method involves a heavy water shielded exposure to minimize (n,p) reactions and a sodium iodide shielded high purity germanium counter for the gamma ray assay. Preliminary results on assays for U/Th/K contamination in titaniumwill be presented.

Protein deficiency lowers resistance of Mormon crickets to the pathogenic fungus Beauveria bassiana

USDA-ARS?s Scientific Manuscript database

Little is known about the effects of dietary macronutrients on the capacity of insects to ward off a fungal pathogen. Here we tested the hypothesis that Mormon crickets fed restricted protein diets have lower enzymatic assays of generalized immunity, slower rates of encapsulation of foreign bodies,...

Antioxidant activity of black bean (Phaseolus vulgaris L.) protein hydrolysates

USDA-ARS?s Scientific Manuscript database

The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by e...

The Most Proficient Enzyme as the Central Theme in an Integrated, Research-based Biochemistry Laboratory Course

ERIC Educational Resources Information Center

Smiley, Jeffrey A.

2002-01-01

The enzyme orotidine-5'-monophosphate decarboxylase is an attractive choice for the central theme of an integrated, research-based biochemistry laboratory course. A series of laboratory exercises common to most instructional laboratories, including enzyme assays, protein purification, enzymatic characterization, elementary kinetics, and…

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

Biosensors and Bio-Bar Code Assays Based on Biofunctionalized Magnetic Microbeads

PubMed Central

Jaffrezic-Renault, Nicole; Martelet, Claude; Chevolot, Yann; Cloarec, Jean-Pierre

2007-01-01

This review paper reports the applications of magnetic microbeads in biosensors and bio-bar code assays. Affinity biosensors are presented through different types of transducing systems: electrochemical, piezo electric or magnetic ones, applied to immunodetection and genodetection. Enzymatic biosensors are based on biofunctionalization through magnetic microbeads of a transducer, more often amperometric, potentiometric or conductimetric. The bio-bar code assays relie on a sandwich structure based on specific biological interaction of a magnetic microbead and a nanoparticle with a defined biological molecule. The magnetic particle allows the separation of the reacted target molecules from unreacted ones. The nanoparticles aim at the amplification and the detection of the target molecule. The bio-bar code assays allow the detection at very low concentration of biological molecules, similar to PCR sensitivity.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.

PubMed

Navarro, David; Couturier, Marie; da Silva, Gabriela Ghizzi Damasceno; Berrin, Jean-Guy; Rouau, Xavier; Asther, Marcel; Bignon, Christophe

2010-07-16

To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU.

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Glomerular filtration rate estimation from plasma creatinine after inhibition of tubular secretion: relevance of the creatinine assay.

PubMed

Kemperman, F A; Silberbusch, J; Slaats, E H; van Zanten, A P; Weber, J A; Krediet, R T; Arisz, L

1999-05-01

Estimation of glomerular filtration rate (GFR) from plasma creatinine concentration after inhibition of tubular creatinine secretion with cimetidine provides a good assessment in patients with various nephropathies and with non-insulin-dependent diabetes mellitus (NIDDM). The aim of this study was to compare cimetidine-aided GFR estimations using various creatinine assays. In 30 outpatients with NIDDM GFR was measured as the urinary clearance of continuously infused [125I]iothalamate. Plasma creatinine concentration was analysed after oral cimetidine with an alkaline picrate (AP) method, with an enzymatic (PAP) assay and with HPLC. GFR estimations were calculated with the Cockcroft Gault formula (CG). AP creatinine concentrations were significantly higher than PAP or HPLC values. GFR estimations by AP (CG(AP) 66 +/- 19 ml/min/1.73 m2, mean SD) were significantly lower than GFR (89 +/- 30), whereas CG(PAP) (85 +/- 30) and CG(HPLC) (84 +/- 34 ml/min/1.73 m2) were not. Bland and Altman analysis showed a difference between CG(AP) and GFR of -22.4 +/- 17.7 ml/min/1.73 m2; this difference becomes larger when the GFR increases. The difference between CG and GFR was only -3.8 +/- 14.8 ml/min/1.73 m2 for PAP and -4.4 +/- 17.5 ml/min/1.73 m2 for HPLC, without any systematic difference. A good assessment of the GFR from plasma creatinine after cimetidine administration is possible when creatinine is measured with an enzymatic assay or with the less convenient HPLC method. The more widespread and cheaper alkaline picrate assay is not suitable for GFR-estimation.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

PubMed

Greiner, Mark A; Burckart, Kimberlee A; Wagoner, Michael D; Schmidt, Gregory A; Reed, Cynthia R; Liaboe, Chase A; Flamme-Wiese, Miles J; Zimmerman, M Bridget; Mullins, Robert F; Kardon, Randy H; Goins, Kenneth M; Aldrich, Benjamin T

2015-05-01

We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Enzymatic measurement of free and esterified cholesterol levels in plasma and other biological preparations using the oxygen electrode in a modified glucose analyzer.

PubMed

Dietschy, J M; Weeks, L E; Delente, J J

1976-12-01

A method is described for assaying free and esterified cholesterol using the oxygen electrode in a modified glucose analyzer to measure the relative amount of oxygen utilization taking place during oxydation of free cholesterol by the enzyme, cholesterol oxidase. A second enzyme, cholesterol ester hydrolase, is utilized to generate free cholesterol from cholesterol esters. This assay procedure is rapid, specific, reproducible and applicable to the measurement of free and esterified cholesterol carried in the major plasma lipoprotein fractions of man and the rat and, in addition, it can be utilized for the assay of sterols in subcellular fractions of cells.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Remote enzyme activation using gold coated magnetite as antennae for radio frequency fields

NASA Astrophysics Data System (ADS)

Collins, Christian B.; Ackerson, Christopher J.

2018-02-01

The emerging field of remote enzyme activation, or the ability to remotely turn thermophilic increase enzyme activity, could be a valuable tool for understanding cellular processes. Through exploitation of the temperature dependence of enzymatic processes and high thermal stability of thermophilic enzymes these experiments utilize nanoparticles as `antennae' that convert radiofrequency (RF) radiation into local heat, increasing activity of the enzymes without increasing the temperature of the surrounding bulk solution. To investigate this possible tool, thermolysin, a metalloprotease was covalently conjugated to 4nm gold coated magnetite particles via peptide bond formation with the protecting ligand shell. RF stimulated protease activity at 17.76 MHz in a solenoid shaped antenna, utilizing both electric and magnetic field interactions was investigated. On average 40 percent higher protease activity was observed in the radio frequency fields then when bulk heating the sample to the same temperature. This is attributed to electrophoretic motion of the nanoparticle enzyme conjugates and local regions of heat generated by the relaxation of the magnetite cores with the oscillating field. Radio frequency local heating of nanoparticles conjugated to enzymes as demonstrated could be useful in the activation of specific enzymes in complex cellular environments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swarup, S.; Chatterjea, J.B.; Hosain, P.

Several Hb. E-thalassemia and a few other cases of hematological interest were subjected to radio-iron tests using tracer doses of only 0.035 plus or minus 0.005 mu c/kg. of bodyweight and gas-flow counter for assaying the radio-activity of the samples. Serum iron and hemoglobin iron concentrations; curves for plasma iron clearance and appearance of radio-iron in red cells, and the plasma iron and the red cell iron turnover rates were obtained by standard methods. The results showed an increased rate of plasma clearance but decreased utilization of iron by erythrocytes in Hb. E-thalassemia disease; plasma iron-clearance pattern was similar tomore » iron deficiency anemia but the red cell radio-iron uptake pattern bore similarity to aplastic anemia. This suggests that ineffective erythropoiesis contributes significantly to the causation of anemia in Hb. E-thalassemia disease. (auth)« less

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

PubMed Central

Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

2015-01-01

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway. PMID:26177797

Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

1988-07-01

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

Comparison of cell wall polysaccharide hydrolysis by a dilute acid/enzymatic saccharification process and rumen microorganisms

USDA-ARS?s Scientific Manuscript database

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance of biomass in the bioenergy conversion process. Cell wall polysaccharide hydrolysis by dilute sulfuric acid pretreatment at 121 degrees C followed by cellulase hydrolysis for 72 h (CONV) and in v...

Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

PubMed

Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

2018-01-01

Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

MicroRNA-9 functions as a tumor suppressor and enhances radio-sensitivity in radio-resistant A549 cells by targeting neuropilin 1.

PubMed

Xiong, Kai; Shao, Li Hong; Zhang, Hai Qin; Jin, Linlin; Wei, Wei; Dong, Zhuo; Zhu, Yue Quan; Wu, Ning; Jin, Shun Zi; Xue, Li Xiang

2018-03-01

Radiotherapy is commonly used to treat lung cancer but may not kill all cancer cells, which may be attributed to the radiotherapy resistance that often occurs in non-small cell lung cancer (NSCLC). At present, the molecular mechanism of radio-resistance remains unclear. Neuropilin 1 (NRP1), a co-receptor for vascular endothelial growth factor (VEGF), was demonstrated to be associated with radio-resistance of NSCLC cells via the VEGF-phosphoinositide 3-kinase-nuclear factor-κB pathway in our previous study. It was hypothesized that certain microRNAs (miRs) may serve crucial functions in radio-sensitivity by regulating NRP1. Bioinformatics predicted that NRP1 was a potential target of miR-9, and this was validated by luciferase reporter assays. Functionally, miR-9-transfected A549 cells exhibited a decreased proliferation rate, increased apoptosis rate and attenuated migratory and invasive abilities. Additionally, a high expression of miR-9 also significantly enhanced the radio-sensitivity of A549 cells in vitro and in vivo . These data improve understanding of the mechanisms of cell radio-resistance, and suggest that miR-9 may be a molecular target for the prediction of radio-sensitivity in NSCLC.

The improvement of competitive saprophytic capabilities of Trichoderma species through the use of chemical mutagens.

PubMed

Rashmi, Singh; Maurya, Sudarshan; Upadhyay, Ram Sanmukh

2016-01-01

The antagonistic potential of Trichoderma strains was assayed by studying the effect of their culture filtrate on the radial growth of Sclerotium rolfsii, the causal agent of chickpea collar rot. Trichoderma harzianum-1432 (42.2%) and Trichoderma atroviride (40.3%) were found to be strong antagonists. To enhance their antagonistic potential, mutagenesis of these two selected strains was performed. Two mutants, Th-m1 and T. atroviride m1, were found to be more effective than their parent strains. The enzymatic activities of the selected parent and mutant strains were assayed, and although both mutants were found to have enhanced enzymatic activities compared to their respective parent strains, Th-m1 possessed the maximum cellulase (5.69U/mL) and β-1,3-glucanase activity (61.9U/mL). Th-m1 also showed high competitive saprophytic ability (CSA) among all of the selected parent and mutant strains, and during field experiments, Th-m1 was found to successfully possess enhanced disease control (82.9%). Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

The improvement of competitive saprophytic capabilities of Trichoderma species through the use of chemical mutagens

PubMed Central

Rashmi, Singh; Maurya, Sudarshan; Upadhyay, Ram Sanmukh

2016-01-01

The antagonistic potential of Trichoderma strains was assayed by studying the effect of their culture filtrate on the radial growth of Sclerotium rolfsii, the causal agent of chickpea collar rot. Trichoderma harzianum-1432 (42.2%) and Trichoderma atroviride (40.3%) were found to be strong antagonists. To enhance their antagonistic potential, mutagenesis of these two selected strains was performed. Two mutants, Th-m1 and T. atroviride m1, were found to be more effective than their parent strains. The enzymatic activities of the selected parent and mutant strains were assayed, and although both mutants were found to have enhanced enzymatic activities compared to their respective parent strains, Th-m1 possessed the maximum cellulase (5.69 U/mL) and β-1,3-glucanase activity (61.9 U/mL). Th-m1 also showed high competitive saprophytic ability (CSA) among all of the selected parent and mutant strains, and during field experiments, Th-m1 was found to successfully possess enhanced disease control (82.9%). PMID:26887221

Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

NASA Astrophysics Data System (ADS)

Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

2015-09-01

Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

The Purity of Radioiodide-I131 Assessed by in Vivo and in Vitro Methods

PubMed Central

Fawcett, D. M.; Olde, G. L.; McLeod, L. E.

1962-01-01

Between 41 and 94% of the radioactivity of 24 preparations of I131 supplied without cysteine preservative was non-iodide on chromatographic analysis. Extraneous radio-activity was essentially absent from I131 supplied with cysteine. It was converted to iodide-I131 by 10-3 M cysteine or iodide but not by incubation at pH 2. The average thyroid uptake of I131 containing extraneous radioactivity was significantly lower than the uptake of I131 free from non-iodide impurity in 16 human subjects measured under controlled conditions and in a random group of 669 patients. Incubation of samples of I131 containing non-iodide radioactivity with tyrosine and cupric chloride resulted in the non-enzymatic formation of monoiodotyrosine-I131 either in the presence or absence of thyroid homogenate. Enzymatic formation of monoiodotyrosine-I131 by thyroid homogenates could be demonstrated only when I131 free from extraneous activity was used. ImagesFig. 1Fig. 2 PMID:13891874

Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain.

PubMed

Nonato, L F; Rocha-Vieira, E; Tossige-Gomes, R; Soares, A A; Soares, B A; Freitas, D A; Oliveira, M X; Mendonça, V A; Lacerda, A C; Massensini, A R; Leite, H R

2016-09-29

Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (P<0.05) and increased the activity of the antioxidant enzyme superoxide dismutase (SOD) (P<0.05) with no effect on brain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (P<0.05) and maintenance of body weight. In this context, the reduced TBARS content and increased SOD antioxidant activity induced by 8 weeks of swimming training are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

Guaiacol peroxidase zymography for the undergraduate laboratory.

PubMed

Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M; Kurz, Liliana

2014-01-01

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

PubMed

Rodrigues, Marili V N; Barbosa, Alexandre F; da Silva, Júlia F; dos Santos, Deborah A; Vanzolini, Kenia L; de Moraes, Marcela C; Corrêa, Arlene G; Cass, Quezia B

2016-01-15

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

PubMed

Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-15

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA.

PubMed

Arakawa, Hidetoshi; Nakabayashi, Shigeo; Ohno, Ken-Ichi; Maeda, Masako

2012-04-01

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10 -18 to 1.0×10 -15  mol/assay, with a detection limit of 1.0×10 -18  mol/assay. The coefficient of variation (CV, n =8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and themore » IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.« less

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

PubMed

Coady, Katherine K; Kan, H Lynn; Schisler, Melissa R; Gollapudi, B Bhaskar; Neal, Barbara; Williams, Amy; LeBaron, Matthew J

2014-08-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system. Copyright © 2014 Elsevier Ltd. All rights reserved.

A facile low-cost enzymatic paper-based assay for the determination of urine creatinine.

PubMed

Talalak, Kwanrutai; Noiphung, Julaluk; Songjaroen, Temsiri; Chailapakul, Orawon; Laiwattanapaisal, Wanida

2015-11-01

Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

Removing small non-enzymatic molecules for biochemical assay of redox regulatory enzymes; An exemplary comments on "Antioxidant responses in gills and digestive gland of oyster Crassostrea madrasensis (Preston) under lead exposure.

PubMed

Paital, Biswaranjan

2018-06-15

For biochemical assay of every enzyme including redox regulatory enzymes, any interfering small molecules (ISM) that may cross react with substrates or indirectly influence the reaction, must be removed. Such ISM(s) if present, need to be either neutralized or filtered out both from the sample or from the reaction mixture. This is a standard protocol adapted worldwide and fundamental rule in biochemistry. Without such approach, results obtained from a study that includes enzymatic assays seem to be inaccurate. Such inaccuracy raises question on the use of such data in future, especially as ecotoxic biomarkers. Tissue specific seasonal variation in natural titre of such ISM(s) in organisms leads to give rise counterfeit results. Such a case is highlighted in this correspondence article in relation to assay of redox regulatory enzymes including superoxide dismutase, catalase, especially the enzymes of glutathione system in presence of glutathione (GSH) in sample. This fact is discussed considering a recent publication doi.org/10.1016/j.ecoenv.2017.03.056 in which the authors have measured glutathione enzymes in tissues without removing GSH, that acts as ISM, from the sample. It is inferred that logical and sound scientific practices need to be followed for measuring biochemical activity of all enzymes in general and enzymes of glutathione system in particular. The main objective of this article was to make an alert in scientific society to avoid such mistakes in future. Copyright © 2018 Elsevier Inc. All rights reserved.

Label-free fluorescent enzymatic assay of citrate synthase by CoA-Au(I) co-ordination polymer and its application in a multi-enzyme logic gate cascade.

PubMed

Li, Yong; Wang, Huixia; Dai, Futao; Li, Pei; Jin, Xin; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

2016-12-15

Citrate synthase (CS) is one of the key metabolic enzymes in the Krebs tricarboxylic acid (TCA) cycle. It regulates energy generation in mitochondrial respiration by catalysing the reaction between oxaloacetic acid (OAA) and acetyl coenzyme A (Ac-CoA) to generate citrate and coenzyme A (CoA). CS has been shown to be a biomarker of neurological diseases and various kinds of cancers. Here, a label-free fluorescent assay has been developed for homogeneously detecting CS and its inhibitor based on the in situ generation of CoA-Au(I) co-ordination polymer (CP) and the fluorescence signal-on by SYBR Green II-stained CoA-Au(I) CP. Because of the unique property of the CoA-Au(I) CP, this CS activity assay method could achieve excellent selectivity and sensitivity, with a linear range from 0.0033 U/μL to 0.264 U/μL and a limit of detection to be 0.00165 U/μL. Meanwhile, this assay method has advantages of being facile and cost effective with quick detection. Moreover, based on this method, a biomimetic logic system was established by rationally exploiting the cascade enzymatic interactions in TCA cycle for chemical information processing. In the TCA cycle-derived logic system, an AND-AND-AND-cascaded gate was rigorously operated step by step in one pot, and is outputted by a label-free fluorescent signal with visualized readout. Copyright © 2016 Elsevier B.V. All rights reserved.

A disposable tear glucose biosensor--part 3: assessment of enzymatic specificity.

PubMed

Lan, Kenneth; McAferty, Kenyon; Shah, Pankti; Lieberman, Erica; Patel, Dharmendra R; Cook, Curtiss B; La Belle, Jeffrey T

2011-09-01

A concept for a tear glucose sensor based on amperometric measurement of enzymatic oxidation of glucose was previously presented, using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) as the enzyme. Glucose dehydrogenase flavin adenine dinucleotide is further characterized in this article and evaluated for suitability in glucose-sensing applications in purified tear-like saline, with specific attention to the effect of interfering substances only. These interferents are specifically saccharides that could interact with the enzymatic activity seen in the sensor's performance. Bench top amperometric glucose assays were performed using an assay solution of GDH-FAD and ferricyanide redox mediator with samples of glucose, mannose, lactose, maltose, galactose, fructose, sucrose, and xylose at varying concentrations to evaluate specificity, linear dynamic range, signal size, and signal-to-noise ratio. A comparison study was done by substituting an equivalent activity unit concentration of glucose oxidase (GOx) for GDH-FAD. Glucose dehydrogenase flavin adenine dinucleotide was found to be more sensitive than GOx, producing larger oxidation currents than GOx on an identical glucose concentration gradient, and GDH-FAD exhibited larger slope response (-5.65 × 10(-7) versus -3.11 × 10(-7) A/mM), signal-to-noise ratio (18.04 versus 2.62), and linear dynamic range (0-30 versus 0-10 mM), and lower background signal (-7.12 versus -261.63 nA) than GOx under the same assay conditions. GDH-FAD responds equally to glucose and xylose but is otherwise specific for glucose. Glucose dehydrogenase flavin adenine dinucleotide compares favorably with GOx in many sensor-relevant attributes and may enable measurement of glucose concentrations both higher and lower than those measurable by GOx. GDH-FAD is a viable enzyme to use in the proposed amperometric tear glucose sensor system and perhaps also in detecting extreme hypoglycemia or hyperglycemia in blood. © 2011 Diabetes Technology Society.

Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

NASA Astrophysics Data System (ADS)

Jindo, K.; Matsumoto, K.; García Izquierdo, C.; Sonoki, T.; Sanchez-Monedero, M. A.

2014-07-01

Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere with colorimetric determination of the hydrolysed PNP, either by the sorption of the substrate or the reaction product of hydrolysis into biochar surface. The aim of the present work is to study the biochar sorption capacity for PNP in biochar-blended composting mixtures in order to assess its impact on the estimation of the colorimetric-based enzymatic assays. A retention test was conducted by adding a solution of known amounts of PNP in universal buffer solution (pH = 5, 6.5 and 11, corresponding to the β-glucosidase, acid and alkaline phosphatase activity assays, respectively), in samples taken at the initial stage and after maturation stage from four different composting piles (two manure composting piles; PM: poultry manure, CM: cow manure and two other similar piles containing 10% of additional biochar (PM + B, CM + B)). The results show that biochar-blended composts (PM + B, CM + B) generally exhibited low enzymatic activities, compared to manure compost without biochar (PM, CM). In terms of the difference between the initial and maturation stage of composting process, the PNP retention in biochar was shown higher at maturation stage, caused most probably by an enlarged proportion of biochar inside compost mixture after the selective degradation of easily decomposable organic matter. TThe retention of PNP on biochar was influenced by pH dependency of sorption capacity of biochar and/or PNP solubility, since PNP was more efficiently retained by biochar at low pH values (5 and 6.5) than at high pH values (11).

A Disposable Tear Glucose Biosensor—Part 3: Assessment of Enzymatic Specificity

PubMed Central

Lan, Kenneth; McAferty, Kenyon; Shah, Pankti; Lieberman, Erica; Patel, Dharmendra R; Cook, Curtiss B; La Belle, Jeffrey T

2011-01-01

Background A concept for a tear glucose sensor based on amperometric measurement of enzymatic oxidation of glucose was previously presented, using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) as the enzyme. Glucose dehydrogenase flavin adenine dinucleotide is further characterized in this article and evaluated for suitability in glucose-sensing applications in purified tear-like saline, with specific attention to the effect of interfering substances only. These interferents are specifically saccharides that could interact with the enzymatic activity seen in the sensor's performance. Methods Bench top amperometric glucose assays were performed using an assay solution of GDH-FAD and ferricyanide redox mediator with samples of glucose, mannose, lactose, maltose, galactose, fructose, sucrose, and xylose at varying concentrations to evaluate specificity, linear dynamic range, signal size, and signal-to-noise ratio. A comparison study was done by substituting an equivalent activity unit concentration of glucose oxidase (GOx) for GDH-FAD. Results Glucose dehydrogenase flavin adenine dinucleotide was found to be more sensitive than GOx, producing larger oxidation currents than GOx on an identical glucose concentration gradient, and GDH-FAD exhibited larger slope response (-5.65 × 10-7 versus -3.11 × 10-7 A/mM), signal-to-noise ratio (18.04 versus 2.62), and linear dynamic range (0–30 versus 0–10 mM), and lower background signal (-7.12 versus -261.63 nA) than GOx under the same assay conditions. GDH-FAD responds equally to glucose and xylose but is otherwise specific for glucose. Conclusion Glucose dehydrogenase flavin adenine dinucleotide compares favorably with GOx in many sensor-relevant attributes and may enable measurement of glucose concentrations both higher and lower than those measurable by GOx. GDH-FAD is a viable enzyme to use in the proposed amperometric tear glucose sensor system and perhaps also in detecting extreme hypoglycemia or hyperglycemia in blood. PMID:22027303

Poly β-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

PubMed

Yan, Huijuan; He, Leiliang; Zhao, Wenjie; Li, Jishan; Xiao, Yue; Yang, Ronghua; Tan, Weihong

2014-11-18

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

2016-01-01

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

NASA Astrophysics Data System (ADS)

Jindo, K.; Matsumoto, K.; García Izquierdo, C.; Sonoki, T.; Sanchez-Monedero, M. A.

2014-03-01

Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere colorimetric determination of the hydrolysed PNP, either by the sorption of the substrate or the reaction-product of hydrolysis into biochar surface. The aim of the present work is to study the biochar sorption capacity for PNP in biochar-blended composting mixtures in order to assess its impact on the estimation of the colorimetric-based enzymatic assays. A retention test was conducted by adding a solution of known amounts of PNP in universal buffer solution (pH = 5, 6.5 and 11, corresponding to the β-glucosidase, acid and alkaline phosphatase activity assays, respectively), in samples taken at the initial stage and after maturation stage from 4 different composting piles (two manure composting piles (PM: poultry manure, CM: cow manure) and two other similar piles containing 10% of additional biochar (PM + B, CM + B)). The results show that biochar blended composts (PM + B, CM + B) generally exhibited low enzymatic activities, compared to manure compost without biochar (PM, CM). In terms of the difference between the initial and maturation stage of composting process, the PNP retention in biochar was shown more clearly at maturation stage, caused by an enlarged proportion of biochar inside compost mixture after the selective degradation of easily decomposable organic matter. The retention of PNP was more pronounced at low pH (5 and 6.5) than at high pH (11), 3 reflecting on pH dependency of sorption 49 capacity of biochar and/or PNP 50 solubility.

Falsely Elevated Plasma Creatinine Due to an Immunoglobulin M Paraprotein.

PubMed

McGill, Mitchell R; Vijayan, Anitha; Trulock, Elbert P; Witt, Chad A; Kohler, Giselle D; Scott, Mitchell G

2016-11-01

The most common method for measuring plasma creatinine is based on its reaction with picric acid. However, enzymatic methods are becoming more popular due to improved specificity. We present a case of falsely elevated plasma creatinine values obtained by an enzymatic method that turned out to be due to a monoclonal immunoglobulin M (IgM) paraprotein. A 63-year-old woman evaluated for lung transplantation had falsely increased plasma creatinine levels (1.54-1.71mg/dL; corresponding to estimated glomerular filtration rates of 32-36 mL/min/1.73m 2 ) as measured by the Roche Creatinine plus enzymatic assay when compared with the picric acid-based procedure and several other enzymatic methods, which gave plasma creatinine values of 0.7 to 0.8mg/dL. Serum protein electrophoresis revealed an IgM κ light chain paraprotein. Removal of high-molecular-weight (>30kDa) proteins by ultrafiltration reduced the patient's plasma creatinine level by the Roche enzymatic method to 0.7mg/dL. Addition of the patient's immunoglobulin fraction to plasma from other patients with normal plasma creatinine levels resulted in values that were increased by 0.58 to 0.62mg/dL. Furthermore, removal of non-IgM immunoglobulins with protein G-coupled beads did not eliminate the interference from the patient's plasma. Taken together, these studies demonstrate that falsely elevated plasma creatinine values by the Roche enzymatic method can be due to an IgM paraprotein. Copyright © 2016 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

PubMed

Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

2018-08-01

Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction. © 2018 Wiley Periodicals, Inc.

Naringin suppresses cell metastasis and the expression of matrix metalloproteinases (MMP-2 and MMP-9) via the inhibition of ERK-P38-JNK signaling pathway in human glioblastoma.

PubMed

Aroui, Sonia; Aouey, Bakhta; Chtourou, Yassine; Meunier, Annie-Claire; Fetoui, Hamadi; Kenani, Abderraouf

2016-01-25

Naringin (4',5,7-trihydroxyflavanone 7-rhamnoglucoside), a natural flavonoid, has pharmacological properties. In the present study, we investigated the anti-metastatic activity of naringin and its molecular mechanism(s) of action in human glioblastoma cells. Naringin exhibits inhibitory effects on the invasion and adhesion of U87 cells in a concentration-dependent manner by Matrigel Transwell and cell adhesion assays. Naringin also inhibited the migration of U87 cells in a concentration-dependent manner by wound-healing assay. Additional experiments showed that naringin treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase (MMP)-2 and MMP-9 using a gelatin zymography assay and western blot analyses. Furthermore, naringin was able to reduce the protein phosphorylation of extracellular signal-regulated kinase ERK, p38 mitogen-activated protein kinase and c-Jun N-terminal kinase by western blotting. Collectively, our data showed that naringin attenuated the MAPK signaling pathways including ERK, JNK and p38 and resulted in the downregulation of the expression and enzymatic activities of MMP-2, MMP-9, contributing to the inhibition of metastasis in U87 cells. These findings proved that naringin may offer further application as an antimetastatic agent. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification.

PubMed

Yin, Huanshun; Wang, Mo; Li, Bingchen; Yang, Zhiqing; Zhou, Yunlei; Ai, Shiyun

2015-01-15

A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn(2+) through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Multicenter evaluation of an enzymatic method for glycated albumin.

PubMed

Paleari, Renata; Bonetti, Graziella; Callà, Cinzia; Carta, Mariarosa; Ceriotti, Ferruccio; Di Gaetano, Nicola; Ferri, Marilisa; Guerra, Elena; Lavalle, Gabriella; Cascio, Claudia Lo; Martino, Francesca Gabriela; Montagnana, Martina; Moretti, Marco; Santini, Gabriele; Scribano, Donata; Testa, Roberto; Vero, Anna; Mosca, Andrea

2017-06-01

The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA 1c test is not reliable. We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

Selective ultrasound-enhanced enzymatic hydrolysis of oleuropein to its aglycon in olive (Olea europaea L.) leaf extracts.

PubMed

Delgado-Povedano, María Del Mar; Priego-Capote, Feliciano; Luque de Castro, María Dolores

2017-04-01

Hydrolysis of oleuropein, the main phenol in olive (Olea europaea L.) leaf extracts, to oleuropein aglycon and other subsequent products in the hydrolytic pathway can be catalyzed by different enzymes. Three of the most used hydrolases were assayed to catalyze the process, and β-glucosidase from Aspergillus niger was selected. Acceleration of the enzymatic hydrolysis by ultrasound (US) was studied using a Box-Behnken design (duty cycle, amplitude, cycle time) and an oleuropein standard, and the optimum US conditions for achieving maximum yield of oleuropein aglycon were 0.5s/s duty cycle, 50% amplitude and 45s cycle. The method was applied to obtain oleuropein aglycon from commercial and laboratory extracts from olive leaves, which may have a pharmacological use as deduced by its healthy properties. The kinetics of the US-assisted enzymatic hydrolysis was monitored by analysis of the target compounds using liquid chromatography-tandem mass spectrometry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of Soybean Proteins for All Allergic Subjects.

PubMed

Panda, Rakhi; Tetteh, Afua O; Pramod, Siddanakoppalu N; Goodman, Richard E

2015-11-04

Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of β-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

PubMed Central

2011-01-01

Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy. PMID:21864341

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

PubMed

Sobolewski, Ireneusz; Polska, Katarzyna; Zylicz-Stachula, Agnieszka; Jeżewska-Frąckowiak, Joanna; Rak, Janusz; Skowron, Piotr

2011-08-24

Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Jasmonic acid-amino acid conjugation enzyme assays.

PubMed

Rowe, Martha L; Staswick, Paul E

2013-01-01

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase.

PubMed

Kim, Jihoon; Chang, Ji-Youn; Kim, Yoon-Young; Kim, Moon-Jong; Kho, Hong-Seop

2018-05-01

To investigate the effects of the molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase in solution and on the hydroxyapatite surface. Hyaluronic acids of four different molecular weights (10 kDa, 100 kDa, 1 MDa, and 2 MDa), hen egg-white lysozyme, bovine lactoperoxidase, and human whole saliva were used. Viscosity values of hyaluronic acids were measured using a cone-and-plate viscometer at six different concentrations (0.1-5.0 mg/mL). Enzymatic activities of lysozyme and peroxidase were examined by hydrolysis of fluorescein-labeled Micrococcus lysodeikticus and oxidation of fluorogenic 2',7'-dichlorofluorescein to fluorescing 2',7'-dichlorofluorescein, respectively. In solution assays, only 2 MDa-hyaluronic acid significantly inhibited lysozyme activities in saliva. In surface assays, hyaluronic acids inhibited lysozyme and peroxidase activities; the inhibitory activities were more apparent with high-molecular-weight ones in saliva than in purified enzymes. The 100 kDa-hyaluronic acid at 5.0 mg/mL, 1 MDa-one at 0.5 mg/mL, and 2 MDa-one at 0.2 mg/mL showed viscosity values similar to those of human whole saliva at a shear rate range required for normal oral functions. The differences among the influences of the three conditions on the enzymatic activities were not statistically significant. High-molecular-weight hyaluronic acids at low concentration and low-molecular-weight ones at high concentration showed viscosity values similar to those of human whole saliva. Inhibitory effects of hyaluronic acids on lysozyme and peroxidase activities were more significant with high-molecular-weight ones on the surface and in saliva compared with in solution and on purified enzymes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sensitive high-throughput screening for the detection of reducing sugars.

PubMed

Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

2012-01-01

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

Determining Glutathione Levels in Plants.

PubMed

Sahoo, Smita; Awasthi, Jay Prakash; Sunkar, Ramanjulu; Panda, Sanjib Kumar

2017-01-01

Upon exposure to abiotic stresses, plants tend to accumulate excessive amounts of reactive oxygen species (ROS) that inturn react with cellular lipids, proteins, and DNA. Therefore, decreasing ROS accumulation is indispensible to survive under stress, which is accomplished by inducing enzymatic and nonenzymatic antioxidant defense pathways. Glutathione, particularly reduced glutathione (GSH), represents a principal anitioxidant that could decrease ROS through scavenging them directly or indirectly through ascorbate-glutathione cycle or GSH peroxidases. Glutathione content can be determined using HPLC or spectrophotometric assays. In this chapter, we provided detailed assays to determine total, reduced, and oxidized gluathione using spectrophotometric method.

Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

2015-04-01

On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC bacteria. Therefore the applicability of on-site enzymatic activity determination as a direct surrogate or proxy parameter for microbiological standard assays and quantification of fecal indicator bacteria (FIB) concentration could not be approved and further research in this field is necessary. Presently we conclude that rapid on-site detection of enzymatic activity is applicable for surface water monitoring and that it constitutes a complementary on-site monitoring parameter with high potential. Selection of the type of measured enzymatic activities has to be done on a catchment-specific basis and further work is needed to learn more about its detailed information characteristics in different habitats. The accomplishment of this method detecting continuous data of enzymatic activity in high temporal resolution caused by a target bacterial member is on the way of becoming a powerful tool for water quality monitoring, health related water quality- and early warning requirements.

Deceiving proteins! A case of lymphoma and high creatinine.

PubMed

Metraiah, El Hakem Abdelkarim; Regan, Helen; Louw, Johanna; Kidder, Dana

2017-01-23

Estimation of kidney function by measuring serum creatinine is one the commonest laboratory tests conducted in clinical practice. Enzymatic methods are often used to measure serum creatinine. Clinicians should be aware of the limitations of these methods, such as test interference with paraproteins.We present a case of falsely elevated serum creatinine in a patient referred for renal biopsy. The combination of fluctuating creatinine and normal blood urea level was unusual. Serum protein electrophoresis revealed the presence of an IgM paraprotein. Further investigations confirmed an underlying diagnosis of lymphoplasmacytoid lymphoma. This case highlights how IgM paraprotein can interfere with creatinine estimation by enzymatic assay and the utility of alternative methods of estimating serum creatinine. 2017 BMJ Publishing Group Ltd.

Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reindl, W.; Deng, K.; Gladden, J.M.

2011-05-01

The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation ofmore » the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.« less

Heterogeneous glycation of cancellous bone and its association with bone quality and fragility.

PubMed

Karim, Lamya; Vashishth, Deepak

2012-01-01

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage.

Heterogeneous Glycation of Cancellous Bone and Its Association with Bone Quality and Fragility

PubMed Central

Karim, Lamya; Vashishth, Deepak

2012-01-01

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage. PMID:22514706

Screening of plants used in the European traditional medicine to treat memory disorders for acetylcholinesterase inhibitory activity and anti amyloidogenic activity.

PubMed

Lobbens, Eva S B; Vissing, Karina J; Jorgensen, Lene; van de Weert, Marco; Jäger, Anna K

2017-03-22

Plants used in the traditional medicine of Europe to treat memory dysfunction and/or to enhance memory were investigated for activity against the underlying mechanisms of Alzheimer's disease. To investigate 35 ethanolic extracts of plants, selected using an ethnopharmacological approach, for anti-amyloidogenic activity as well as an ability to inhibit the enzymatic activity of acetylcholinesterase. The anti-amyloidogenic activity of the extracts against amyloid beta was investigated by Thioflavin T fibrillation assays and the ability to inhibit the enzymatic activity of acetylcholinesterase was evaluated monitoring the hydrolysis of acetylthiocholine RESULTS: Under the experimental conditions investigated, extracts of two plants, Carum carvi and Olea sylvestris, inhibited amyloid beta fibrillation considerably, eight plant extracts inhibited amyloid beta fibrillation to some extent, 16 plant extracts had no effect on amyloid beta fibrillation and nine extracts accelerated fibrillation of amyloid beta. Furthermore, five plant extracts from Corydalis species inhibited the enzymatic activity of acetylcholinesterase considerably, one plant extract inhibited the enzymatic activity of acetylcholinesterase to some extent and 29 plant extract had no effect on the enzymatic activity of acetylcholinesterase. An optimal extract in this study would possess acetylcholinesterase inhibitory activity as well as anti-amyloidogenic activity in order to address multiple facets of Alzheimer's disease, until the molecular origin of the disease is unraveled. Unfortunately no such extract was found. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Fabrication and electrochemistry characteristics of nickel-doped diamond-like carbon film toward applications in non-enzymatic glucose detection

NASA Astrophysics Data System (ADS)

Liu, Chi-Wen; Chen, Wei-En; Sun, Yin Tung Albert; Lin, Chii-Ruey

2018-04-01

This research work focused on the fabrication of nickel-doped diamond-like carbon (DLC) films and their characteristics including of surface morphology, microstructure, and electrochemical aiming at applications in non-enzymatic glucose detection. Novel nanodiamond target was employed in unbalanced magnetron radio-frequency co-sputtering process to prepared high quality Ni-doped DLC thin film at room temperature. TEM analysis reveals a highly uniform distribution of Ni crystallites in amorphous carbon matrix with fraction ranged from 3 to 11.5 at.% which is considered as active sites for the glucose detection. Our cyclic voltammetry measurements using 0.1 M H2SO4 solution demonstrated that the as-prepared Ni-doped DLC films possess large electrochemical potential window of 2.12 V, and this was also observed to be significantly reduced at high Ni doping level owing to lower sp3 fraction. The non-enzymatic glucose detection investigation indicates that the Ni-doped DLC thin film electrode prepared under 7 W of DC sputtering power on Ni target possesses good detecting performance, high stability, and high sensitivity to glucose concentration up to 10 mM, even with the existence of uric acid and ascorbic acid. The peak current was observed to be proportional to glucose concentration and scanning rate, demonstrating highly reversibility redox process of the film electrode and glucose.

Microplate-based filter paper assay to measure total cellulase activity.

PubMed

Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian

2004-12-30

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%. 2004 Wiley Periodicals, Inc.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

PubMed Central

2010-01-01

Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU PMID:20637080

Evaluation of in vitro enzymatic and non-enzymatic antioxidant properites of leaf extract from Alpinia Purpurata (Vieill.) K. Schum.

PubMed

Raj, Chinthamony Arul; Ragavendran, Paramasivam; Sophia, Dominic; Starlin, Thangarajan; Rathi, Muthian Ahalliya; Gopalakrishnan, Velliyur Kanniappan

2016-09-01

To evaluate the enzymatic and non-enzymatic antioxidants of leaf extract from Alpinia purpurata. One gram of fresh leaf of Alpinia purpurata was grinded in 2 mL of 50% ethanol and centrifuged at 10,000×g at 4°C for 10 min. The supernatant obtained was used within 4 h for various enzymatic antioxidants assays like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), ascorbate oxidase, peroxidase, polyphenol oxidase (PPO) and non-enzymatic antioxidants such as vitamin C, total reduced glutathione (TRG) and lipid peroxidation (LPO). The leaf extract of Alpinia purpurata possess antioxidants like vitamin C 472.92±6.80 μg/mg protein, GST 372.11±5.70 μmol of 1-chloro 2,4 dinitrobenzene (CDNB)-reduced glutathione (GSH) conjugate formed/min/mg protein, GPx 281.69±6.43 μg of glutathione oxidized/min/mg protein, peroxidases 173.12±9.40 μmol/g tissue, TRG 75.27±3.55 μg/mg protein, SOD 58.03±2.11 U/mg protein, CAT 46.70±2.35 μmol of H2O2 consumed/min/mg protein in high amount whereas ascorbate oxidase 17.41±2.46 U/g tissue, LPO 2.71±0.14 nmol/L of malondialdehyde formed/min/mg protein and PPO 1.14±0.11 μmol/g tissue in moderate amount. Alpinia purpurata has the potential to scavenge the free radicals and protect against oxidative stress causing diseases. In future, Alpinia purpurata may serve as a good pharmacotherapeutic agent.

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Preliminary studies on the activities of spin traps as scavengers of free radicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogunbiyi, P.O.; Washington, I.

1991-03-15

The spin trapping agents, N-t-Butyl-a-phenyl-nitrone (PBN) and 5,5-Dimethyl-1-pyroline-N-oxide (DMPO) have been used to investigate the primary free radicals involved in various tissue injuries. Also, PBN and DMPO can provide some protection against free radical-induced lung injuries. However, their therapeutic potentials as free radical scavengers remained unexamined. In this study, the effects of PBN and DMPO on guinea pig lung microsomal lipid peroxidation were investigated using thiobarbituric acid-reactive substance assay. Superoxide anions (O{sup 2}{minus}) were generated in an enzymatic and a non-enzymatic system. PBN and DMPO each, significantly inhibited NADPH-stimulated lipid peroxidation irrespective of the presence of Fe{sup 3+}. Cytochrome cmore » reduction by the enzymatic and nitro blue tetrazolium reduction by the non-enzymatic O{sup 2}{minus} generating systems were both inhibited by PBN and DMPO as well as superoxide dismutase and dimethyl sulfoxide when compared with the controls. The spin traps exhibited lower potencies in these systems than the reference compounds, SOD and DMSO, which are well established as O{sup 2}{minus} and hydroxyl radical scavengers respectively. Results demonstrate the free radical scavenging properties of PBN and DMPO. This is an indication of their possible usefulness as antioxidants.« less

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

The effect of phosphomonoesterases on the oxygen isotope composition of phosphate

NASA Astrophysics Data System (ADS)

von Sperber, Christian; Kries, Hajo; Tamburini, Federica; Bernasconi, Stefano M.; Frossard, Emmanuel

2014-01-01

Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi). These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. During the enzymatic hydrolysis an isotopic fractionation (ε) occurs leaving an imprint on the oxygen isotope composition of the released Pi which might be used to trace phosphorus in the environment. Therefore, enzymatic assays with acid phosphatases from wheat germ and potato tuber and alkaline phosphatase from Escherichia coli were prepared in order to determine the oxygen isotope fractionation caused by these enzymes. Adenosine 5‧ monophosphate and glycerol phosphate were used as substrates. The oxygen isotope fractionation caused by acid phosphatases is 20-30‰ smaller than for alkaline phosphatases, resulting in a difference of 5-7.5‰ in δ18O of Pi depending on the enzyme. We attribute the enzyme dependence of the isotopic fractionation to distinct reaction mechanisms of the two types of phosphatases. The observed difference is large enough to distinguish between the two enzymatic processes in environmental samples. These findings show that the oxygen isotope composition of Pi can be used to trace different enzymatic processes, offering an analytical tool that might contribute to a better understanding of the P-cycle in the environment.

Norepinephrine metabolism in neuron: dissociation between 3,4-dihydroxyphenylglycol and 3,4-dihydroxymandelic acid pathways.

PubMed

Dong, Wen-Xin; Ni, Xiang-Lian

2002-01-01

To investigate the pre-synaptic metabolism of norepinephrine (NE), judged by variations in plasma concentration of 3,4-dihydroxyphenylglycol (DHPG) and 3,4-dihydroxymandelic acid (DOMA). Pithed and electrically stimulated (2.5 Hz) rats were given intravenous infusion of exogenous NE (6 nmol . kg-1 . min-1). Plasma NE, DHPG, DOMA, and the activities of mono- amine oxidases (MAO) were measured with the radio-enzymatic assay. Exogenous NE induces an about 100-fold increase in plasma NE concentration while blood pressure remained within normal limits. A 12-fold increase in plasma DHPG and 1.2-fold increase for DOMA were observed. When NE transportation across the pre-synaptic membrane was inhibited by desipramine (2 mg/kg, iv), a great reduction in plasma DHPG concentration (about 25 % of control) was observed while DOMA remained unchanged. When MAO-A activity was inhibited to 25 % of control by clorgyline (2 mg/kg, iv) and MAO-B to 30 % by deprenyl, the plasma DHPG and DOMA concentrations were reduced to 15 % and 70 % of controls, and to 26 % and 76 % of controls, respectively. When clorgyline and deprenyl were combined, plasma DHPG was vanished (less than 2 % of control) while plasma DOMA remained in the same range (72 % of control). The metabolizing system of NE in pre-synapse, associating with the pre-synaptic reuptake plus oxidative deamination on the external membrane of mitochondria, is predominant for the reduction to DHPG.

Development of a thyroperoxidase inhibition assay for high ...

EPA Pesticide Factsheets

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR, LifeTechnologies), were employed in an endpoint assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics including Z’, dynamic range, and activity using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z’ score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2’,4,4’-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negati

Comparative hepatic in vitro depletion and metabolite formation of major perfluorooctane sulfonate precursors in Arctic polar bear, beluga whale, and ringed seal.

PubMed

Letcher, Robert J; Chu, Shaogang; McKinney, Melissa A; Tomy, Gregg T; Sonne, Christian; Dietz, Rune

2014-10-01

Perfluorooctane sulfonate (PFOS) has been reported to be among the most concentrated persistent organic pollutants in Arctic marine wildlife. The present study examined the in vitro depletion of major PFOS precursors, N-ethyl-perfluorooctane sulfonamide (N-EtFOSA) and perfluorooctane sulfonamide (FOSA), as well as metabolite formation using an assay based on enzymatically viable liver microsomes for three top Arctic marine mammalian predators, polar bear (Ursus maritimus), beluga whale (Delphinapterus leucas), and ringed seal (Pusa hispida), and in laboratory rat (Rattus rattus) serving as a general mammalian model and positive control. Rat assays showed that N-EtFOSA (38 nM or 150 ng mL(-1)) to FOSA metabolism was >90% complete after 10 min, and at a rate of 23 pmol min(-1) mg(-1) protein. Examining all species in a full 90 min incubation assay, there was >95% N-EtFOSA depletion for the rat active control and polar bear microsomes, ∼65% for ringed seals, and negligible depletion of N-EtFOSA for beluga whale. Concomitantly, the corresponding in vitro formation of FOSA from N-EtFOSA was also quantitatively rat≈polar bear>ringed seal>beluga whale. A lack of enzymatic ability and/or a rate too slow to be detected likely explains the lack of N-EtFOSA to FOSA transformation for beluga whale. In the same assays, the depletion of the FOSA metabolite was insignificant (p>0.01) and with no concomitant formation of PFOS metabolite. This suggests that, in part, a source of FOSA is the biotransformation of accumulated N-EtFOSA in free-ranging Arctic ringed seal and polar bear. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

PubMed

Monteiro, S; Santos, R

2018-04-01

To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. © 2017 The Society for Applied Microbiology.

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

PubMed

Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-01-15

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

Antioxidant and cytotoxic activities of three species of tropical seaweeds.

PubMed

Chia, Yin Yin; Kanthimathi, M S; Khoo, Kong Soo; Rajarajeswaran, Jayakumar; Cheng, Hwee Ming; Yap, Wai Sum

2015-09-29

Three species of seaweeds (Padina tetrastromatica, Caulerpa racemosa and Turbinaria ornata) are widely consumed by Asians as nutraceutical food due to their antioxidant properties. Studies have shown that these seaweeds exhibit bioactivities which include antimicrobial, antiviral, anti-hypertensive and anticoagulant activities. However, investigations into the mechanisms of action pertaining to the cytotoxic activity of the seaweeds are limited. The aim of this study was to determine the antioxidant and cytotoxic activities of whole extracts of P. tetrastromatica, C. racemosa and T. ornata, including the cellular events leading to the apoptotic cell death of the extract treated-MCF-7 cells. Bioassay guided fractionation was carried out and the compounds identified. Powdered samples were sequentially extracted for 24 h. Their antioxidant activities were assessed by the DPPH radical, superoxide, nitric oxide and hydroxyl radical scavenging assays. The cytotoxic activity of the extract-treated MCF-7cells was assessed using the MTT assay. The most potent fraction was subjected to bioassay guided fractionation with column chromatography. All the fractions were tested for cytotoxic activity, caspase activity and effect on DNA fragmentation. All three seaweeds showed potent radical scavenging activities in the various assays. The activity of the cellular antioxidant enzymes, superoxide dismutase, catalase and glutathione reductase, in MCF-7 cells, decreased in a time-dependent manner. The partially purified fractions exhibited higher cytotoxic activity, as assessed by the MTT assay, than the whole extracts in the breast adenocarcinoma cell line, MCF-7. LC-MS analysis revealed the presence of bioactive alkaloids such as camptothecin, lycodine and pesudopelletierine. Based on the results obtained, all three seaweeds are rich sources of enzymatic and non-enzymatic antioxidants which could contribute to their reported medicinal benefits.

Liquid chromatography-tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma.

PubMed

Christner, Susan; Guo, Jianxia; Parise, Robert A; Ringeval, Melanie; Hoye, Adam T; Wipf, Peter; Epperly, Michael W; Greenberger, Joel S; Beumer, Jan H; Eiseman, Julie L

2018-02-20

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50μL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions.

PubMed

Zehender, Hartmut; Mayr, Lorenz M

2007-10-01

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.

Enzymatic Comparisons of Aspergillus niger PhyA and Escherichia coli AppA2 Phytases

USDA-ARS?s Scientific Manuscript database

This study was to compare three phytase activity assays and kinetics of Aspergillus niger PhyA and Escherichia coli AppA2 phytases expressed in Pichia pastoris at the observed stomach pH of 3.5. In Experiment 1, equivalent phytase activities in the crude preparations of PhyA and AppA2 were tested ...

Isolation of Tannin-Degrading Lactobacilli from Humans and Fermented Foods

PubMed Central

Osawa, Ro; Kuroiso, Keiko; Goto, Satoshi; Shimizu, Akira

2000-01-01

Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species. PMID:10877812

Method of detecting and counting bacteria in body fluids

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Picciolo, G. L. (Inventor)

1973-01-01

A novel method is reported for determining bacterial levels in urine samples, which method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of non-bacterial ATP. After the removal of non-bacterial ATP, the bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay using an enzyme obtained from the firefly.

Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase

PubMed Central

Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

1999-01-01

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors. PMID:10051623

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase.

PubMed

Guimarães, Rafaela; Calhelha, Ricardo C; Froufe, Hugo J C; Abreu, Rui M V; Carvalho, Ana Maria; Queiroz, Maria João R P; Ferreira, Isabel C F R

2016-01-01

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine.

PubMed

Liu, Qingtao; Chen, Yuqi; Yuan, Minglai; Du, Guocheng; Chen, Jian; Kang, Zhen

2017-09-01

Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, ureABCEFGDH , in Bacillus paralicheniformis ATCC 9945a and characterized it in Escherichia coli Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes. IMPORTANCE An iron-containing, oxygen-stable acid urease from B. paralicheniformis ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry. Copyright © 2017 American Society for Microbiology.

A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine

PubMed Central

Liu, Qingtao; Chen, Yuqi; Yuan, Minglai; Chen, Jian

2017-01-01

ABSTRACT Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, ureABCEFGDH, in Bacillus paralicheniformis ATCC 9945a and characterized it in Escherichia coli. Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes. IMPORTANCE An iron-containing, oxygen-stable acid urease from B. paralicheniformis ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry. PMID:28646111

Characterization of gossypol biosynthetic pathway

PubMed Central

Tian, Xiu; Ruan, Ju-Xin; Huang, Jin-Quan; Fang, Xin; Chen, Zhi-Wen; Hong, Hui; Wang, Ling-Jian; Mao, Ying-Bo; Lu, Shan; Zhang, Tian-Zhen; Chen, Xiao-Ya

2018-01-01

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion. PMID:29784821

Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis

PubMed Central

Gril, Brunilde; Palmieri, Diane; Qian, Yong; Smart, DeeDee; Ileva, Lilia; Liewehr, David J.; Steinberg, Seth M.; Steeg, Patricia S.

2010-01-01

Purpose Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved anti-angiogenic drug that targets VEGFR1-3, PDGFRβ, PDGFRα and c-kit, for prevention of experimental brain metastases and mechanism of action. Experimental Design In vitro assays included B-Raf enzymatic assays, western blots and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 post-injection. Brain metastases were counted histologically, imaged and immunostained. Results Treatment with 100 mg/kg pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (p<0.0001) and 39% decline in micrometastases (p=0.004). In vitro, pazopanib was directly anti-proliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib treated brain metastases while blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon MRI imaging by 55% (p=0.067), without affecting brain metastasis vascular density. Conclusions The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor, and suggest its potential for prevention of brain metastatic colonization of HER2+ breast cancer. PMID:21081656

Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.

PubMed

Gril, Brunilde; Palmieri, Diane; Qian, Yong; Smart, DeeDee; Ileva, Lilia; Liewehr, David J; Steinberg, Seth M; Steeg, Patricia S

2011-01-01

Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved antiangiogenic drug that targets VEGFR1, VEGFR2, VEGFR3, PDGFRβ, PDGFRα, and c-kit, for prevention of experimental brain metastases and mechanism of action. In vitro assays included B-Raf enzymatic assays, Western blots, and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 postinjection. Brain metastases were counted histologically, imaged, and immunostained. Treatment with 100 mg/kg of pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (P < 0.0001) and a 39% decline in micrometastases (P = 0.004). In vitro, pazopanib was directly antiproliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib-treated brain metastases whereas blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon magnetic resonance imaging (MRI) by 55% (P = 0.067), without affecting brain metastasis vascular density. The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor and suggest its potential for prevention of brain metastatic colonization of HER2(+) breast cancer. ©2010 AACR.

Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

PubMed

Turgeon, Nathalie; Toulouse, Marie-Josée; Ho, Jim; Li, Dongqing; Duchaine, Caroline

2017-02-01

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis μ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

A rhodium(III)-based inhibitor of autotaxin with antiproliferative activity.

PubMed

Kang, Tian-Shu; Wang, Wanhe; Zhong, Hai-Jing; Liang, Jia-Xin; Ko, Chung-Nga; Lu, Jin-Jian; Chen, Xiu-Ping; Ma, Dik-Lung; Leung, Chung-Hang

2017-02-01

Cancer of the skin is by far the most common of all cancers. Melanoma accounts for only about 1% of skin cancers but causes a large majority of skin cancer deaths. Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), regulates physiological and pathological functions of lysophosphatidic acid (LPA), and is thus an important therapeutic target. We synthesized ten metal-based complexes and a novel cyclometalated rhodium(III) complex 1 was identified as an ATX enzymatic inhibitor using multiple methods, including ATX enzymatic assay, thermal shift assay, western immunoblotting and so on. Protein thermal shift assays showed that 1 increased the melting temperature (T m ) of ATX by 3.5°C. 1 also reduced ATX-LPA mediated downstream survival signal pathway proteins such as ERK and AKT, and inhibited the activation of the transcription factor nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3). 1 also exhibited strong anti-proliferative activity against A2058 melanoma cells (IC 50 =0.58μM). Structure-activity relationship indicated that both the rhodium(III) center and the auxiliary ligands of complex 1 are important for bioactivity. 1 represents a promising scaffold for the development of small-molecule ATX inhibitors for anti-tumor applications. To our knowledge, complex 1 is the first metal-based ATX inhibitor reported to date. Rhodium complexes will have the increased attention in therapeutic and bioanalytical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and validation of an automated enzyme assay for paracetamol (acetaminophen).

PubMed

Morris, H C; Overton, P D; Ramsay, J R; Campbell, R S; Hammond, P M; Atkinson, T; Price, C P

1990-02-28

A rapid, enzymatic assay for serum or plasma paracetamol has been developed with the potential for adaptation to a wide range of clinical analysers. The method involves the action of an amidase enzyme to produce 4-aminophenol from paracetamol, which in turn reacts with 8-hydroxyquinoline in the presence of manganese ions to form a blue dye. Two stable reagents are used and excellent precision is achieved over the drug concentration range 0-2.5 mmol/l. The method, which is complete within 6 min, has been validated using a Monarch centrifugal analyser and shows no significant interference from endogenous serum compounds, drugs or paracetamol metabolites.

Treatment of supermarket vegetable wastes to be used as alternative substrates in bioprocesses.

PubMed

Díaz, Ana Isabel; Laca, Amanda; Laca, Adriana; Díaz, Mario

2017-09-01

Fruits and vegetables have the highest wastage rates at retail and consumer levels. These wastes have promising potential for being used as substrates in bioprocesses. However, an effective hydrolysis of carbohydrates that form these residues has to be developed before the biotransformation. In this work, vegetable wastes from supermarket (tomatoes, green peppers and potatoes) have been separately treated by acid, thermal and enzymatic hydrolysis processes in order to maximise the concentration of fermentable sugars in the final broth. For all substrates, thermal and enzymatic processes have shown to be the most effective. A new combined hydrolysis procedure including these both treatments was also assayed and the enzymatic step was successfully modelled. With this combined hydrolysis, the percentage of reducing sugars extracted was increased, in comparison with the amount extracted from non-hydrolysed samples, approximately by 30% in the case of tomato and green peeper wastes. For potato wastes this percentage increased from values lower than 1% to 77%. In addition, very low values of fermentation inhibitors were found in the final broth. Copyright © 2017. Published by Elsevier Ltd.

Removal of bisphenol A in canned liquid food by enzyme-based nanocomposites

NASA Astrophysics Data System (ADS)

Tapia-Orozco, Natalia; Meléndez-Saavedra, Fanny; Figueroa, Mario; Gimeno, Miquel; García-Arrazola, Roeb

2018-02-01

Laccase from Trametes versicolor was immobilized on TiO2 nanoparticles; the nanocomposites obtained were used for the removal of bisphenol A (BPA) in a liquid food matrix. To achieve a high enzymatic stability over a wide pH range and at temperatures above 50 °C, the nanocomposite structures were prepared by both physical adsorption and covalent linking of the enzyme onto the nanometric support. All the nanocomposite structures retained 40% of their enzymatic activity after 60 days of storage. Proof-of-concept experiments in aqueous media using the nanocomposites resulted on a > 60% BPA removal after 48 h and showed that BPA was depleted within 5 days. The nanocomposites were tested in canned liquid food samples; the removal reached 93.3% within 24 h using the physically adsorbed laccase. For the covalently linked enzyme, maximum BPA removal was 91.3%. The formation of BPA dimers and trimers was observed in all the assays. Food samples with sugar and protein contents above 3 and 4 mg mL-1 showed an inhibitory effect on the enzymatic activity.

Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

PubMed

Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

2016-09-01

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

[HTLV-1 in a Mapuche population].

PubMed

Inostroza, J; Díaz, P; Saunier, C

1990-12-01

The seroprevalence of HTLV-1 in 405 serum samples from South American Indians, Mapuches, from the IXth region of Chile was investigated using enzyme linked immunosorbent assay (ELISA). Six samples were positive and only 3 of them were also positive by western blot and radio immuno precipitation assay. This corresponds to a seroprevalence of 0.74% for HTLV-1 in healthy Mapuches, which differs from that observed in other populations throughout the world. Additional studies are necessary to evaluate the real magnitudes of HTLV-1 infection in Chile.

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

Highly Sensitive Biosensing with Solid-State Nanopores Displaying Enzymatically Reconfigurable Rectification Properties.

PubMed

Pérez-Mitta, Gonzalo; Peinetti, Ana S; Cortez, M Lorena; Toimil-Molares, María Eugenia; Trautmann, Christina; Azzaroni, Omar

2018-05-09

Molecular design of biosensors based on enzymatic processes taking place in nanofluidic elements is receiving increasing attention by the scientific community. In this work, we describe the construction of novel ultrasensitive enzymatic nanopore biosensors employing "reactive signal amplifiers" as key elements coupled to the transduction mechanism. The proposed framework offers innovative design concepts not only to amplify the detected ionic signal and develop ultrasensitive nanopore-based sensors but also to construct nanofluidic diodes displaying specific chemo-reversible rectification properties. The integrated approach is demonstrated by electrostatically assembling poly(allylamine) on the anionic pore walls followed by the assembly of urease. We show that the cationic weak polyelectrolyte acts as a "reactive signal amplifier" in the presence of local pH changes induced by the enzymatic reaction. These bioinduced variations in proton concentration ultimately alter the protonation degree of the polyamine resulting in amplifiable, controlled, and reproducible changes in the surface charge of the pore walls, and consequently on the generated ionic signals. The "iontronic" response of the as-obtained devices is fully reversible, and nanopores are reused and assayed with different urea concentrations, thus ensuring reliable design. The limit of detection (LOD) was 1 nM. To the best of our knowledge, this value is the lowest LOD reported to date for enzymatic urea detection. In this context, we envision that this approach based on the use of "reactive signal amplifiers" into solid-state nanochannels will provide new alternatives for the molecular design of highly sensitive nanopore biosensors as well as (bio)chemically addressable nanofluidic elements.

Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow's milk proteins.

PubMed

Morisawa, Y; Kitamura, A; Ujihara, T; Zushi, N; Kuzume, K; Shimanouchi, Y; Tamura, S; Wakiguchi, H; Saito, H; Matsumoto, K

2009-06-01

Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood. We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay. Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3-4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested beta-lactoglobulin or alpha-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay. Heat treatment alone did not alter the molecular weight of beta-lactoglobulin or alpha-casein. Heat treatment of beta-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for alpha-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both beta-lactoglobulin and alpha-casein to induce histamine release from passively sensitized basophils. Heat treatment reduced the allergenicity of beta-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of alpha-casein.

STUDIES ON ORGANIC ACID METABOLISM,

DTIC Science & Technology

Lipoic acid metabolism: The acetyl and succinyl thio esters of civinyl dimercapto were prepared by chemical and enzymatic means. The oxidation...reduction reactions of the disulfide-dimercapto groups in pyrimidine nucleotide-linked reactions were explored in the initial lipoic acid assay organiam...disulfide couple. The studies appeared to indicate a bound form of lipoic acid to be the coenzyme, and suggested that an amide or possibly another

Phospholipids and their degrading enzyme in the tears of soft contact lens wearers.

PubMed

Yamada, Masakazu; Mochizuki, Hiroshi; Kawashima, Motoko; Hata, Seiichiro

2006-12-01

Low tear phospholipids levels are associated with tear film instability in soft contact lens wearers. We assayed levels of phospholipids and their degrading enzyme secretory phospholipase A2 (sPLA2) both in tears and deposited on contact lenses composed of 2 hydrophilic materials after 1 day of routine use. Polymacon (Medalist; FDA group 1, low water/nonionic) and Etafilcon A (One Day Acuvue; group 4, high water/ionic) contact lenses were worn for 12 hours by 16 experienced contact lens wearers. Phospholipids in tear fluids and deposited on contact lenses were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Double-antibody sandwich ELISA was used to determine group IIa sPLA2 concentrations, and sPLA2 activity was assayed using 1,2-diheptanoyl thio-phosphatidylcholine as substrate. Phospholipids concentrations in tears with Polymacon and Etafilcon A were 186 +/- 39 and 162 +/- 33 microg/mL, respectively. The latter concentration was significantly lower than that observed in the same subjects when not wearing contact lenses (P = 0.0023). In tears, both group IIa sPLA2 concentrations and enzymatic activity remained unchanged, regardless of lens wearing. However, Etafilcon A (0.57 +/- 0.09 microg/lens) showed more group IIa sPLA2 deposition than Polymacon (0.01 +/- 0.01 microg/lens; P < 0.001). Furthermore, group IIa sPLA2 deposited on Etafilcon A but not on Polymacon lenses retained its enzymatic activity. Significant differences of group IIa sPLA2 deposition were found in the 2 lenses tested. Such deposition might induce phospholipid hydrolysis in tears and thereby promote tear film instability in hydrophilic contact lens wearers.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

PubMed

Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

2005-10-01

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Elimination of Isoxazolyl-Penicillins antibiotics in waters by the ligninolytic native Colombian strain Leptosphaerulina sp. considerations on biodegradation process and antimicrobial activity removal.

PubMed

Copete-Pertuz, Ledys S; Plácido, Jersson; Serna-Galvis, Efraím A; Torres-Palma, Ricardo A; Mora, Amanda

2018-07-15

In this work, Leptosphaerulina sp. (a Colombian native fungus) significantly removed three Isoxazolyl-Penicillin antibiotics (IP): oxacillin (OXA, 16000 μg L -1 ), cloxacillin (CLX, 17500 μg L -1 ) and dicloxacillin (DCX, 19000 μg L -1 ) from water. The biological treatment was performed at pH 5.6, 28 °C, and 160 rpm for 15 days. The biotransformation process and lack of toxicity of the final solutions (antibacterial activity (AA) and cytotoxicity) were tested. The role of enzymes in IP removal was analysed through in vitro studies with enzymatic extracts (crude and pre-purified) from Leptosphaerulina sp., commercial enzymes and enzymatic inhibitors. Furthermore, the applicability of mycoremediation process to a complex matrix (simulated hospital wastewater) was evaluated. IP were considerably abated by the fungus, OXA was the fastest degraded (day 6), followed by CLX (day 7) and DCX (day 8). Antibiotics biodegradation was associated to laccase and versatile peroxidase action. Assays using commercial enzymes (i.e. laccase from Trametes versicolor and horseradish peroxidase) and inhibitors (EDTA, NaCl, sodium acetate, manganese (II) ions) confirmed the significant role of enzymatic transformation. Whereas, biomass sorption was not an important process in the antibiotics elimination. Evaluation of AA against Staphylococcus aureus ATCC 6538 revealed that Leptosphaerulina sp. also eliminated the AA. In addition, the cytotoxicity assay (MTT) on the HepG2 cell line demonstrated that the IP final solutions were non-toxic. Finally, Leptosphaerulina sp. eliminated OXA and its AA from synthetic hospital wastewater at 6 days. All these results evidenced the potential of Leptosphaerulina sp. mycoremediation as a novel environmentally friendly process for the removal of IP from aqueous systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

PubMed

Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

2016-01-01

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.

PubMed

Lynch, James T; Cockerill, Mark J; Hitchin, James R; Wiseman, Daniel H; Somervaille, Tim C P

2013-11-01

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays. Copyright © 2013 Elsevier Inc. All rights reserved.

A Fluorescent G-quadruplex Sensor for Chemical RNA Copying.

PubMed

Giurgiu, Constantin; Wright, Tom; O'Flaherty, Derek; Szostak, Jack

2018-06-25

Non-enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Here, we present a highly parallel assay for template directed RNA synthesis that relies on the intrinsic fluorescence of a 2-aminopurine modified G-quadruplex. We demonstrate the application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopolan,G.; Thwin, M.; Gopalakrishnakone, P.

Russell's viper (Vipera russelli, also known as Daboia russelli) is one of the major causes of fatal snakebites. To date, five Daboia russelli subspecies have been recognized. Daboiatoxin (DbTx) is the main lethal phospholipase A{sub 2} (PLA{sub 2}) toxin in the venom of D. russelli siamensis (Myanmar viper) and has strong neurotoxic, myotoxic and cytotoxic activities. DbTx and its homologous neurotoxins viperotoxin F from D. russelli formosensis (Taiwan viper) and vipoxin from the Bulgarian sand viper V. ammodytes meridionalis consist of complexes between a nontoxic acidic PLA2 protein and an enzymatically active basic PLA2. DbTx and viperotoxin F are presynapticmore » toxins, while vipoxin is postsynaptic. The two chains of DbTx have been separated and their PLA2 enzymatic activity has been measured using the secretory PLA2 assay kit. The enzymatic activity of DbTx chain B is reduced by 30% of its original activity by chain A in a unimolar ratio, thus indicating that DbTx chain A acts as an inhibitor. The lethal activity of the two chains has also been studied in male albino mice and chain A is less lethal than chain B. The crystal structure of DbTx has also been determined and its structural details are compared with those of the two homologues. Furthermore, an attempt is made to correlate the sequence and structural determinants of these toxins with their enzymatic activities and their pharmacological effects.« less

Application of Nanoparticle Technology to Reduce the Anti-Microbial Resistance through β-Lactam Antibiotic-Polymer Inclusion Nano-Complex.

PubMed

Salamanca, Constain H; Yarce, Cristhian J; Roman, Yony; Davalos, Andrés F; Rivera, Gustavo R

2018-02-10

Biocompatible polymeric materials with potential to form functional structures in association with different therapeutic molecules have a high potential for biological, medical and pharmaceutical applications. Therefore, the capability of the inclusion of nano-Complex formed between the sodium salt of poly(maleic acid- alt -octadecene) and a β-lactam drug (ampicillin trihydrate) to avoid the chemical and enzymatic degradation and enhance the biological activity were evaluated. PAM-18Na was produced and characterized, as reported previously. The formation of polymeric hydrophobic aggregates in aqueous solution was determined, using pyrene as a fluorescent probe. Furthermore, the formation of polymer-drug nano-complexes was characterized by Differential Scanning Calorimetry-DSC, viscometric, ultrafiltration/centrifugation assays, zeta potential and size measurements were determined by dynamic light scattering-DLS. The PAM-18Na capacity to avoid the chemical degradation was studied through stress stability tests. The enzymatic degradation was evaluated from a pure β-lactamase, while the biological degradation was determined by different β-lactamase producing Staphylococcus aureus strains. When ampicillin was associated with PAM-18Na, the half-life time in acidic conditions increased, whereas both the enzymatic degradation and the minimum inhibitory concentration decreased to a 90 and 75%, respectively. These results suggest a promissory capability of this polymer to protect the β-lactam drugs against chemical, enzymatic and biological degradation.

Application of Nanoparticle Technology to Reduce the Anti-Microbial Resistance through β-Lactam Antibiotic-Polymer Inclusion Nano-Complex

PubMed Central

Yarce, Cristhian J.; Roman, Yony; Davalos, Andrés F.; Rivera, Gustavo R.

2018-01-01

Biocompatible polymeric materials with potential to form functional structures in association with different therapeutic molecules have a high potential for biological, medical and pharmaceutical applications. Therefore, the capability of the inclusion of nano-Complex formed between the sodium salt of poly(maleic acid-alt-octadecene) and a β-lactam drug (ampicillin trihydrate) to avoid the chemical and enzymatic degradation and enhance the biological activity were evaluated. PAM-18Na was produced and characterized, as reported previously. The formation of polymeric hydrophobic aggregates in aqueous solution was determined, using pyrene as a fluorescent probe. Furthermore, the formation of polymer-drug nano-complexes was characterized by Differential Scanning Calorimetry-DSC, viscometric, ultrafiltration/centrifugation assays, zeta potential and size measurements were determined by dynamic light scattering-DLS. The PAM-18Na capacity to avoid the chemical degradation was studied through stress stability tests. The enzymatic degradation was evaluated from a pure β-lactamase, while the biological degradation was determined by different β-lactamase producing Staphylococcus aureus strains. When ampicillin was associated with PAM-18Na, the half-life time in acidic conditions increased, whereas both the enzymatic degradation and the minimum inhibitory concentration decreased to a 90 and 75%, respectively. These results suggest a promissory capability of this polymer to protect the β-lactam drugs against chemical, enzymatic and biological degradation. PMID:29439391

Receptor-based screening assays for the detection of antibiotics residues - A review.

PubMed

Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui

2017-05-01

Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

Micropollutant degradation via extracted native enzymes from activated sludge.

PubMed

Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

2016-05-15

A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the full variety of indigenous enzymatic activity of the activated sludge source material could not be restored, experimental modifications, e.g. different lysate filtration, significantly enhanced specific enzyme activities (e.g. >96% removal of the antibiotic erythromycin). Therefore, the approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Antimicrobial and antioxidant effect of methanolic Crinum jagus bulb extract in wound healing

PubMed Central

Udegbunam, Sunday Ositadimma; Udegbunam, Rita Ijeoma; Nnaji, Theophilus Okafor; Anyanwu, Madubuike Umunna; Kene, Raphel Okoli Chukwujekwu; Anika, Silavanus Maduka

2015-01-01

Aim: The aim of this study was to evaluate the antimicrobial and antioxidant effects of Crinum jagus (J. Thomps.) Dandy methanolic bulb extract in wound healing. Materials and Methods: Phytochemical screening revealed the presence of alkaloids, glycosides, tannins, and saponins in the extract. In vitro antimicrobial activity of the extract was determined by agar well diffusion method. In vivo antimicrobial activity of the extract was determined by microbial assay of excision wound in rats contaminated with Staphylococcus aureus, Bacillus subtilis, Pseudomonas areuginosa, and Candida albicans and treated with 300 mg/kg body weight (bw) of 10 and 5% methanolic C. jagus bulb extract ointment (MCJBEO), respectively. Enzymatic antioxidant effect of the extract was determined in vivo by assaying superoxide dismutase (SOD) and catalase (CAT) activity, and malondialdehyde (MDA) level in excision wound biopsies of rats treated with 10 and 5% MCJBEO, respectively, following standard methods. Non-enzymatic antioxidant effect of the extract was determined in vitro using diphenylpicrylhydrazyl (DPPH) method following standard procedure. Results: The extract exhibited in vitro antimicrobial effect in a concentration-dependent manner with one hundred (100) mg/ml concentration of the extract having the highest inhibitory zone diameter for B. subtilis (25 mm), S. aureus (21 mm), and C. albicans (14 mm) followed by the 50, 25 and 12.5 mg/ml concentrations, respectively. B. subtilis, S. aureus, and C. albicans were not isolated from wounds of animals treated with both extract concentrations 10% and 5% MCJBEO, and reference drug (framycetin sulfate/clotrimazole). Activities of the enzymatic antioxidants SOD and CAT in wound biopsies treated with 10% MCJBEO were significantly (P < 0.05) higher when compared with those treated with 5% MCJBEO. Significantly (P < 0.05) decreased MDA level of wound biopsies from extract-treated rats was observed. The extract exhibited non-enzymatic antioxidant (DPPH) effect in a concentration-dependent manner. Conclusion: This study has shown that an anti-microbial and antioxidant effects could possibly be part of mechanism by which C. jagus bulb extract promote wound healing process. PMID:26401415

A fluorescent microplate assay for diarrheic shellfish toxins.

PubMed

Vieytes, M R; Fontal, O I; Leira, F; Baptista de Sousa, J M; Botana, L M

1997-06-01

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

PubMed

Chung, Thomas D Y

2013-01-01

Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates.

PubMed

Mirazizi, Fatemeh; Bahrami, Azita; Haghbeen, Kamahldin; Shahbani Zahiri, Hossein; Bakavoli, Mehdi; Legge, Raymond L

2016-12-01

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.

Security Assessment of Cyberphysical Digital Microfluidic Biochips.

PubMed

Ali, Sk Subidh; Ibrahim, Mohamed; Sinanoglu, Ozgur; Chakrabarty, Krishnendu; Karri, Ramesh

2016-01-01

A digital microfluidic biochip (DMFB) is an emerging technology that enables miniaturized analysis systems for point-of-care clinical diagnostics, DNA sequencing, and environmental monitoring. A DMFB reduces the rate of sample and reagent consumption, and automates the analysis of assays. In this paper, we provide the first assessment of the security vulnerabilities of DMFBs. We identify result-manipulation attacks on a DMFB that maliciously alter the assay outcomes. Two practical result-manipulation attacks are shown on a DMFB platform performing enzymatic glucose assay on serum. In the first attack, the attacker adjusts the concentration of the glucose sample and thereby modifies the final result. In the second attack, the attacker tampers with the calibration curve of the assay operation. We then identify denial-of-service attacks, where the attacker can disrupt the assay operation by tampering either with the droplet-routing algorithm or with the actuation sequence. We demonstrate these attacks using a digital microfluidic synthesis simulator. The results show that the attacks are easy to implement and hard to detect. Therefore, this work highlights the need for effective protections against malicious modifications in DMFBs.

Evaluating In Vitro DNA Damage Using Comet Assay.

PubMed

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

In vitro radiosensitizing effects of ultrasmall gadolinium based particles on tumour cells.

PubMed

Mowat, P; Mignot, A; Rima, W; Lux, F; Tillement, O; Roulin, C; Dutreix, M; Bechet, D; Huger, S; Humbert, L; Barberi-Heyob, M; Aloy, M T; Armandy, E; Rodriguez-Lafrasse, C; Le Duc, G; Roux, S; Perriat, P

2011-09-01

Since radiotherapy is widely used in cancer treatment, it is essential to develop strategies which lower the irradiation burden while increasing efficacy and become efficient even in radio resistant tumors. Our new strategy is relying on the development of solid hybrid nanoparticles based on rare-earth such as gadolinium. In this paper, we then evidenced that gadolinium-based particles can be designed to enter efficiently into the human glioblastoma cell line U87 in quantities that can be tuned by modifying the incubation conditions. These sub-5 nm particles consist in a core of gadolinium oxide, a shell of polysiloxane and are functionalized by diethylenetriaminepentaacetic acid (DTPA). Although photoelectric effect is maximal in the [10-100 keV] range, such particles were found to possess efficient in-vitro radiosensitizing properties at an energy of 660 keV by using the "single-cell gel electrophoresis comet assay," an assay that measures the number of DNA damage that occurs during irradiation. Even more interesting, the particles have been evidenced by MTT assays to be also efficient radiosensitizers at an energy of 6 MeV for doses comprised between 2 and 8 Gy. The properties of the gadolinium-based particles give promising opening to a particle-assisted radio-therapy by using irradiation systems already installed in the majority of hospitals.

Bio-barcode gel assay for microRNA

NASA Astrophysics Data System (ADS)

Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

2014-02-01

MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

Fabrication and optimisation of a fused filament 3D-printed microfluidic platform

NASA Astrophysics Data System (ADS)

Tothill, A. M.; Partridge, M.; James, S. W.; Tatam, R. P.

2017-03-01

A 3D-printed microfluidic device was designed and manufactured using a low cost (2000) consumer grade fusion deposition modelling (FDM) 3D printer. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication. However, in this work, the optical transparency of the device was improved through manufacture optimisation to such a point that optical colorimetric assays can be performed in a 50 µl device. A colorimetric enzymatic cascade assay was optimised using glucose oxidase and horseradish peroxidase for the oxidative coupling of aminoantipyrine and chromotropic acid to produce a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of glucose in solution. For comparison the assay was run in standard 96 well plates with a commercial plate reader. The results show the accurate and reproducible quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device with performance comparable to that of a plate reader assay.

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

PubMed

Lin, Li-Ching; Chen, Chi-Fen; Ho, Chun-Te; Liu, Jun-Jen; Liu, Tsan-Zon; Chern, Chi-Liang

2018-04-01

This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ‑glutamylcysteine synthetase heavy chain (γ‑GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ‑GCSh activity were measured by HPLC methods. γ‑GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ‑GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ‑GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. Our data unequivocally demonstrate that γ‑GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Radio-sensitization by Piper longumine of human breast adenoma MDA-MB-231 cells in vitro.

PubMed

Yao, Jian-Xin; Yao, Zhi-Feng; Li, Zhan-Feng; Liu, Yong-Biao

2014-01-01

The current study investigated the effects of Piper longumine on radio-sensitization of human breast cancer MDA-MB-231 cells and underlying mechanisms. Human breast cancer MDA-MB-231 cells were cultured in vitro and those in logarithmic growth phase were selected for experiments divided into four groups: control, X-ray exposed, Piper longumine, and Piper longumine combined with X-rays. Conogenic assays were performed to determine the radio-sensitizing effects. Cell survival curves were fitted by single-hit multi-target model and then the survival fraction (SF), average lethal dose (D0), quasi-threshold dose (Dq) and sensitive enhancement ratio (SER) were calculated. Cell apoptosis was analyzed by flow cytometry (FCM).Western blot assays were employed for expression of apoptosis-related proteins (Bc1-2 and Bax) after treatment with Piper longumine and/or X-ray radiation. The intracellular reactive oxygen species (ROS) level was detected by FCM with a DCFH-DA probe. The cloning formation capacity was decreased in the group of piperlongumine plus radiation, which displayed the values of SF2, D0, Dq significantly lower than those of radiation alone group and the sensitive enhancement ratio (SER) of D0 was1.22 and 1.29, respectively. The cell apoptosis rate was increased by the combination treatment of Piper longumine and radiation. Piper longumine increased the radiation-induced intracellular levels of ROS. Compared with the control group and individual group, the combination group demonstrated significantly decreased expression of Bcl-2 with increased Bax. Piper longumine at a non-cytotoxic concentration can enhance the radio-sensitivity of MDA- MB-231cells, which may be related to its regulation of apoptosis-related protein expression and the increase of intracellular ROS level, thus increasing radiation-induced apoptosis.

Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

PubMed

Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

2017-08-15

Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

An HTRF® Assay for the Protein Kinase ATM.

PubMed

Adams, Phillip; Clark, Jonathan; Hawdon, Simon; Hill, Jennifer; Plater, Andrew

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase that plays a key role in the regulation of DNA damage pathways and checkpoint arrest. In recent years, there has been growing interest in ATM as a therapeutic target due to its association with cancer cell survival following genotoxic stress such as radio- and chemotherapy. Large-scale targeted drug screening campaigns have been hampered, however, by technical issues associated with the production of sufficient quantities of purified ATM and the availability of a suitable high-throughput assay. Using a purified, functionally active recombinant ATM and one of its physiological substrates, p53, we have developed an in vitro FRET-based activity assay that is suitable for high-throughput drug screening.

Phosphorescent inner filter effect-based sensing of xanthine oxidase and its inhibitors with Mn-doped ZnS quantum dots.

PubMed

Tang, Dandan; Zhang, Jinyi; Zhou, Rongxin; Xie, Ya-Ni; Hou, Xiandeng; Xu, Kailai; Wu, Peng

2018-05-10

Overexpression and crystallization of uric acid have been recognized as the course of hyperuricemia and gout, which is produced via xanthine oxidase (XOD)-catalyzed oxidation of xanthine. Therefore, the medicinal therapy of hyperuricemia and gout is majorly based on the inhibition of the XOD enzymatic pathway. The spectroscopic nature of xanthine and uric acid, namely both absorption (near the ultraviolet region) and emission (non-fluorescent) characteristics, hinders optical assay development for XOD analysis. Therefore, the state-of-the-art analysis of XOD and the screening of XOD inhibitors are majorly based on chromatography. Here, we found the near ultraviolet absorption of uric acid overlapped well with the absorption of a large bandgap semiconductor quantum dots, ZnS. On the other hand, the intrinsic weak fluorescence of ZnS QDs can be substantially improved via transition metal ion doping. Therefore, herein, we developed an inner filter effect-based assay for XOD analysis and inhibitor screening with Mn-doped ZnS QDs. The phosphorescence of Mn-doped ZnS QDs could be quenched by uric acid generated from xanthine catabolism by XOD, leading to the phosphorescence turn-off detection of XOD with a limit of detection (3σ) of 0.02 U L-1. Furthermore, the existence of XOD inhibitors could inhibit the XOD enzymatic reaction, resulting in weakened phosphorescence quenching. Therefore, the proposed assay could also be explored for the facile screening analysis of XOD inhibitors, which is important for the potential medicinal therapy of hyperuricemia and gout.

A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)

PubMed Central

Deshpande, Kanchanmala; Mishra, Rupesh K.; Bhand, Sunil

2010-01-01

A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg·mL−1 with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%. PMID:22163555

A Novel Chemical Compound for Inhibition of SARS Coronavirus Helicase.

PubMed

Lee, Jin-Moo; Cho, Jin-Beom; Ahn, Hee-Chul; Jung, Woong; Jeong, Yong-Joo

2017-11-28

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)- N -(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC 50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

Production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins by anaerobic gram-positive cocci.

PubMed Central

Marshall, R; Kaufman, A K

1981-01-01

Clinical isolates of Peptococcus and Peptostreptococcus species and Streptococcus intermedius strains were obtained from local hospitals. After confirmed identification, each isolate was tested for the in vitro production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins. Of the 60 strains studied, 18 had enzymatic activity. The variability of enzyme production suggests that such assays are not suitable as an aid to identification of these organisms. PMID:7229018

An Evaluation of the NIDS (registered trademark) ACE (trademark) Test

DTIC Science & Technology

2014-06-30

included chemicals commonly used for drinking water disinfection (chlorine and chloramine), byproducts of cyanobacteria blooms (geosmin and 2...duration field toxicity test for Army drinking water . One component of the ESB the ACE Test) is an enzymatic assay designed to detect neurotoxicants...adverse health effects is the presence of toxic industrial chemicals (TICs) in drinking water . The current field water test kit – the water quality

Cosmeceutical Effects of Galactomannan Fraction from Arenga pinnata Fruits In vitro

PubMed Central

Yanti; Madriena; Ali, Soegianto

2017-01-01

Background: Cosmeceuticals refer to natural cosmetics with medical-like benefits due to their bioactive contents. Sugar palm fruit (Arenga pinnata) extract has been claimed for its anti-aging effect in vitro. However, its active compounds for cosmeceuticals is still unclear. Objective: This study was aimed to extract galactomannan from A. pinnata fruits and test its efficacy for tyrosinase inhibition, antioxidant, and anti-photoaging activities in vitro. Materials and Methods: Galactomannan from A. pinnata fruits was extracted by freeze drying and identified for its chemical compounds by using pyrolysis gas chromatography-mass spectrometry (py-GC/MS). Galactomannan was tested for its tyrosinase inhibition in both cell-based (melanocytes) and enzymatic assays, antioxidant activity using ferrous ion chelating assay (FCA) assay, and anti-photoaging activity for inhibiting the gene expression of matrix metalloproteinase-1 (MMP-1) and MMP-13 in macrophages using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Results: Identification of galactomannan fraction from A. pinnata fruits by py-GC/MS mainly consisted of oxonium ion and glucosides. For cellular assay, galactomannan at 5 μg/mL inhibited >50% of tyrosinase activity in melanocytes induced by phorbol myristate acetate. At the enzymatic level, galactomannan at similar concentration showed less tyrosinase activity inhibition (~20%). FCA results showed that galactomannan at 10 μg/mL exerted >50% of antioxidant activity. The qRT-PCR data indicated that galactomannan at 5 μg/mL inhibited >50% of MMP-1 and MMP-13 gene expressions in ultraviolet B-treated macrophages. Conclusion: Galactomannan fraction from A. pinnata fruits has efficacy for enlightening effect, antioxidant, and anti-photoaging activity in the dose-independent pattern, indicating its cosmeceutical effects for skin healthcare. SUMMARY A. pinnata fruit containing galactomannan has cosmeceutical potentials through enlightening effect, antioxidant, and anti-photoaging activity in vitro.Galactomannan fraction has inhibitory effect on tyrosinase activity in both cellular melanocytes and enzymatic systems.Galactomannan fraction has strong protection against UVB-irradiation effect by inhibiting collagenase genes (MMP-1 and MMP-13) in macrophages. Abbreviations Used: Py-GC/MS: Pyrolysis-Gas Chromatography-Mass Spectrometry; FCA: Ferrous chelating activity; MMP: Matrix metalloproteinase; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction; PMA: Phorbol myristate acetate; UV: Ultraviolet; RPMI: Roswell Park Memorial Institute; DMEM: Dulbecco's modified eagle media; FBS: Fetal bovine serum; PBS: Phosphate buffered saline; MTT: 3-(4,5-diethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide; L-DOPA: L-3,4-dihydroxyphenylalanine; EDTA: Ethylenediaminetetraacetic acid; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; DPPH: 1,1-diphenyl-2-picryl-hydrazyl; SPF: Sun protection factor PMID:28250652

Multiphoton manipulations of enzymatic photoactivity in aspartate aminotransferase.

PubMed

Hill, Melissa P; Freer, Lucy H; Vang, Mai C; Carroll, Elizabeth C; Larsen, Delmar S

2011-04-21

The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal 5'-phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc.2010, 132 (47), 16953-16961]. This increases the net catalytic activity (k(cat)) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.

Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.

PubMed

Yoshida, Akihiro; Yoshimura, Mamiko; Ohara, Naoya; Yoshimura, Shigeru; Nagashima, Shiori; Takehara, Tadamichi; Nakayama, Koji

2009-11-01

Hydrogen sulfide is one of the predominant volatile sulfur compounds (VSCs) produced by oral bacteria. This study developed and evaluated a system for detecting hydrogen sulfide production by oral bacteria. L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (METase) and beta carbon-sulfur (beta C-S) lyase were used to degrade homocysteine and cysteine, respectively, to produce hydrogen sulfide. Enzymatic reactions resulting in hydrogen sulfide production were assayed by reaction with bismuth trichloride, which forms a black precipitate when mixed with hydrogen sulfide. The enzymatic activities of various oral bacteria that result in hydrogen sulfide production and the capacity of bacteria from periodontal sites to form hydrogen sulfide in reaction mixtures containing L-cysteine or DL-homocysteine were assayed. With L-cysteine as the substrate, Streptococcus anginosus FW73 produced the most hydrogen sulfide, whereas Porphyromonas gingivalis American Type Culture Collection (ATCC) 33277 and W83 and Fusobacterium nucleatum ATCC 10953 produced approximately 35% of the amount produced by the P. gingivalis strains. Finally, the hydrogen sulfide found in subgingival plaque was analyzed. Using bismuth trichloride, the hydrogen sulfide produced by oral bacteria was visually detectable as a black precipitate. Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

High-throughput selection for cellulase catalysts using chemical complementation.

PubMed

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

PubMed Central

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

Cholinesterase assay for monitoring the kinetics of the JD6. 5 organophosphorus acid anhydrase in detoxification of diisopropylfluorophosphate. Final report, January-June 1988

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, H.R.; Cheng, T.C.; DeFrank, J.J.

1992-06-01

In the present studies, cholinesterase was used for monitoring the enzymatic activities of the JD6.5 organophosphorus acid anhydrase. The kinetic data indicated that: (1) the first order of kinetic constants (k) and Vmax values of the enzymatic reactions increased as the concentrations of the enzyme increased; (2) while the half-life (tl/2) of diisopropylfluorophosphate (DFP) hydrolysis decreased as the enzyme concentrations increased; (3) the minimum time required for hydrolysis of 9mM of DFP was 3 min at the concentrations of the enzyme present; Km values of DFP were found to be in range of 5mM; and (4) both MnCl2 and NaClmore » were found to be required for the optimal activity of the enzyme.« less

Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system

USDA-ARS?s Scientific Manuscript database

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyc...

Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation.

PubMed

Mathy-Hartert, M; Hogge, L; Sanchez, C; Deby-Dupont, G; Crielaard, J M; Henrotin, Y

2008-07-01

Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.

CD73 promotes proliferation and migration of human cervical cancer cells independent of its enzyme activity.

PubMed

Gao, Zhao-Wei; Wang, Hui-Ping; Lin, Fang; Wang, Xi; Long, Min; Zhang, Hui-Zhong; Dong, Ke

2017-02-15

CD73 has both enzymatic and non-enzymatic functions in cells. As a nucleotidase, CD73 plays its enzymatic function by catalyzing the hydrolysis of AMP into adenosine and phosphate. In addition to this, accumulating data have shown that CD73 is a key regulatory molecule involved in cancer growth and metastasis, but this non-enzymatic function of CD73 in cervical cancer cells has not been well studied. CD73 was overexpressed by pcDNA-NT5E expression vector transfection in Hela and SiHa cells. Cell's proliferation and migration were evaluated by MTT and scratch healing assay. The CD73 specific antagonist -APCP was used to inhibit CD73 enzymatic activity. And the effect of APCP on CD73 activity was determined by high performance liquid chromatography (HPLC). Expression level was assessed by qRT-PCR and western blotting. In the present study, we used Hela and SiHa cell lines to evaluate the effects of CD73 on cervical cancer cells proliferation and migration, and further explore the potential regulating mechanisms. Our data showed that CD73 overexpression significantly promoted cervical cancer cells proliferation and migration, and this promotive effect was not reverted by blocking CD73 enzymatic activity, both in Hela and SiHa cells. On the other hand, our data also showed that high concentration of adenosine inhibited Hela and SiHa cells proliferation and migration. These results demonstrated that the promotive effect of CD73 on cervical cancer cells proliferation and migration in vitro was independent from its enzymatic activity (i.e. production of adenosine). Furthermore, the expressions of EGFR, VEGF and Akt were significantly increased in CD73 overexpression Hela and SiHa cells. Our data suggested that CD73 might promote proliferation and migration via potentiating EGFR/Akt and VEGF/Akt pathway, which was independent of CD73 enzyme activity. These data provide a novel insight into the regulating function of CD73 in cancer cells and suggest that CD73 may be promising therapeutic target in cervical cancer.

Protein immobilization techniques for microfluidic assays

PubMed Central

Kim, Dohyun; Herr, Amy E.

2013-01-01

Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

Protective effects of azelaic acid against high-fat diet-induced oxidative stress in liver, kidney and heart of C57BL/6J mice.

PubMed

Muthulakshmi, Shanmugam; Saravanan, Ramalingam

2013-05-01

Excess fat intake induces hyperinsulinaemia, increases nutrient uptake and lipid accumulation, amplifies ROS generation, establishes oxidative stress and morphological changes leading to tissue injury in the liver, kidney and heart of high-fat diet (HFD)-fed mice. The effect of azelaic acid (AzA), a C9 α,ω-dicarboxylic acid, against HFD-induced oxidative stress was investigated by assaying the activities and levels of antioxidants and oxidative stress markers in the liver, kidney and heart of C57BL/6J mice. Mice were segregated into two groups, one fed standard diet (NC) and the other fed high-fat diet (HFD) for 15 weeks. HFD-fed mice were subjected to intragastric administration of AzA (80 mg/kg BW)/RSG (10 mg/kg BW) during 11-15 weeks. Glucose, insulin, triglycerides, hepatic and nephritic markers were analysed in the plasma and the activity of enzymatic, non-enzymatic antioxidants and lipid peroxidation markers were examined in the plasma/erythrocytes, liver, kidney and heart of normal and experimental mice. We inferred significant decrease in enzymatic and non-enzymatic antioxidants along with significant increase in glucose, insulin, hepatic and nephritic markers, triglycerides and lipid peroxidation markers in HFD-fed mice. Administration of AzA could positively restore the levels of plasma glucose, insulin, triglycerides, hepatic and nephritic markers to near normal. AzA increased the levels of enzymatic and nonenzymatic antioxidants with significant reduction in the levels of lipid peroxidation markers. Histopathological examination of liver, kidney and heart substantiated these results. Hence, we put forward that AzA could counteract the potential injurious effects of HFD-induced oxidative stress in C57BL/6J mice.

Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of Mammalian cell substrate derived biotherapeutics.

PubMed

Zhan, Dejin; Roy, Margaret R; Valera, Christine; Cardenas, Jesse; Vennari, Joann C; Chen, Janice W; Liu, Shengjiang

2002-12-01

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Structural Requirements For Bone Sialoprotein Binding And Modulation Of Matrix Metalloproteinase-2

PubMed Central

Jain, Alka; Karadag, Abdullah; Fisher, Larry W.; Fedarko, Neal S.

2008-01-01

Bone sialoprotein (BSP) has been shown to induce limited gelatinase activity in latent matrix metalloproteinase-2 (MMP-2) without removal of the propeptide and to restore enzymatic activity to MMP-2 previously inhibited by tissue inhibitor of matrix metalloproteinase-2 (TIMP2). The current study identifies structural domains in human BSP and MMP-2 that contribute to these interactions. The 26 amino acid domain encoded by exon 4 of BSP is shown by a series of binding and activity assays to be involved in the displacement of MMP-2′s propeptide from the active site and thereby inducing the protease activity. Binding assays in conjunction with enzyme activity assays demonstrate that both amino- and carboxy-terminal domains of BSP contribute to restoration of activity to TIMP2-inhibited MMP-2, while the MMP-2 hemopexin domain is not required for reactivation. PMID:18729384

Structural requirements for bone sialoprotein binding and modulation of matrix metalloproteinase-2.

PubMed

Jain, Alka; Karadag, Abdullah; Fisher, Larry W; Fedarko, Neal S

2008-09-23

Bone sialoprotein (BSP) has been shown to induce limited gelatinase activity in latent matrix metalloproteinase-2 (MMP-2) without removal of the propeptide and to restore enzymatic activity to MMP-2 previously inhibited by tissue inhibitor of matrix metalloproteinase-2 (TIMP2). The current study identifies structural domains in human BSP and MMP-2 that contribute to these interactions. The 26 amino acid domain encoded by exon 4 of BSP is shown by a series of binding and activity assays to be involved in the displacement of MMP-2's propeptide from the active site and thereby inducing the protease activity. Binding assays in conjunction with enzyme activity assays demonstrate that both amino- and carboxy-terminal domains of BSP contribute to restoration of activity to TIMP2-inhibited MMP-2, while the MMP-2 hemopexin domain is not required for reactivation.

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

PubMed Central

Eady, J. J.; Orta, T.; Dennis, M. F.; Stratford, M. R.; Peacock, J. H.

1995-01-01

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines. PMID:7577452

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor.

PubMed

Vargas, E; Ruiz, M A; Campuzano, S; Reviejo, A J; Pingarrón, J M

2016-03-31

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h(-1)) and ease of automation. Copyright © 2016 Elsevier B.V. All rights reserved.

The status of glutathione peroxidase, superoxide dismutase, vitamins A, C, E and malondialdehyde in patients with cardiovascular disease in Zahedan, Southeast Iran.

PubMed

Karajibani, Mansour; Hashemi, Mohammad; Montazerifar, Farzaneh; Bolouri, Ahmad; Dikshit, Madhurima

2009-08-01

Growing evidence has demonstrated that oxidative stress and increased altered oxygen utilization contribute to atherogenesis and cardiovascular disease (CVD) progression. Antioxidants protect the body from damage caused by free radicals. The objective of this study was to determine antioxidants status in CVD patients. This cross-sectional study was performed on 71 patients clinically diagnosed with CVD and 63 healthy individuals. Plasma malondialdehyde (MDA) level was measured for lipid peroxidation product and erythrocyte SOD and GPx activities as enzymatic antioxidants. The serum levels of vitamins A and E were assayed using HPLC and vitamin C by the photometric method. Total antioxidant capacity (TAC) was measured using the ferric reducing ability of plasma (FRAP) method. The results showed a significant reduction in antioxidant status (enzymatic and non-enzymatic) with a concomitant increase in the concentrations of lipid peroxidation products in CVD patients. There was a significant inverse correlation among TAC, SOD, GPx and vitamin C with MDA. It can be concluded that the antioxidant defense system plays an important role in preventing the development and progression of CVD with the ability to control oxidative stress.

Comparison of Enzymatic Assay for HBA1C Measurement (Abbott Architect) With Capillary Electrophoresis (Sebia Minicap Flex Piercing Analyser).

PubMed

Tesija Kuna, Andrea; Dukic, Kristina; Nikolac Gabaj, Nora; Miler, Marijana; Vukasovic, Ines; Langer, Sanja; Simundic, Ana-Maria; Vrkic, Nada

2018-03-08

To compare the analytical performances of the enzymatic method (EM) and capillary electrophoresis (CE) for hemoglobin A1c (HbA1c) measurement. Imprecision, carryover, stability, linearity, method comparison, and interferences were evaluated for HbA1c via EM (Abbott Laboratories, Inc) and CE (Sebia). Both methods have shown overall within-laboratory imprecision of less than 3% for International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units (<2% National Glycohemoglobin Standardization Program [NGSP] units). Carryover effects were within acceptable criteria. The linearity of both methods has proven to be excellent (R2 = 0.999). Significant proportional and constant difference were found for EM, compared with CE, but were not clinically relevant (<5 mmol/mol; NGSP <0.5%). At the clinically relevant HbA1c concentration, stability observed with both methods was acceptable (bias, <3%). Triglyceride levels of 8.11 mmol per L or greater showed to interfere with EM and fetal hemoglobin (HbF) of 10.6% or greater with CE. The enzymatic method proved to be comparable to the CE method in analytical performances; however, certain interferences can influence the measurements of each method.

Two new mutations in the glucose-6-phosphatase gene cause glycogen storage disease in Hungarian patients.

PubMed

Parvari, R; Lei, K J; Szonyi, L; Narkis, G; Moses, S; Chou, J Y

1997-01-01

Glycogen storage disease type 1a (von Gierke disease, GSD-1A) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the identification of the mutations causing GSD-1a. This, in turn, allows the development of non-invasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. Here we report on two new mutations E110Q and D38V causing GSD-1a in two Hungarian patients. The analyses of these mutations by site-directed mutagenesis followed by transient expression assays demonstrated that E110Q retains 17% of G6Pase enzymatic activity while the D38V abolishes the enzymatic activity. The patient with the E110Q has G222R as his other mutation. G222R was also shown to preserve about 4% of the G6Pase enzymatic activity. Nevertheless, the patient presented with the classical severe symptomatology of the GSD-1a.

Ceramic microsystem incorporating a microreactor with immobilized biocatalyst for enzymatic spectrophotometric assays.

PubMed

Baeza, Mireia; López, Carmen; Alonso, Julián; López-Santín, Josep; Alvaro, Gregorio

2010-02-01

Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

Early phenylpropanoid biosynthetic steps in Cannabis sativa: link between genes and metabolites.

PubMed

Docimo, Teresa; Consonni, Roberto; Coraggio, Immacolata; Mattana, Monica

2013-06-28

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

PubMed

Yoneda, Juliana Sakamoto; Scanavachi, Gustavo; Sebinelli, Heitor Gobbi; Borges, Júlio Cesar; Barbosa, Leandro R S; Ciancaglini, Pietro; Itri, Rosangela

2016-08-01

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity. Copyright © 2016. Published by Elsevier B.V.

"Signal-on" photoelectrochemical biosensor for sensitive detection of human T-Cell lymphotropic virus type II DNA: dual signal amplification strategy integrating enzymatic amplification with terminal deoxynucleotidyl transferase-mediated extension.

PubMed

Shen, Qingming; Han, Li; Fan, Gaochao; Zhang, Jian-Rong; Jiang, Liping; Zhu, Jun-Jie

2015-01-01

A novel "signal-on" photoelectrochemical (PEC) biosensor for sensitive detection of human T-cell lymphotropic virus type II (HTLV-II) DNA was developed on the basis of enzymatic amplification coupled with terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. The intensity of the photocurrent signal was proportional to the concentration of the HTLV-II DNA-target DNA (tDNA) by dual signal amplification. In this protocol, GR-CdS:Mn/ZnS nanocomposites were used as photoelectric conversion material, while pDNA was used as the tDNA recognizing unit. Moreover, the TdT-mediated extension and the enzymatic signal amplification technique were used to enhance the sensitivity of detection. Using this novel dual signal amplification strategy, the prototype of PEC DNA sensor can detect as low as ∼0.033 fM of HTLV-II DNA with a linear range of 0.1-5000 fM, with excellent differentiation ability even for single-base mismatches. This PEC DNA assay opens a promising platform to detect various DNA targets at ultralow levels for early diagnoses of different diseases.

Early Phenylpropanoid Biosynthetic Steps in Cannabis sativa: Link between Genes and Metabolites

PubMed Central

Docimo, Teresa; Consonni, Roberto; Coraggio, Immacolata; Mattana, Monica

2013-01-01

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data. PMID:23812081

Automated saccharification assay for determination of digestibility in plant materials.

PubMed

Gomez, Leonardo D; Whitehead, Caragh; Barakate, Abdellah; Halpin, Claire; McQueen-Mason, Simon J

2010-10-27

Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Enzymatic testing sensitivity, variability and practical diagnostic algorithm for pyruvate dehydrogenase complex (PDC) deficiency.

PubMed

Shin, Ha Kyung; Grahame, George; McCandless, Shawn E; Kerr, Douglas S; Bedoyan, Jirair K

2017-11-01

Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia in children. Prompt and correct diagnosis of PDC deficiency and differentiating between specific vs generalized, or secondary deficiencies has important implications for clinical management and therapeutic interventions. Both genetic and enzymatic testing approaches are being used in the diagnosis of PDC deficiency. However, the diagnostic efficacy of such testing approaches for individuals affected with PDC deficiency has not been systematically investigated in this disorder. We sought to evaluate the diagnostic sensitivity and variability of the various PDC enzyme assays in females and males at the Center for Inherited Disorders of Energy Metabolism (CIDEM). CIDEM data were filtered by lactic acidosis and functional PDC deficiency in at least one cell/tissue type (blood lymphocytes, cultured fibroblasts or skeletal muscle) identifying 186 subjects (51% male and 49% female), about half were genetically resolved with 78% of those determined to have a pathogenic PDHA1 mutation. Assaying PDC in cultured fibroblasts in cases where the underlying genetic etiology is PDHA1, was highly sensitive irrespective of gender; 97% (95% confidence interval [CI]: 90%-100%) and 91% (95% CI: 82%-100%) in females and males, respectively. In contrast to the fibroblast-based testing, the lymphocyte- and muscle-based testing were not sensitive (36% [95% CI: 11%-61%, p=0.0003] and 58% [95% CI: 30%-86%, p=0.014], respectively) for identifying known PDC deficient females with pathogenic PDHA1 mutations. In males with a known PDHA1 mutation, the sensitivity of the various cell/tissue assays (75% lymphocyte, 91% fibroblast and 88% muscle) were not statistically different, and the discordance frequency due to the specific cell/tissue used for assaying PDC was 0.15±0.11. Based on this data, a practical diagnostic algorithm is proposed accounting for current molecular approaches, enzyme testing sensitivity, and variability due to gender, cell/tissue type used for testing, and successive repeat testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.

PubMed

Adhikari, Sanjay; Karmahapatra, Soumendra K; Elias, Hadi; Dhopeshwarkar, Priyanka; Williams, R Scott; Byers, Stephen; Uren, Aykut; Roy, Rabindra

2011-09-01

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5'-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3'- and 5'-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3'- and 5'-phosphotyrosyl linkage at the 3' and 5' ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5'-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5' activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC(50)=40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner. Copyright © 2011 Elsevier Inc. All rights reserved.

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

In situ nanoplasmonic probing of enzymatic activity of monolayer-confined glucose oxidase on colloidal nanoparticles.

PubMed

He, Haili; Xu, Xiaolong; Wu, Haoxi; Zhai, Yujuan; Jin, Yongdong

2013-05-07

In situ probing protein-particle interactions and activities of proteins on colloidal nanoparticle (NP) surfaces is a long-standing key challenge in understanding the nanobio interfaces and virtually important for a variety of biological and biomedical applications. The interactions of NPs with proteins, for instance, are known to form NP bioconjugates or protein coronas; protein surface immobilization and molecular layer-by-layer deposition techniques are widely used, but a clear understanding of the confinement effect on protein activity by molecular coating, at the monolayer level, remains poorly understood. We explore here a novel approach, using colloidal plasmonic nanocomplexes coated with glucose oxidase (GOx) as self-sensing nanoprobes for in situ optical probing of surface-confined enzymatic activity, which is at least 1-2 orders of magnitude more sensitive than standard colorimetric assays for detecting GOx activity. We found that enzymatic activity of monolayer-confined GOx on colloidal NPs was significantly enhanced as compared with free GOx (also proved by conformational changes from circular dichroism studies), with a low apparent Michaelis-Menten constant Km of ~0.115 mM and high turnover kcat/Km of ~8394 M(-1)·s(-1); compared with the "anchored-type" suspending GOx, the outmost polyelectrolyte monolayer-protected "sandwiched-type" GOx exhibits significantly improved enzymatic activities toward higher temperatures and wider pH range. This finding is of fundamental important and instructive for safe use of such nanomaterials for bioapplications.

Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain

PubMed Central

Garrett, Christopher K.; Broadwell, Lindsey J.; Hayne, Cassandra K.; Neher, Saskia B.

2015-01-01

Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme. PMID:26270534

Production of wheat gluten hydrolysates with reduced antigenicity employing enzymatic hydrolysis combined with downstream unit operations.

PubMed

Merz, Michael; Kettner, Lucas; Langolf, Emma; Appel, Daniel; Blank, Imre; Stressler, Timo; Fischer, Lutz

2016-08-01

Due to allergies or other health disorders a certain segment of the population is not able to safely consume some plant proteins, which are the main protein support in human nutrition. Coeliac disease is a prominent autoimmune disorder and requires a strict adherence to a gluten-free diet. The aim of this study was to identify suitable combinations of enzymatic hydrolysis and common unit operations in food processing (centrifugation, ultra-filtration) to produce gluten-free wheat gluten hydrolysates for food application. To analyse the hydrolysates, a simple and cheap competitive ELISA protocol was designed and validated in this study as well. The competitive ELISA was validated using gliadin spiked skim milk protein hydrolysates, due to the latter application of the assay. The limit of quantification was 4.19 mg kg(-1) , which allowed the identification of gluten-free (<20 mg kg(-1) ) hydrolysates. Enzymatic hydrolysis, including the type of peptidase, and the downstream processing greatly affected the antigenicity of the hydrolysates. Enzymatic hydrolysis and downstream processing operations, such as centrifugation and ultra-filtration, reduced the antigenicity of wheat gluten hydrolysates. Gluten-free hydrolysates were obtained with Flavourzyme after centrifugation (25 g L(-1) substrate) and after 1 kDa ultra-filtration (100 g L(-1) substrate). A multiple peptidase complex, such as Flavourzyme, seems to be required for the production of gluten-free hydrolysates. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

The Cytoskeleton & ATP in Sulfur Mustard-Mediated Injury to Endothelial Cells & Keratinocytes.

DTIC Science & Technology

1996-12-01

platelets. J. Cell. Biol. 86:77-86, 1980 . 5. Cassimeris, L, McNeill, H, and Zigmond, SH. Chemoattractant-stimulated polymorphonuclear leukocytes contain two...Arch. Biochem. Biophys. 175:627- 634, 1976. 20. Schraufstatter, IU, Hinshaw, DB, Hyslop , PA, Spragg, RG, and Cochrane, CG: Oxidant injury of cells: DNA... 1980 . 22. Brehe, JE, and Burch, HB. Enzymatic assay for glutathione. Anal Biochem. 74:189, 1976. 23. Griffith, OW. Determination of glutathione and

Asymmetric distribution in the biosynthesis of cotton cellulose-[U-14C

Treesearch

R. Riffer; A. Broido

1974-01-01

Autoradiographs of mature cotton bolls which earlier had radioglucose introduced via a thin incision into their peduncles show a marked asymmetry in distribution of the label. Radio-assay shows the specific activity of the cotton fibres on the treated side to be as much as 30-fold that on the opposite side.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice

PubMed Central

Burns, David P.; Ali, Izza; Rieux, Clement; Healy, James; Jasionek, Greg; O’Halloran, Ken D.

2017-01-01

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease. PMID:29210997

Urine benzodiazepines screening of involuntarily drugged and robbed or raped patients.

PubMed

Boussairi, A; Dupeyron, J P; Hernandez, B; Delaitre, D; Beugnet, L; Espinoza, P; Diamant-Berger, O

1996-01-01

This study involved 35 patients who claimed to have been drugged before being robbed or raped, despite urine negative toxicologic screening by immunoenzymatic methods. The urines were frozen for further investigations, including enzymatic hydrolysis of urinary conjugates, liquid-solid extraction and, finally, immunoenzymatic screening of concentrated urine extract. Urine benzodiazepines were analyzed by immunoenzymatic assay before and after enzymatic hydrolysis combined with extraction. On direct immunoenzymatic screening, 17 of the 35 urine samples were benzodiazepine positive. Enrichment of preserved specimens improved the detection threshold from 200 ng/mL to 50 ng/mL and 10 of the 18 negative urines became positive. This method allowed us to demonstrate the benzodiazepines in half of previously negative urine samples. Benzodiazepine screening is particularly problematic because of low dosage, rapid elimination, failure to detect conjugated metabolites by immunoenzymatic reagents and high threshold of sensitivity for certain substances.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

PubMed

Mashimo, Yasumasa; Mie, Masayasu; Kobatake, Eiry

2018-04-01

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Multiplex tandem mass spectrometry assay for newborn screening of X-linked adrenoleukodystrophy, biotinidase deficiency, and galactosemia with flexibility to assay other enzyme assays and biomarkers.

PubMed

Hong, Xinying; Kumar, Arun Babu; Ronald Scott, C; Gelb, Michael H

2018-03-29

All States screen for biotinidase deficiency and galactosemia, and X-linked adrenoleukodystrophy (X-ALD) has recently been added to the Recommended Uniform Screening Panel (RUSP).We sought to consolidate these tests by combining them into a single multiplex tandem mass spectrometry assay as well as to improve the current protocol for newborn screening of galactosemia.A 3 mm punch of a dried blood spot (DBS) was extracted with organic solvent for analysis of the C26:0-lysophosphatidylcholine biomarker for X-ALD.An additional punch was used to assay galactose-1-phosphate uridyltransferase (GALT) and biotinidase.All assays were combined for a single injection for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (2.3 min per sample).The GALT LC-MS/MS assay does not give a false positive for galactosemia if glucose-6-phosphate dehydrogenase is deficient.The multiplex assay shows acceptable reproducibility and provides for rapid analysis of X-ALD, biotinidase deficiency, and galactosemia.The throughput and ease of sample preparation are acceptable for newborn screening laboratories.We also show that the LC-MS/MS assay is expandable to include several other diseases including Pompe and Hurler diseases (enzymatic activities and biomarkers).Because of consolidation of assays, less manpower is needed compared to running individual assays on separate platforms.The flexibility of the LC-MS/MS platform allows each newborn screening laboratory to analyze the set of diseases offered in their panel. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of the protease activity of the light chain of type A botulinum neurotoxin by aqueous extract from stinging nettle (Urtica dioica) leaf.

PubMed

Gul, Nizamettin; Ahmed, S Ashraf; Smith, Leonard A

2004-11-01

We investigated the inhibitory effect of stinging nettle leaf extract on the protease activity of botulinum neurotoxin type A and B light chains. The nettle leaf infusion was fractionated and HPLC-based enzymatic assays were performed to determine the capacity of each fraction to inhibit the protease activity of botulinum neurotoxin type A and B light chains. Assay results demonstrated that a water-soluble fraction obtained from the nettle leaf infusion inhibited type A, but did not inhibit type B light chain protease activity. The inhibition mode of water soluble fraction against protease activity of type A light chain was analyzed and found to be a non-competitive.

Methods for Determining Spontaneous Mutation Rates

PubMed Central

Foster, Patricia L.

2007-01-01

Spontaneous mutations arise as a result of cellular processes that act upon or damage DNA. Accurate determination of spontaneous mutation rates can contribute to our understanding of these processes and the enzymatic pathways that deal with them. The methods that are used to calculate mutation rates are based on the model for the expansion of mutant clones originally described by Luria and Delbrück and extended by Lea and Coulson. The accurate determination of mutation rates depends on understanding the strengths and limitations of these methods and how to optimize a fluctuation assay for a given method. This chapter describes the proper design of a fluctuation assay, several of the methods used to calculate mutation rates, and ways to evaluate the results statistically. PMID:16793403

Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors.

PubMed

Németh, Gábor; Greff, Zoltán; Sipos, Anna; Varga, Zoltán; Székely, Rita; Sebestyén, Mónika; Jászay, Zsuzsa; Béni, Szabolcs; Nemes, Zoltán; Pirat, Jean-Luc; Volle, Jean-Noël; Virieux, David; Gyuris, Ágnes; Kelemenics, Katalin; Ay, Eva; Minarovits, Janos; Szathmary, Susan; Kéri, György; Orfi, László

2014-05-22

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

Homogeneous screening assay for human tankyrase.

PubMed

Narwal, Mohit; Fallarero, Adyary; Vuorela, Pia; Lehtiö, Lari

2012-06-01

Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

PubMed

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

PubMed

Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

2010-05-26

Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

PubMed Central

Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

2015-01-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

Development of a new microtiter plate format for clinically relevant assays.

PubMed

Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

2012-02-21

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

PubMed

Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

2012-10-01

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

Magneto immunofluorescence assay for diagnosis of celiac disease.

PubMed

Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

2013-10-10

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit. Copyright © 2013 Elsevier B.V. All rights reserved.

Antioxidant Properties of Brazilian Tropical Fruits by Correlation between Different Assays

PubMed Central

Pereira Lima, Giuseppina Pace; Fabris, Sabrina

2013-01-01

Four different assays (the Folin-Ciocalteu, DPPH, enzymatic method, and inhibitory activity on lipid peroxidation) based on radically different physicochemical principles and normally used to determine the antioxidant activity of food have been confronted and utilized to investigate the antioxidant activity of fruits originated from Brazil, with particular attention to more exotic and less-studied species (jurubeba, Solanum paniculatum; pequi, Caryocar brasiliense; pitaya, Hylocereus undatus; siriguela, Spondias purpurea; umbu, Spondias tuberosa) in order to (i) verify the correlations between results obtained by the different assays, with the final purpose to obtain more reliable results avoiding possible measuring-method linked mistakes and (ii) individuate the more active fruit species. As expected, the different methods give different responses, depending on the specific assay reaction. Anyhow all results indicate high antioxidant properties for siriguela and jurubeba and poor values for pitaya, umbu, and pequi. Considering that no marked difference of ascorbic acid content has been detected among the different fruits, experimental data suggest that antioxidant activities of the investigated Brazilian fruits are poorly correlated with this molecule, principally depending on their total polyphenolic content. PMID:24106692

New and highly sensitive assay for L-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography-voltammetry.

PubMed

Rahman, M K; Nagatsu, T; Kato, T

1980-12-12

This paper describes a new, inexpensive and highly sensitive assay for aromatic L-amino acid decarboxylase (AADC) activity, using L-5-hydroxytryptophan (L-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. L-5-HTP was used as substrate and D-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from L-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using L-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

PubMed

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

SNAP Assay Technology.

PubMed

O'Connor, Thomas P

2015-12-01

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The monocarboxylate carrier from rat liver mitochondria. Purification and kinetic characterization in a reconstituted system.

PubMed

Capuano, F; Di Paola, M; Azzi, A; Papa, S

1990-02-12

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Method of detecting and counting bacteria

NASA Technical Reports Server (NTRS)

Picciolo, G. L.; Chappelle, E. W. (Inventor)

1976-01-01

An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids. The method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of nonbacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

PubMed

Yang, Jin Kuk; Park, Min S; Waldo, Geoffrey S; Suh, Se Won

2003-01-21

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans

PubMed Central

Wang, Yufei; Wang, Xiuqing; Jiang, Wentao; Wang, Kun; Luo, Junyuan; Li, Wei; Zhou, Xuedong; Zhang, Linglin

2018-01-01

ABSTRACT Cariogenic virulence factors of Streptococcus mutans include acidogenicity, aciduricity, and extracellular polysaccharides (EPS) synthesis. The de novo designed antimicrobial peptide GH12 has shown bactericidal effects on S. mutans, but its interaction with virulence and regulatory systems of S. mutans remains to be elucidated. The objectives were to investigate the effects of GH12 on virulence factors of S. mutans, and further explore the function mechanisms at enzymatic and transcriptional levels. To avoid decrease in bacterial viability, we limited GH12 to subinhibitory levels. We evaluated effects of GH12 on acidogenicity of S. mutans by pH drop, lactic acid measurement and lactate dehydrogenase (LDH) assay, on aciduricity through survival rate at pH 5.0 and F1F0-ATPase assay, and on EPS synthesis using quantitative measurement, morphology observation, vertical distribution analyses and biomass calculation. Afterwards, we conducted quantitative real-time PCR to acquire the expression profile of related genes. GH12 at 1/2 MIC (4 mg/L) inhibited acid production, survival rate, EPS synthesis, and biofilm formation. The enzymatic activity of LDH and F1F0-ATPase was inhibited, and ldh, gtfBCD, vicR, liaR, and comDE genes were significantly downregulated. In conclusion, GH12 inhibited virulence factors of S. mutans, through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems. PMID:29503706

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

PubMed

Winter, Martin; Bretschneider, Tom; Kleiner, Carola; Ries, Robert; Hehn, Jörg P; Redemann, Norbert; Luippold, Andreas H; Bischoff, Daniel; Büttner, Frank H

2018-07-01

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC 50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

Impact of immobilizing of low molecular weight hyaluronic acid within gelatin-based hydrogel through enzymatic reaction on behavior of enclosed endothelial cells.

PubMed

Khanmohammadi, Mehdi; Sakai, Shinji; Taya, Masahito

2017-04-01

The hydrogels having the ability to promote migration and morphogenesis of endothelial cells (ECs) are useful for fabricating vascularized dense tissues in vitro. The present study explores the immobilization of low molecular weight hyaluronic acid (LMWHA) derivative within gelatin-based hydrogel to stimulate migration of ECs. The LMWHA derivative possessing phenolic hydroxyl moieties (LMWHA-Ph) was bound to gelatin-based derivative hydrogel through the horseradish peroxidase-catalyzed reaction. The motility of ECs was analyzed by scratch migration assay and microparticle-based cell migration assay. The incorporated LMWHA-Ph molecules within hydrogel was found to be preserved stably through covalent bonds during incubation. The free and immobilized LMWHA-Ph did not lose an inherent stimulatory effect on human umbilical vein endothelial cells (HUVECs). The immobilized LMWHA-Ph within gelatin-based hydrogel induced the high motility of HUVECs, accompanied by robust cytoskeleton extension, and cell subpopulation expressing CD44 cell receptor. In the presence of immobilized LMWHA-Ph, the migration distance and the number of existing HUVECs were demonstrated to be encouraged in dose-dependent and time-dependent manners. Based on the results obtained in this work, it was concluded that the enzymatic immobilization of LMWHA-Ph within gelatin-based hydrogel represents a promising approach to promote ECs' motility and further exploitation for vascular tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Gold nanocluster-based ratiometric fluorescent probes for hydrogen peroxide and enzymatic sensing of uric acid.

PubMed

Yang, Dongqin; Luo, Minchuan; Di, Junwei; Tu, Yifeng; Yan, Jilin

2018-05-18

A method is described for ratiometric fluorometric assays of H 2 O 2  by using two probes that have distinct response profiles. Under the catalytic action of ferrous ion, the 615 nm emission of protein-stabilized gold nanoclusters (under 365 nm photoexcitation) is quenched by H 2 O 2 , while an increased signal is generated with a peak at 450 nm by oxidizing coumarin with the H 2 O 2 /Fe(II) system to form a blue emitting fluorophore. These decrease/increase responses give a ratiometric signal. The ratio of the fluorescences at the two peaks are linearly related to the concentration of H 2 O 2 in the range from 0.05 to 10 μM, with a 7.7 nM limit of detection. The detection scheme was further coupled to the urate oxidase catalyzed oxidation of uric acid which proceeds under the formation of H 2 O 2 . This method provides an simple and effective means for the construction of ratiometric fluorometric (enzymatic) assays that involve the detection of H 2 O 2 . Graphical abstract Under catalysis by ferrous ion, hydrogen peroxide quenches the luminescence of gold nanoclusters (AuNCs) and oxidizes coumarin into a fluorescent derivative, which rendered fluorescence ON and OFF at two distinct wavelengths for ratiometric measurements.

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

PubMed

Huber, Alexander; France, Richard M; Riccalton-Banks, Lisa; McLaren, Jane; Cox, Helen; Quirk, Robin A; Shakesheff, Kevin M; Thompson, David; Panjwani, Naveed; Shipley, Sarah; Pickett, Andy

2008-05-01

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

The stability of prostatic acid phosphatase, as measured by a capture immunoenzyme assay.

PubMed

Lin, T M; Chin-See, M W; Halbert, S P

1984-03-27

A capture immunoenzyme assay (CIEA) for prostatic acid phosphatase (PAP) was developed and used to study the stability of this isoenzyme. Immunospecifically purified goat antibodies to PAP were covalently bound to special discs and used to capture the enzyme in serum samples in a weakly acidic medium during the first incubation (2 h) at 37 degrees C. The capture enzyme was then measured by its catalytic activity with p-nitrophenyl phosphate as substrate during the second incubation (1 h) at 37 degrees C. As much as 98% of the PAP in test specimens was captured and measured by this CIEA. The test results were expressed as enzymatic activity (U/l), extrapolated from a standard curve which was linear between 0.026 and 70 U/l. In test sera stored at 4 degrees C, the PAP was variably stable for 7 to 70 days, but the enzyme was quite stable in serum when stored at -20 degrees C for at least 156 days. At room temperature, when the sera were appropriately acidified, there was no loss of enzymatic activity for periods of 15 days, and in some cases, a large proportion of activity was still intact after 70 days. At 4 degrees C, as well as -20 degrees C, acidified serum and the partially purified PAP standard showed complete stability for at least 7 months. The CIEA reactivity of positive test specimens was inhibited by L(+)-tartaric acid, but not by cupric sulfate. The acid phosphatases of blood cell extracts were non-reactive in the CIEA procedure. The CIEA results of 224 serum samples from patients with and without prostate cancer correlated very well with those obtained by two direct enzymatic and two commercial RIA procedures, with correlation coefficients between 0.960 and 0.993, and diagnostic agreement between 86% and 100%.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients.

PubMed

Bentaleb, El Mehdi; Abid, Mohammed; El Messaoudi, My Driss; Lakssir, Brahim; Ressami, El Mostafa; Amzazi, Saaïd; Sefrioui, Hassan; Ait Benhassou, Hassan

2016-09-27

Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries. In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB. Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA. To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines.

PubMed

Riebel, Matthias; Sabel, Andrea; Claus, Harald; Fronk, Petra; Xia, Ning; Li, Huige; König, Helmut; Decker, Heinz

2015-09-18

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells) delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

Degradation and protection of DNAzymes on human skin.

PubMed

Marquardt, Kay; Eicher, Anna-Carola; Dobler, Dorota; Höfer, Frank; Schmidts, Thomas; Schäfer, Jens; Renz, Harald; Runkel, Frank

2016-10-01

DNAzymes are catalytic nucleic acid based molecules that have become a new class of active pharmaceutical ingredients (API). Until now, five DNAzymes have entered clinical trials. Two of them were tested for topical application, whereby dermally applied DNAzymes had been prone to enzymatic degradation. To protect the DNAzymes the enzymatic activity of human skin has to be examined. Therefore, the enzymatic activity of human skin was qualitatively and quantitatively analyzed. Activity similar to that of DNase II could be identified and the specific activity was determined to be 0.59Units/mg. These results were used to develop an in vitro degradation assay to screen different kinds of protective systems on human skin. The chosen protective systems consisted of biodegradable chitosans or polyethylenimine, which forms polyplexes when combined with DNAzymes. The polyplexes were characterized in terms of particle size, zeta potential, stability and degree of complexation. The screening revealed that the protective efficiency of the polyplexes depended on the polycation and the charge ratio (ξ). At a critical ξ ratio between 1.0 and 4.1 and at a maximal zeta potential, sufficient protection of the DNAzyme was achieved. The results of this study will be helpful for the development of a protective dermal drug delivery systems using polyplexes. Copyright © 2016 Elsevier B.V. All rights reserved.

Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

2012-01-01

A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.

Effect of endogenous and exogenous enzymatic treatment of green vanilla beans on extraction of vanillin and main aromatic compounds.

PubMed

Pardío, Violeta T; Flores, Argel; López, Karla M; Martínez, David I; Márquez, Ofelia; Waliszewski, Krzysztof N

2018-06-01

Endogenous and exogenous enzymatic hydrolysis carried out to obtain vanilla extracts with higher concentrations of vanillin using green vanilla beans. Sequences initiated with freezing of green vanilla beans at - 1 °C for 24 h, followed by endogenous hydrolysis under optimal β-glucosidase activity at 4.2 and 35 °C for 96 h, exogenous hydrolysis with Crystalzyme PML-MX at pH 5.0 and 40 °C for 72 h, and ethanol extraction at 40% (v v -1 ) for 30 days. In the proposed method, 200 g of fresh green vanilla beans with 84% moisture (32 g dry base) were used to obtain a liter of single fold vanilla extract. This method allowed the release of 82.57% of the theoretically available vanillin from its precursor glucovanillin with 5.78 g 100 g -1 green vanilla beans (dry base). Vanillic acid, p -hydroxybenzaldehyde and vanillyl alcohol were also released and found in commercial and enzymatic extracts. Glucovanillin was detected in commercial and traditional extracts but was absent in enzymatic extracts, indicating incomplete hydrolysis during the curing process. An in vitro assay was conducted to determine if the presence of peroxidase during hydrolysis might affect overall vanillin concentration. Results showed that POD can use vanillin as a substrate under conditions similar to those in which hydrolysis was conducted (pH 5.0 and 50 °C), possibly explaining why vanillin concentration was not complete at the end of the process.

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

PubMed

Kalb, Suzanne R; Boyer, Anne E; Barr, John R

2015-08-31

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

PubMed Central

Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

2015-01-01

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway.

PubMed

Zhao, Feng; Wang, Lu; Liu, Ke

2009-04-21

Arctigenin, a bioactive constituent from dried seeds of Arctium lappa L. (Compositae) which has been widely used as a Traditional Chinese Medicine for dispelling wind and heat included in Chinese Pharmacophere, was found to exhibit anti-inflammatory activities but its molecular mechanism remains unknown yet. To investigate the anti-inflammatory mechanism of arctigenin. Cultured macrophage RAW 264.7 cells and THP-1 cells were used for the experiments. Griess assay was used to evaluate the inhibitory effect of arctigenin on the overproduction of nitric oxide (NO). ELISA was used to determine the level of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). The inhibitory effect on the enzymatic activity of cyclooxygenase-2 (COX-2) was tested by colorimetric method. Western blot was used to detect the expression of inducible nitric oxide synthase (iNOS) and COX-2. Arctigenin suppressed lipopolysaccharide (LPS)-stimulated NO production and pro-inflammatory cytokines secretion, including TNF-alpha and IL-6 in a dose-dependent manner. Arctigenin also strongly inhibited the expression of iNOS and iNOS enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected by arctigenin. These results indicated that potent inhibition on NO, TNF-alpha and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of arctigenin. Arctigenin suppressed the overproduction of NO through down-regulation of iNOS expression and iNOS enzymatic activity in LPS-stimulated macrophage.

Electrical stimulation of the hypothalamic nucleus paraventricularis mimics the effects of light on pineal melatonin synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olcese, J.; Reuss, S.; Steinlechner, S.

In an attempt to clarify further the role of the hypothalamic paraventricular nuclei (PVN) in the control of pineal function, the effects of 2 min electrical stimulation of these nuclei were investigated in acutely blinded, adult, male Sprague-Dawley rats. Pineal serotonin-N-acetyltransferase (NAT) activity, melatonin content and catecholamine levels were measured by means of radio-enzymatic, radioimmunoassay and high-performance liquid-chromatography methods, respectively. All three pineal parameters underwent significant declines following brief PVN stimulation during the night time. These observations lend credence to the view that the neural pathways transmitting light information to the sympathetic innervation controlling pineal melatonin synthesis. 22 references, 1more » figure.« less

Pre-analytic and analytic sources of variations in thiopurine methyltransferase activity measurement in patients prescribed thiopurine-based drugs: A systematic review.

PubMed

Loit, Evelin; Tricco, Andrea C; Tsouros, Sophia; Sears, Margaret; Ansari, Mohammed T; Booth, Ronald A

2011-07-01

Low thiopurine S-methyltransferase (TPMT) enzyme activity is associated with increased thiopurine drug toxicity, particularly myelotoxicity. Pre-analytic and analytic variables for TPMT genotype and phenotype (enzyme activity) testing were reviewed. A systematic literature review was performed, and diagnostic laboratories were surveyed. Thirty-five studies reported relevant data for pre-analytic variables (patient age, gender, race, hematocrit, co-morbidity, co-administered drugs and specimen stability) and thirty-three for analytic variables (accuracy, reproducibility). TPMT is stable in blood when stored for up to 7 days at room temperature, and 3 months at -30°C. Pre-analytic patient variables do not affect TPMT activity. Fifteen drugs studied to date exerted no clinically significant effects in vivo. Enzymatic assay is the preferred technique. Radiochemical and HPLC techniques had intra- and inter-assay coefficients of variation (CVs) below 10%. TPMT is a stable enzyme, and its assay is not affected by age, gender, race or co-morbidity. Copyright © 2011. Published by Elsevier Inc.

Novel enzymatic assay predicts minoxidil response in the treatment of androgenetic alopecia.

PubMed

Goren, Andy; Castano, Juan Antonio; McCoy, John; Bermudez, Fernando; Lotti, Torello

2014-01-01

Topical minoxidil is the most common drug used for the treatment of androgenetic alopecia (AGA) in men and women. Although topical minoxidil exhibits a good safety profile, the efficacy in the overall population remains relatively low at 30-40%. To observe significant improvement in hair growth, minoxidil is typically used daily for a period of at least 3-4 months. Due to the significant time commitment and low response rate, a biomarker for predicting patient response prior to therapy would be advantageous. Minoxidil is converted in the scalp to its active form, minoxidil sulfate, by the sulfotransferase enzyme SULT1A1. We hypothesized that SULT1A1 enzyme activity in the hair follicle correlates with minoxidil response for the treatment of AGA. Our preliminary retrospective study of a SULT1A1 activity assay demonstrates 95% sensitivity and 73% specificity in predicting minoxidil treatment response for AGA. A larger prospective study is now under way to further validate this novel assay. © 2013 Wiley Periodicals, Inc.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity

PubMed Central

Wahba, Alexander S.; Esmaeili, Abbasali; Damha, Masad J.; Hudson, Robert H. E.

2010-01-01

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H. PMID:19933258

Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

NASA Astrophysics Data System (ADS)

Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

2016-12-01

We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Excessive S-Adenosyl-L-Methionine-Dependent Methylation Increases Levels of Methanol, Formaldehyde and Formic Acid in Rat Brain Striatal Homogenates: Possible role in S-adenosyl-L-methionine-induced Parkinson’s disease-like disorders

PubMed Central

Lee, Eun-Sook; Chen, Hongtao; Hardman, Chadwick; Simm, Anthony; Charlton, Clivel

2009-01-01

Aims Excessive methylation may be a precipitating factor for Parkinson’s disease (PD) since S-adenosylmethionine (SAM), the endogenous methyl donor, induces PD-like changes when injected into the rat brain. The hydrolysis of the methyl ester bond of the methylated proteins produces methanol. Since methanol is oxidized into formaldehyde, and formaldehyde into formic acid in the body, we investigated the effects of SAM on the production of methanol, formaldehyde and formic acid in rat brain striatal homogenates and the toxicity of these products in PC12 cells. Main methods radio-enzymatic and colorimetric assays, cell viability, Western blot. Key findings SAM increased the formation of methanol, formaldehyde and formic acid in a concentration and time-dependent manner. Concentrations of [3H-methyl]-SAM at 0.17, 0.33, 0.67 and 1.34 nM produced 3.8, 8.0, 18.3 and 34.4 fmol/mg protein/h of [3H] methanol in rat striatal homogenates, respectively. SAM also significantly generated formaldehyde and formic acid in striatal homogenates. Formaldehyde was the most toxic metabolite to differentiated PC12 pheochromocytoma cells in cell culture studies, indicating that formaldehyde formed endogenously may contribute to neuronal damage in excessive methylation conditions. Subtoxic concentration of formaldehyde decreased the expression of tyrosine hydroxylase, the limiting factor in dopamine synthesis. Formaldehyde was more toxic to catecholaminergic PC12 cells than C6 glioma cells, indicating that neurons are more vulnerable to formaldehyde than glia cells. Significance We suggest that excessive carboxylmethylation of proteins might be involved in the SAM-induced PD-like changes and in the aging process via the toxic effects of formaldehyde. PMID:18930743

Peptide-based biosensors: From self-assembled interfaces to molecular probes in electrochemical assays.

PubMed

Puiu, Mihaela; Bala, Camelia

2018-04-01

Redox-tagged peptides have emerged as functional materials with multiple applications in the area of sensing and biosensing applications due to their high stability, excellent redox properties and versatility of biomolecular interactions. They allow direct observation of molecular interactions in a wide range of affinity and enzymatic assays and act as electron mediators. Short helical peptides possess the ability to self-assemble in specific configurations with the possibility to develop in highly-ordered, stable 1D, 2D and 3D architectures in a hierarchical controlled manner. We provide here a brief overview of the electrochemical techniques available to study the electron transfer in peptide films with particular interest in developing biosensors with immobilized peptide motifs, for biological and clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

PubMed

Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

2011-11-01

An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Application of microstructural optical waveguides with hollow core for enzyme immunoassay

NASA Astrophysics Data System (ADS)

Pidenko, Pavel S.; Pidenko, Sergei A.; Burmistrova, Natalia A.; Shuvalov, Andrei A.; Chibrova, Anastasiya A.; Skibina, Yulia S.; Goryacheva, Irina Y.

2018-04-01

Microstructural optical waveguides with the hollow core are actively studied as a promising support for heterogeneous immunoassay in development of new optical biosensor elements for medicine and biology. Overcoming of the limitations associated with the low sorption capacity of glass used for the waveguides production is a crucial step for this assay format. In this work the possibility of silanization of microstructural optical waveguides with the hollow core using (3-glycidyloxypropyl) trimethoxysilane and their further application to enzymatic immunoassay was studied.

Chemical composition of ground pearl (Eurhizococcus colombianus) cysts.

PubMed

Quiñones, Winston; Vicente, Bernardo; Torres, Fernando; Archbold, Rosendo; Murillo, Walter; Londoño, Martha; Echeverri, Fernando

2008-01-29

Ground pearl (Eurhizococcus colombianus) is a crop pest in Colombia, with special impact on fig, grass, rubus and tomato plants. The insect is resistant to external insecticide application because it produces a thick waxy shell that isolates it from the environment. The composition of this shell was determined by NMR and MS as a triglyceride, whose fatty acid is transformed into other products with the metamorphosis of the insect. Additionally, several enzymatic inhibitors were assayed to control the insect with negative results.

Endotoxin removal by radio frequency gas plasma (glow discharge)

NASA Astrophysics Data System (ADS)

Poon, Angela

2011-12-01

Contaminants remaining on implantable medical devices, even following sterilization, include dangerous fever-causing residues of the outer lipopolysaccharide-rich membranes of Gram-negative bacteria such as the common gut microorganism E. coli. The conventional method for endotoxin removal is by Food & Drug Administration (FDA)-recommended dry-heat depyrogenation at 250°C for at least 45 minutes, an excessively time-consuming high-temperature technique not suitable for low-melting or heat-distortable biomaterials. This investigation evaluated the mechanism by which E. coli endotoxin contamination can be eliminated from surfaces during ambient temperature single 3-minute to cumulative 15-minute exposures to radio-frequency glow discharge (RFGD)-generated residual room air plasmas activated at 0.1-0.2 torr in a 35MHz electrodeless chamber. The main analytical technique for retained pyrogenic bio-activity was the Kinetic Chromogenic Limulus Amebocyte Lysate (LAL) Assay, sufficiently sensitive to document compliance with FDA-required Endotoxin Unit (EU) titers less than 20 EU per medical device by optical detection of enzymatic color development corresponding to < 0.5 EU/ml in sterile water extracts of each device. The main analytical technique for identification of chemical compositions, amounts, and changes during sequential reference Endotoxin additions and subsequent RFGD-treatment removals from infrared (IR)-transparent germanium (Ge) prisms was Multiple Attenuated Internal Reflection (MAIR) infrared spectroscopy sensitive to even monolayer amounts of retained bio-contaminant. KimaxRTM 60 mm x 15 mm and 50mm x 15mm laboratory glass dishes and germanium internal reflection prisms were inoculated with E. coli bacterial endotoxin water suspensions at increments of 0.005, 0.05, 0.5, and 5 EU, and characterized by MAIR-IR spectroscopy of the dried residues on the Ge prisms and LAL Assay of sterile water extracts from both glass and Ge specimens. The Ge prism MAIR-IR measurements were repeated after employing 3-minute RFGD treatments sequentially for more than 10 cycles to observe removal of deposited matter that correlated with diminished EU titers. The results showed that 5 cycles, for a total exposure time of 15 minutes to low-temperature gas plasma, was sufficient to reduce endotoxin titers to below 0.05 EU/ml, and correlated with concurrent reduction of major endotoxin reference standard absorption bands at 3391 cm-1, 2887 cm-1, 1646 cm -1 1342 cm-1, and 1103 cm-1 to less than 0.05 Absorbance Units. Band depletion varied from 15% to 40% per 3-minute cycle of RFGD exposure, based on peak-to-peak analyses. In some cases, 100% of all applied biomass was removed within 5 sequential 3-minute RFGD cycles. The lipid ester absorption band expected at 1725 cm-1 was not detectable until after the first RFGD cycle, suggesting an unmasking of the actual bacterial endotoxin membrane induced within the gas plasma environment. Future work must determine the applicability of this low-temperature, quick depyrogenation process to medical devices of more complicated geometry than the flat surfaces tested here.

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Ultramicro-fluorometric assay for the diagnosis of Gaucher disease in dried blood spots on filter paper.

PubMed

Herrera, D; Monaga, M; Campos, D; Pampín, Y; González, E C; Lavaut, K

2013-01-01

Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency of the lysosomal acid β-D-glucosidase (GBA). The aim of this study was to develop an ultramicro-fluorometric assay based on the method of Chamoles et al. for determining GBA activity in dried blood spots on filter paper (DBS). The assay used 3-mm diameter blood spot and 8 mmol/l of 4-methylumbelliferyl-β-D-glucoside as enzymatic substrate. The reaction occurred in plates incubated at 37°C for 20 hours and the enzyme activity was expressed in μmol hydrolysed substrate/l blood/h. The fluorescence of the enzyme product was automatically measured in a fluorometer-photometer reader (SUMA Technology). The intra and inter-assay coefficients of variation were lower than 9 and 12%, respectively, and the recovery range was 97-109%.Three patients with GD were correctly diagnosed using the ultramicroassay. Healthy newborn DBS samples (n = 3003) from the National Neonatal Screening Program were analyzed, and the mean GBA activity was 5.7 μmol/l blood/h. Our assay showed high Pearson (n = 26; r = 0.99) and concordance correlations (ρc = 0.99) with the traditional method described by Chamoles et al. The analytical performance characteristics of our ultramicro-fluorometric assay suggest that it can be used in the diagnosis of GD in newborns and adults.

Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants.

PubMed

Smirnova, Julia; Fernie, Alisdair R; Spahn, Christian M T; Steup, Martin

2017-09-01

Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify β-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. Copyright © 2017. Published by Elsevier Inc.

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.

PubMed

Lin, Sansan; Fischl, Anthony S; Bi, Xiahui; Parce, Wally

2003-03-01

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

Digital Assays Part I: Partitioning Statistics and Digital PCR.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error. The remainder focuses on digital PCR (dPCR) for quantification of nucleic acids. We discuss five commercial instruments that partition samples into physically isolated chambers (cdPCR) or droplet emulsions (ddPCR). We compare the strengths of dPCR (absolute quantitation, precision, and ability to detect rare or mutant targets) with those of its predecessor, quantitative real-time PCR (dynamic range, larger sample volumes, and throughput). Lastly, we describe several promising applications of dPCR, including copy number variation, quantitation of circulating tumor DNA and viral load, RNA/miRNA quantitation with reverse transcription dPCR, and library preparation for next-generation sequencing. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows. Part II focuses on digital protein and cell assays.

Antioxidative and anticholinesterase activity of Cyphomandra betacea fruit.

PubMed

Ali Hassan, Siti Hawa; Abu Bakar, Mohd Fadzelly

2013-01-01

Cyphomandra betacea is one of the underutilized fruits which can be found in tropical and subtropical countries. This study was conducted to determine the antioxidant activity and phytochemical contents in different parts (i.e., flesh and peel) of the fruits. Antioxidants were analyzed using DPPH and ABTS free radical scavenging assays as well as FRAP assay. Anticholinesterase activity was determined using enzymatic assay using acetyl cholinesterase enzyme. For 80% methanol extract, the peel of the fruit displayed higher antioxidant activity in both FRAP and ABTS free radical scavenging assays while the flesh displayed higher antioxidant activity in the DPPH assay. Total phenolic and total flavonoid content were higher in the peel with the values of 4.89 ± 0.04 mg gallic acid equivalent (GAE)/g and 3.36 ± 0.01 mg rutin equivalent (RU)/g, respectively. Total anthocyanin and carotenoid content were higher in the flesh of the fruit with the values of 4.15 ± 0.04 mg/100 g and 25.13 ± 0.35 mg/100 g. The anticholinesterase was also higher in the peel of C. betacea. The same trends of phytochemicals, antioxidant, and anticholinesterase were also observed in the distilled water extracts. These findings suggested that C. betacea has a potential as natural antioxidant-rich nutraceutical products.

Antioxidative and Anticholinesterase Activity of Cyphomandra betacea Fruit

PubMed Central

Ali Hassan, Siti Hawa; Abu Bakar, Mohd Fadzelly

2013-01-01

Cyphomandra betacea is one of the underutilized fruits which can be found in tropical and subtropical countries. This study was conducted to determine the antioxidant activity and phytochemical contents in different parts (i.e., flesh and peel) of the fruits. Antioxidants were analyzed using DPPH and ABTS free radical scavenging assays as well as FRAP assay. Anticholinesterase activity was determined using enzymatic assay using acetyl cholinesterase enzyme. For 80% methanol extract, the peel of the fruit displayed higher antioxidant activity in both FRAP and ABTS free radical scavenging assays while the flesh displayed higher antioxidant activity in the DPPH assay. Total phenolic and total flavonoid content were higher in the peel with the values of 4.89 ± 0.04 mg gallic acid equivalent (GAE)/g and 3.36 ± 0.01 mg rutin equivalent (RU)/g, respectively. Total anthocyanin and carotenoid content were higher in the flesh of the fruit with the values of 4.15 ± 0.04 mg/100 g and 25.13 ± 0.35 mg/100 g. The anticholinesterase was also higher in the peel of C. betacea. The same trends of phytochemicals, antioxidant, and anticholinesterase were also observed in the distilled water extracts. These findings suggested that C. betacea has a potential as natural antioxidant-rich nutraceutical products. PMID:24298210

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin.

PubMed

Camacho-Ruiz, María de Los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A

2015-05-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

PubMed

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

PubMed

Jung, Se-Hui; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Young-Myeong; Ha, Kwon-Soo

2016-05-15

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues.

PubMed

Knudsen, Beatrice S; Allen, April N; McLerran, Dale F; Vessella, Robert L; Karademos, Jonathan; Davies, Joan E; Maqsodi, Botoul; McMaster, Gary K; Kristal, Alan R

2008-03-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

PubMed Central

Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

2008-01-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

Telomere 1 (POT1) gene expression and its association with telomerase activity in colorectal tumor samples with different pathological features.

PubMed

Izgi, Ahu; Gunal, Armagan; Yalcin, Serap; Gunduz, Ufuk

2014-09-01

The ends of chromosoms, telomeres are bound with a number of proteins which protect and stabilize telomeres against degredation, end to end fusion and aberrant recombinations. Telomeric DNA is bound of two groups of proteins, which are double-stranded telomeric DNA bindings proteins, and single stranded telomeric binding proteins. Among telomere binding proteins, protections of telomere 1 protein is a single stranded telomere binding proteins and suggested to be a significant player for telomere elongation and has an association with an enzyme called as telomerase which is an intrinsic reverse transcriptase. Telomerase synthesizes hexameric telomeric repeats onto the chromosomes thereby compansating telomere loss in immortal cells, such as tumor cells, whereas telomeres are shorthened with each division in normal cells. PCR-based TRAP (telomeric repeat amplification protocol) assay is a very sensitive assay for the detection of enzymatic activity of telomerase even if a few numbers of cancerous cells are available. The association between telomerase activity and hPOT1 expression in colorectal cancer is still unclear. Protein extraction was performed from specimens of matched normal and colorectal cancer specimens. Protein concentrations were determined by Bradford assay. Optimized protein concentrations were used for TRAP Assay. TRAP products were seperated by vertical gel electrophoresis on 12.5% polyacrylamide gels and visualized by silver staining. Gene expression of hPOT1 was determined by qPCR analysis. The results demonstrated that all tumor tissues were telomerase positive whereas all corresponding normal tissue was telomerase negative. Among clinicopathological findings, telomerase activity was found to be associated with stage, histology, localization, distant metastasis and lymph node metastasis of tumor in the current study. Although all of the clinicopathological findings differed in the expression of hPOT1 compared to normal tissues, they did not differ from each other significantly, except side of tumor and lymph node metastasis. Telomerase activity and hPOT1 gene expression may serve as a promising tumor marker for colorectal cancer and there is a close association between the enzymatic activty of telomerase and the expression of human protection of telomere 1 gene. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

PubMed Central

Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

2014-01-01

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

Electrochemical detection of microRNAs via gap hybridization assay.

PubMed

Pöhlmann, Christopher; Sprinzl, Mathias

2010-06-01

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection ofmore » target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.« less

Effect-directed analysis of cold-pressed hemp, flax and canola seed oils by planar chromatography linked with (bio)assays and mass spectrometry.

PubMed

Teh, Sue-Siang; Morlock, Gertrud E

2015-11-15

Cold-pressed hemp, flax and canola seed oils are healthy oils for human consumption as these are rich in polyunsaturated fatty acids and bioactive phytochemicals. However, bioactive information on the food intake side is mainly focused on target analysis. For more comprehensive information with regard to effects, single bioactive compounds present in the seed oil extracts were detected by effect-directed assays, like bioassays or an enzymatic assay, directly linked with chromatography and further characterized by mass spectrometry. This effect-directed analysis is a streamlined method for the analysis of bioactive compounds in the seed oil extracts. All effective compounds with regard to the five assays or bioassays applied were detected in the samples, meaning also bioactive breakdown products caused during oil processing, residues or contaminants, aside the naturally present bioactive phytochemicals. The investigated cold-pressed oils contained compounds that exert antioxidative, antimicrobial, acetylcholinesterase inhibitory and estrogenic activities. This effect-directed analysis can be recommended for bioactivity profiling of food to obtain profound effect-directed information on the food intake side. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

PubMed Central

Nakamura, S; Shimooku, K; Akisue, T; Jinnai, H; Hitomi, T; Kiyohara, Y; Ogino, C; Yoshida, K; Nishizuka, Y

1995-01-01

Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low. PMID:8618893

Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity.

PubMed

Popow-Stellmaszyk, Jadwiga; Bajorowicz, Beata; Malankowska, Anna; Wysocka, Magdalena; Klimczuk, Tomasz; Zaleska-Medynska, Adriana; Lesner, Adam

2018-05-16

Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH 2 -terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol-gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

PubMed

Nam, Seung-Hee; Ko, Jin-A; Jun, Woojin; Wee, Young-Jung; Walsh, Marie K; Yang, Kwang-Yeol; Choi, Jin-Ho; Eun, Jon-Bang; Choi, Jeong; Kim, Young-Min; Han, Songhee; Nguyen, Thi Thanh Hanh; Kim, Doman

2017-12-01

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Effects of Oligosaccharides Isolated From Pinewood Hot Water Pre-hydrolyzates on Recombinant Cellulases.

PubMed

Fang, Hong; Kandhola, Gurshagan; Rajan, Kalavathy; Djioleu, Angele; Carrier, Danielle Julie; Hood, Kendall R; Hood, Elizabeth E

2018-01-01

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-β-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%.

Effect of the glass transition temperature on alpha-amylase activity in a starch matrix.

PubMed

Chaudhary, Vinita; Panyoyai, Naksit; Small, Darryl M; Shanks, Robert A; Kasapis, Stefan

2017-02-10

This study optimises a protocol for the estimation of α-amylase activity in a condensed starch matrix in the vicinity of the glass transition region. Enzymatic activity on the vitrified starch system was compared with that of a reference substrate, maltodextrin. The activity was assayed as the rate of release of reducing sugar using a dinitrosalicylic acid procedure. The condensed carbohydrate matrices served the dual purpose of acting as a substrate as well as producing a pronounced effect on the ability to enzymatic hydrolysis. Activation energies were estimated throughout the glass transition region of condensed carbohydrate preparations based on the concept of the spectroscopic shift factor. Results were used to demonstrate a considerable moderation by the mechanical glass transition temperature, beyond the expected linear effect of the temperature dependence, on the reaction rate of starch hydrolysis by α-amylase in comparison with the low-molecular weight chain of maltodextrin. Copyright © 2016. Published by Elsevier Ltd.

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.

PubMed

Botting, Rachel A; Bertram, Kirstie M; Baharlou, Heeva; Sandgren, Kerrie J; Fletcher, James; Rhodes, Jake W; Rana, Hafsa; Plasto, Toby M; Wang, Xin Maggie; Lim, Jake J K; Barnouti, Laith; Kohout, Mark P; Papadopoulos, Tim; Merten, Steve; Olbourne, Norman; Cunningham, Anthony L; Haniffa, Muzlifah; Harman, Andrew N

2017-06-01

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue. © The Author(s).

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes

PubMed Central

Botting, Rachel A.; Bertram, Kirstie M.; Baharlou, Heeva; Sandgren, Kerrie J.; Fletcher, James; Rhodes, Jake W.; Rana, Hafsa; Plasto, Toby M.; Wang, Xin Maggie; Lim, Jake J. K.; Barnouti, Laith; Kohout, Mark P.; Papadopoulos, Tim; Merten, Steve; Olbourne, Norman; Cunningham, Anthony L.; Haniffa, Muzlifah; Harman, Andrew N.

2017-01-01

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue. PMID:28270408

Organic Amendments to Avocado Crops Induce Suppressiveness and Influence the Composition and Activity of Soil Microbial Communities

PubMed Central

Bonilla, Nuria; Vida, Carmen; Martínez-Alonso, Maira; Landa, Blanca B.; Gaju, Nuria; Cazorla, Francisco M.

2015-01-01

One of the main avocado diseases in southern Spain is white root rot caused by the fungus Rosellinia necatrix Prill. The use of organic soil amendments to enhance the suppressiveness of natural soil is an inviting approach that has successfully controlled other soilborne pathogens. This study tested the suppressive capacity of different organic amendments against R. necatrix and analyzed their effects on soil microbial communities and enzymatic activities. Two-year-old avocado trees were grown in soil treated with composted organic amendments and then used for inoculation assays. All of the organic treatments reduced disease development in comparison to unamended control soil, especially yard waste (YW) and almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization. All of the organic amendments assayed in this study were able to suppress white root rot, although their suppressiveness appears to be mediated differentially. PMID:25769825

PROTEIN ADDUCTS AS BIOMAKERS OF EXPOSURE TO ORGANOPHOSPHORUS COMPOUNDS

PubMed Central

Marsillach, Judit; Costa, Lucio G.; Furlong, Clement E.

2013-01-01

Exposure to organophosphorus (OP) compounds can lead to serious neurological damage or death. Following bioactivation by the liver cytochromes P450, the OP metabolites produced are potent inhibitors of serine active-site enzymes including esterases, proteases and lipases. OPs may form adducts on other cellular proteins. Blood cholinesterases (ChEs) have long served as biomarkers of OP exposure in humans. However, the enzymatic assays used for biomonitoring OP exposures have several drawbacks. A more useful approach will focus on multiple biomarkers and avoid problems with the enzymatic activity assays. OP inhibitory effects result from a covalent bond with the active-site serine of the target enzymes. The serine OP adducts become irreversible following a process referred to as aging where one alkyl group dissociates over variable lengths of time depending on the OP adduct. The OP-adducted enzyme then remains in circulation until it is degraded, allowing for a longer window of detection compared with direct analysis of OPs or their metabolites. Mass spectrometry (MS) provides a very sensitive method for identification of post-translational protein modifications. MS analyses of the percentage adduction of the active-site serine of biomarker proteins such as ChEs will eliminate the need for basal activity levels of the individual and will provide for a more accurate determination of OP exposure. MS analysis of biomarker proteins also provides information about the OP that has caused inhibition. Other useful biomarker proteins include other serine hydrolases, albumin, tubulin and transferrin. PMID:23261756

Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation

PubMed Central

Delafontaine, Marie; Villas-Boas, Isadora Maria; Mathieu, Laurence; Josset, Patrice; Blomet, Joël

2017-01-01

Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. PMID:28783135

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E

2008-04-15

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover. Copyright 2008 Wiley Periodicals, Inc.

Scaling down the size and increasing the throughput of glycosyltransferase assays: activity changes on stem cell differentiation.

PubMed

Patil, Shilpa A; Chandrasekaran, E V; Matta, Khushi L; Parikh, Abhirath; Tzanakakis, Emmanuel S; Neelamegham, Sriram

2012-06-15

Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall β(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galβ1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of body temperature on chondroitinase ABC's ability to cleave chondroitin sulfate glycosaminoglycans.

PubMed

Tester, Nicole J; Plaas, Anna H; Howland, Dena R

2007-04-01

Chondroitinase ABC (Ch'ase ABC) is a bacterial lyase that degrades chondroitin sulfate (CS), dermatan sulfate, and hyaluronan glycosaminoglycans (GAGs). This enzyme has received significant attention as a potential therapy for promoting central nervous system and peripheral nervous system repair based on its degradation of CS GAGs. Determination of the stability of Ch'ase ABC activity at temperatures equivalent to normal (37 degrees C) and elevated (39 degrees C) body temperatures is important for optimizing its clinical usage. We report here data obtained from examining enzymatic activity at these temperatures across nine lots of commercially available protease-free Ch'ase ABC. CS GAG degrading activity was assayed by using 1) immunohistochemical detection of unsaturated disaccharide stubs generated by digestion of proteoglycans in tissue sections and 2) fluorophore-assisted carbohydrate electrophoresis (FACE) and/or high-performance liquid chromatography (HPLC) to separate and quantify unsaturated disaccharide digestion products. Our results indicate that there is a significant effect of lot and time on enzymatic thermostability. Average enzymatic activity is significantly decreased at 1 and 3 days at 39 degrees C and 37 degrees C, respectively. Furthermore, the average activity seen after 1 day was significantly different between the two temperatures. Addition of bovine serum albumin as a stabilizer significantly preserved enzymatic activity at 1 day, but not 3 days, at 39 degrees C. These results show that the CS GAG degrading activity of Ch'ase ABC is significantly decreased with incubation at body temperature over time and that all lots do not show equal thermostability. These findings are important for the design and interpretation of experimental and potential clinical studies involving Ch'ase ABC. (c) 2007 Wiley-Liss, Inc.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

PubMed Central

Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

2015-01-01

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

DNA assembly with error correction on a droplet digital microfluidics platform.

PubMed

Khilko, Yuliya; Weyman, Philip D; Glass, John I; Adams, Mark D; McNeil, Melanie A; Griffin, Peter B

2018-06-01

Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl 2 , Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb - 1 . We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

PubMed Central

Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

2014-01-01

ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication. PMID:24920820

Devices Would Detect Drugs In Sweat

NASA Technical Reports Server (NTRS)

Mintz, Fredrick W.; Richards, Gil; Kidwell, David A.; Foster, Conrad; Kern, Roger G.; Nelson, Gregory A.

1996-01-01

Proposed devices worn on skin detect such substances as methamphetamine, morphine, tetrahydrocannabinol (THC), and cocaine in wearers' sweat and transmits radio signals in response to computer queries. Called Remote Biochemical Assay Telemetering System (R-BATS), commonly referred to as "drug badge," attached to wearer by use of adhesive wristband. Used for noninvasive monitoring of levels of prescribed medications in hospital and home-care settings and to detect overdoses quickly.

Design of Potent and Druglike Nonphenolic Inhibitors for Catechol O-Methyltransferase Derived from a Fragment Screening Approach Targeting the S-Adenosyl-l-methionine Pocket.

PubMed

Lerner, Christian; Jakob-Roetne, Roland; Buettelmann, Bernd; Ehler, Andreas; Rudolph, Markus; Rodríguez Sarmiento, Rosa María

2016-11-23

A fragment screening approach designed to target specifically the S-adenosyl-l-methionine pocket of catechol O-methyl transferase allowed the identification of structurally related fragments of high ligand efficiency and with activity on the described orthogonal assays. By use of a reliable enzymatic assay together with X-ray crystallography as guidance, a series of fragment modifications revealed an SAR and, after several expansions, potent lead compounds could be obtained. For the first time nonphenolic and small low nanomolar potent, SAM competitive COMT inhibitors are reported. These compounds represent a novel series of potent COMT inhibitors that might be further optimized to new drugs useful for the treatment of Parkinson's disease, as adjuncts in levodopa based therapy, or for the treatment of schizophrenia.

Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study.

PubMed

Martínez-Castillo, Moisés; Cárdenas-Guerra, Rosa Elena; Arroyo, Rossana; Debnath, Anjan; Rodríguez, Mario Alberto; Sabanero, Myrna; Flores-Sánchez, Fernando; Navarro-Garcia, Fernando; Serrano-Luna, Jesús; Shibayama, Mineko

2017-07-01

The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis.

PubMed

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M; Weissleder, Ralph; Lee, Hakho

2016-02-23

Extracellular vesicles, including exosomes, are nanoscale membrane particles that carry molecular information on parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magneto-electrochemical assay: exosomes are immunomagnetically captured from patient samples and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables (i) highly sensitive, cell-specific exosome detection and (ii) sensor miniaturization and scale-up for high-throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the simultaneous profiling of multiple protein markers within an hour, outperforming conventional methods in assay sensitivity and speed.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis

PubMed Central

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M.; Weissleder, Ralph; Lee, Hakho

2016-01-01

Extracellular vesicles, including exosomes, are nanoscale vesicles that carry molecular information of parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magnetic-electrochemical assay: exosomes are immunomagnetically captured from patient samples, and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables i) highly sensitive, cell-specific exosome detection, and ii) sensor miniaturization and scale-up for high throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device, and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the profiling of multiple protein markers simultaneously within an hour, outperforming conventional methods in assay sensitivity and speed. PMID:26808216

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

PubMed Central

Dai, Meiling; Guo, Hongbo; Dortmans, Jos C. F. M.; Dekkers, Jojanneke; Nordholm, Johan; Daniels, Robert; van Kuppeveld, Frank J. M.; de Vries, Erik

2016-01-01

ABSTRACT Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCE The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with full-length NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used. PMID:27512075

Characterization and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase (MCAT) from Helicobacter pylori.

PubMed

Liu, Weizhi; Han, Cong; Hu, Lihong; Chen, Kaixian; Shen, Xu; Jiang, Hualiang

2006-01-23

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 (HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332 ). Enzymatic characterization of HpMCAT showed that the K(m) value for malonyl-CoA was 21.01+/-2.3 microM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC(50) value at 33.1+/-3.29 microM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.

Bioremediation of cooking oil waste using lipases from wastes

PubMed Central

do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

Comparative evaluation of bioactivity of crystalline trypsin for drying by Fourier-transformed infrared spectroscopy.

PubMed

Otsuka, Makoto; Fukui, Yuya; Ozaki, Yukihiro

2009-03-01

The purpose of this study was to evaluate the enzymatic stability of colloidal trypsin powder during heating in a solid-state by using Fourier transform infrared (FT-IR) spectra with chemoinformatics and generalized two-dimensional (2D) correlation spectroscopy. Colloidal crystalline trypsin powders were heated using differential scanning calorimetry. The enzymatic activity of trypsin was assayed by the kinetic degradation method. Spectra of 10 calibration sample sets were recorded three times with a FT-IR spectrometer. The maximum intensity at 1634cm(-1) of FT-IR spectra and enzymatic activity of trypsin decreased as the temperature increased. The FT-IR spectra of trypsin samples were analyzed by a principal component regression analysis (PCR). A plot of the calibration data obtained was made between the actual and predicted trypsin activity based on a two-component model with gamma(2)=0.962. On the other hand, a 2D method was applied to FT-IR spectra of heat-treated trypsin. The result was consistent with that of the chemoinformetrical method. The results for deactivation of colloidal trypsin powder by heat-treatment indicated that nano-structure of crystalline trypsin changed by heating reflecting that the beta-sheet was mainly transformed, since the peak at 1634cm(-1) decreased with dehydration. The FT-IR chemoinformetrical method allows for a solid-state quantitative analysis of the bioactivity of the bulk powder of trypsin during drying.

Effect of pretreatment severity on accumulation of major degradation products from dilute acid pretreated corn stover and subsequent inhibition of enzymatic hydrolysis of cellulose.

PubMed

Um, Byung-Hwan; van Walsum, G Peter

2012-09-01

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R(o)), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

PubMed

Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

2015-11-01

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Pitfalls in the detection of cholesterol in Huntington's disease models.

PubMed

Marullo, Manuela; Valenza, Marta; Leoni, Valerio; Caccia, Claudio; Scarlatti, Chiara; De Mario, Agnese; Zuccato, Chiara; Di Donato, Stefano; Carafoli, Ernesto; Cattaneo, Elena

2012-10-11

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it.

Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica

PubMed Central

Pinheiro, Guilherme L.; Correa, Raquel F.; Cunha, Raquel S.; Cardoso, Alexander M.; Chaia, Catia; Clementino, Maysa M.; Garcia, Eloi S.; de Souza, Wanderley; Frasés, Susana

2015-01-01

The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes. PMID:26347735

Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

PubMed

Pinheiro, Guilherme L; Correa, Raquel F; Cunha, Raquel S; Cardoso, Alexander M; Chaia, Catia; Clementino, Maysa M; Garcia, Eloi S; de Souza, Wanderley; Frasés, Susana

2015-01-01

The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

Bioremediation of cooking oil waste using lipases from wastes.

PubMed

Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Lignin depolymerization by fungal secretomes and a microbial sink

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salvachúa, Davinia; Katahira, Rui; Cleveland, Nicholas S.

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining,more » and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a “microbial sink” improves the extent of enzymatic lignin depolymerization.« less

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

PubMed Central

Rejón, Juan D.; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J.

2012-01-01

Background and Aims A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. Methods The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Key Results Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. Conclusions In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma. PMID:22922586

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.

PubMed

Liu, Qingsong; Kirubakaran, Sivapriya; Hur, Wooyoung; Niepel, Mario; Westover, Kenneth; Thoreen, Carson C; Wang, Jinhua; Ni, Jing; Patricelli, Matthew P; Vogel, Kurt; Riddle, Steve; Waller, David L; Traynor, Ryan; Sanda, Takaomi; Zhao, Zheng; Kang, Seong A; Zhao, Jean; Look, A Thomas; Sorger, Peter K; Sabatini, David M; Gray, Nathanael S

2012-03-23

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Lignin depolymerization by fungal secretomes and a microbial sink

DOE PAGES

Salvachua, Davinia; Katahira, Rui; Cleveland, Nicholas S.; ...

2016-08-25

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining,more » and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (M w) by 63% and 75% at pH 7 compared to the M w of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe ( Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/methanol/choline (GMC) oxidoreductases and laccases. Altogether, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a 'microbial sink' improves the extent of enzymatic lignin depolymerization.« less

Lignin depolymerization by fungal secretomes and a microbial sink

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salvachua, Davinia; Katahira, Rui; Cleveland, Nicholas S.

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining,more » and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (M w) by 63% and 75% at pH 7 compared to the M w of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe ( Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/methanol/choline (GMC) oxidoreductases and laccases. Altogether, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a 'microbial sink' improves the extent of enzymatic lignin depolymerization.« less

A system utilizing radio frequency identification (RFID) technology to monitor individual rodent behavior in complex social settings.

PubMed

Howerton, Christopher L; Garner, Joseph P; Mench, Joy A

2012-07-30

Pre-clinical investigation of human CNS disorders relies heavily on mouse models. However these show low predictive validity for translational success to humans, partly due to the extensive use of rapid, high-throughput behavioral assays. Improved assays to monitor rodent behavior over longer time scales in a variety of contexts while still maintaining the efficiency of data collection associated with high-throughput assays are needed. We developed an apparatus that uses radio frequency identification device (RFID) technology to facilitate long-term automated monitoring of the behavior of mice in socially or structurally complex cage environments. Mice that were individually marked and implanted with transponders were placed in pairs in the apparatus, and their locations continuously tracked for 24 h. Video observation was used to validate the RFID readings. The apparatus and its associated software accurately tracked the locations of all mice, yielding information about each mouse's location over time, its diel activity patterns, and the amount of time it was in the same location as the other mouse in the pair. The information that can be efficiently collected in this apparatus has a variety of applications for pre-clinical research on human CNS disorders, for example major depressive disorder and autism spectrum disorder, in that it can be used to quantify validated endophenotypes or biomarkers of these disorders using rodent models. While the specific configuration of the apparatus described here was designed to answer particular experimental questions, it can be modified in various ways to accommodate different experimental designs. Copyright © 2012 Elsevier B.V. All rights reserved.

Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness.

PubMed

Menegakis, Apostolos; De Colle, Chiara; Yaromina, Ala; Hennenlotter, Joerg; Stenzl, Arnulf; Scharpf, Marcus; Fend, Falko; Noell, Susan; Tatagiba, Marcos; Brucker, Sara; Wallwiener, Diethelm; Boeke, Simon; Ricardi, Umberto; Baumann, Michael; Zips, Daniel

2015-09-01

To apply our previously published residual ex vivo γH2AX foci method to patient-derived tumour specimens covering a spectrum of tumour-types with known differences in radiation response. In addition, the data were used to simulate different experimental scenarios to simplify the method. Evaluation of residual γH2AX foci in well-oxygenated tumour areas of ex vivo irradiated patient-derived tumour specimens with graded single doses was performed. Immediately after surgical resection, the samples were cultivated for 24h in culture medium prior to irradiation and fixed 24h post-irradiation for γH2AX foci evaluation. Specimens from a total of 25 patients (including 7 previously published) with 10 different tumour types were included. Linear dose response of residual γH2AX foci was observed in all specimens with highly variable slopes among different tumour types ranging from 0.69 (95% CI: 1.14-0.24) to 3.26 (95% CI: 4.13-2.62) for chondrosarcomas (radioresistant) and classical seminomas (radiosensitive) respectively. Simulations suggest that omitting dose levels might simplify the assay without compromising robustness. Here we confirm clinical feasibility of the assay. The slopes of the residual foci number are well in line with the expected differences in radio-responsiveness of different tumour types implying that intrinsic radiation sensitivity contributes to tumour radiation response. Thus, this assay has a promising potential for individualized radiation therapy and prospective validation is warranted. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

New NIR Calibration Models Speed Biomass Composition and Reactivity Characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

2015-09-01

Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. This highlight describes NREL's work to use near-infrared (NIR) spectroscopy and partial least squares multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. This highlight is being developed for the September 2015 Alliance S&T Board meeting.

Markers of Developmentally Regulated Programmed Cell Death and Their Analysis in Cereal Seeds.

PubMed

Domínguez, Fernando; Cejudo, Francisco Javier

2018-01-01

Programmed cell death (PCD) is a key process for the development and differentiation of multicellular organisms, which is characterized by well-defined morphological and biochemical features. These include chromatin condensation, DNA degradation and nuclear fragmentation, with nucleases and proteases playing a relevant function in these processes. In this chapter we describe methods routinely used for the analysis of hallmarks of developmentally regulated PCD in cereal seed tissues, which are based on agarose and polyacrylamide gel electrophoresis, in situ staining of DNA fragmentation, and cell-free assays of relevant enzymatic activities.

Evidence of neutralizing activity against T3 coliphage in oyster Crassostrea gigas hemolymph.

PubMed

Bachère, E; Hervio, D; Mialhe, E; Grizel, H

1990-01-01

To investigate defense reactions of bivalve molluscs against viruses, experimental in vitro assays have been developed using T3 coliphage as a test virus. A native neutralizing factor in oyster Crassostrea gigas serum showed high individual variability and was enhanced significantly by repeated sampling of hemolymph from the same oysters. The responsible factor is apparently thermolabile and sensitive to EDTA treatment. Because of an inhibitory effect by the enzymatic inhibitor, phenylmethylsulphonyl fluoride (PMSF), the T3-neutralizing factor may be related to serine protease.

Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test.

PubMed

Yang, Xinjian; Gao, Zhiqiang

2015-04-25

On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells.

Applications of SHAPES screening in drug discovery.

PubMed

Lepre, Christopher A; Peng, Jeffrey; Fejzo, Jasna; Abdul-Manan, Norzehan; Pocas, Jennifer; Jacobs, Marc; Xie, Xiaoling; Moore, Jonathan M

2002-12-01

The SHAPES strategy combines nuclear magnetic resonance (NMR) screening of a library of small drug-like molecules with a variety of complementary methods, such as virtual screening, high throughput enzymatic assays, combinatorial chemistry, X-ray crystallography, and molecular modeling, in a directed search for new medicinal chemistry leads. In the past few years, the SHAPES strategy has found widespread utility in pharmaceutical research. To illustrate a variety of different implementations of the method, we will focus in this review on recent applications of the SHAPES strategy in several drug discovery programs at Vertex Pharmaceuticals.

Development of a Novel NMR-based Rheb GTPase Assay and Molecular Characterization of TSC2 GAP Activity

DTIC Science & Technology

2010-05-01

GTPase) that belongs to the Ras superfamily and has homologs in yeast, fungi , slime mold, fruit fly, zebra fish, and mammals (1–3). Ge- netic and...characterization of TSC2 disease mutations affecting its GAP activity (months 9-12) While the final aspects of this task are yet to be completed, we have...domain mutants of TSC2 that we examined affected its enzymatic activ- ity. This method can now be applied to study the function and regulation of other

Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

PubMed

Haruta, M; Murata, M; Kadokura, H; Homma, S

1999-03-01

An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

PubMed

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

DOE PAGES

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; ...

2018-05-14

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.

PubMed

Ciossek, Thomas; Julius, Heiko; Wieland, Heike; Maier, Thomas; Beckers, Thomas

2008-01-01

Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.

Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

PubMed

Tran, Nini; Zielke, Ryszard A; Vining, Oliver B; Azevedo, Mark D; Armstrong, Donald J; Banowetz, Gary M; McPhail, Kerry L; Sikora, Aleksandra E

2013-09-01

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

PubMed

Lane, Courtney E; Benton, Michael G

2015-12-01

A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

PubMed

Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

2017-12-01

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

PubMed Central

Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

PubMed

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

PubMed

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

2016-11-15

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. © 2016. Published by The Company of Biologists Ltd.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

PubMed Central

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar

2016-01-01

ABSTRACT Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. PMID:27612509

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

PubMed

Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

2016-04-01

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

PubMed

Dinca, V; Ranella, A; Farsari, M; Kafetzopoulos, D; Dinescu, M; Popescu, A; Fotakis, C

2008-10-01

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Assessment of sperm nucleus integrity in infertile men: a novel research field for anthropology in the molecular era.

PubMed

Lavranos, Giagkos; Manolakou, Panagiota; Katsiki, Evangelia; Angelopoulou, Roxani

2013-12-01

Anthropology has always been particularly interested in the origin of human life and the development towards adulthood. Although originally working with skeletal measurements and bio-morphological markers in modern populations, it has now entered the growing field of applied molecular biology. This relatively recent advance allows the detailed study of major events in human development and senescence. For instance, sperm DNA integrity and chromatin re-organization are crucial factors for fertilization and embryo development. Clinical researchers have developed improved methods for the evaluation of DNA integrity and protaminosis in sperm nuclei, such as the TUNEL and the CMA3 assays. DNA damage in spermatozoal nuclei is detected using the TUNEL assay which depends on the specific enzymatic reaction of TdT with the end strand breaks of DNA. Protaminosis in spermatozoal nucleus is evaluated using CMA3 assay, which is based on the in situ competition between CMA3 and protamines. Such measurements may provide useful data on human reproductive health, aiding the explanation of demographic differences across the world.

Development of a simple, low cost chronoamperometric assay for fructose based on a commercial graphite-nanoparticle modified screen-printed carbon electrode.

PubMed

Nicholas, Phil; Pittson, Robin; Hart, John P

2018-02-15

This paper describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry with a 40µl mixture containing fructose, mediator and FDH, deposited onto the SPCE-G-COOH. The response was linear from 0.1mM to 1.0mM. A commercial fruit juice sample was analysed using the developed assay and the fructose concentration was calculated to be 477mM with a precision of 3.03% (n=5). Following fortification (477mM fructose) the mean recovery was found to be 97.12% with a coefficient of variation of 6.42% (n=5); consequently, the method holds promise for the analysis of commercial fruit juices. Copyright © 2017 Elsevier Ltd. All rights reserved.

The adhesion of blood platelets on fibrinogen surface: comparison of two biochemical microplate assays.

PubMed

Vanícková, Martina; Suttnar, Jirí; Dyr, Jan Evangelista

2006-11-01

The biocompatibility of materials is frequently assessed by blood platelet adhesion, since platelet adhesion plays a considerable role in blood interaction with artificial surfaces. Blood platelets adhesion is an essential event in haemostatic and thrombotic processes. The aim of this study was to simultaneously compare simple biochemical assays widely used for evaluation of platelet static adhesion based on the determination of enzymatic activity of either lactate dehydrogenase (LDH) or acid phosphatase (ACP) in lysates of adhered platelets. Adhesion of platelets from platelet-rich plasma and washed platelets activated by either ADP or thrombin on surfaces covered with fibrinogen and well defined fibrin was studied. The results demonstrated that the amounts of adhered platelets estimated by the LDH method were significantly lower as compared with the amount obtained by ACP method. LDH but not ACP release from platelets during adhesion was shown to take place. It suggests that the LDH method should be used rather as an assay of platelet integrity. The ACP method is much more suitable for quantitative determination of platelet adhesion especially in the development and evaluation of haemocompatibility of new biomaterials.

Endoglucanase (EG) Activity Assays.

PubMed

Rahman, M Shafiqur; Fernando, Sheran; Ross, Brian; Wu, Jiangning; Qin, Wensheng

2018-01-01

Cellulosic biomass, the most common organic compound of primary energy source on earth, is a network of interwoven biopolymers of plant cell walls. Degradation of cellulose is important for global carbon recycling. Moreover, biofuel, a renewable fuel whose energy can be derived from cellulosic biomass by enzymatic hydrolysis of cellulases. Among cellulases are endoglucases that act synergistically for subsequent hydrolytic reactions to break down the polymeric cellulose. However, in cellulolytic enzyme activity endoglucanase plays a prominent role in initiating and sustaining the hydrolytic process. Endoglucanase randomly cleaves the cellulose polymer into smaller sugar and oligomeric polysaccharides. Characterization and quantification of endoglucanase activity is important for industry and in the overall study of cellulose degradation. All assays including those for endoglucanase fall into two broad categories either qualitative or quantitative. Quantitative assays can tell if the enzyme is present, how much and its activity. Measurement can be done indirectly using a secondary colorimetric product like (1) molybdenum blue, (2) 3-amino-5-nitrosalicylic acid, (3) bicinchoninic acid (BCA), and (4) 2-cyanoacetamide or directly using an antibody in an ELISA. In this chapter, we discuss several common protocols for the measurement of endoglucanase activity.

Integrated, Continuous Emulsion Creamer.

PubMed

Cochrane, Wesley G; Hackler, Amber L; Cavett, Valerie J; Price, Alexander K; Paegel, Brian M

2017-12-19

Automated and reproducible sample handling is a key requirement for high-throughput compound screening and currently demands heavy reliance on expensive robotics in screening centers. Integrated droplet microfluidic screening processors are poised to replace robotic automation by miniaturizing biochemical reactions to the droplet scale. These processors must generate, incubate, and sort droplets for continuous droplet screening, passively handling millions of droplets with complete uniformity, especially during the key step of sample incubation. Here, we disclose an integrated microfluidic emulsion creamer that packs ("creams") assay droplets by draining away excess oil through microfabricated drain channels. The drained oil coflows with creamed emulsion and then reintroduces the oil to disperse the droplets at the circuit terminus for analysis. Creamed emulsion assay incubation time dispersion was 1.7%, 3-fold less than other reported incubators. The integrated, continuous emulsion creamer (ICEcreamer) was used to miniaturize and optimize measurements of various enzymatic activities (phosphodiesterase, kinase, bacterial translation) under multiple- and single-turnover conditions. Combining the ICEcreamer with current integrated microfluidic DNA-encoded library bead processors eliminates potentially cumbersome instrumentation engineering challenges and is compatible with assays of diverse target class activities commonly investigated in drug discovery.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

PubMed

Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

2010-01-01

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

Ricin - inhibitor design. Annual report, 15 April 1994-14 April 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schramm, V.L.

1995-05-14

Substrates for ricin A-chain include short RNA stem-loop structures which have been synthesized with radioactive labels for ease of catalytic assay and for kinetic isotope effects. Ricin A-chain from several sources is incapable of completing multiple catalytic cycles using these substrates. A family of ricin substrate analogue molecules have been synthesized and tested which are specific for transition states with oxycarbonium character or for enzymatic mechanisms involving protonation of the adenine leaving group. Formycin analogues were incorporated into RNA oligomeric structures and tested for binding to ricin A-chain or as inhibitors of the ricin-inactivation of in vitro translation using rabbitmore » reticulocyte lysates. Ribo-oxycarbonium ion analogues containing iminoribitol analogues of ribose were synthetically incorporated into RNA oligomeric structures. Neither formycin nor ribo-oxycarbonium analogues, either singly or in RNA oligomers caused significant inhibition of ricin A-chain when assayed in reticulocyte lysate translation assays. The results indicate a novel transition state mechanism for ricin A-chain, or a requirement for additional features of 28s rRNA to bind transition state analogues.« less

Dissolved organic phosphorus (DOP) and its potential role for ecosystem nutrition

NASA Astrophysics Data System (ADS)

Brödlin, Dominik; Hagedorn, Frank; Kaiser, Klaus

2016-04-01

During ecosystem development and soil formation, primary mineral sources of phosphorus are becoming increasingly depleted. Inorganic phosphorus forms tend to be bound strongly to or within secondary minerals, thus, are hardly available to plants and are not leached from soil. What about organic forms of phosphorus? Since rarely studied, little is known about the fluxes of dissolved organic phosphorus (DOP) forms and their role in the P cycle. However, there is evidence that DOP is composed of some plant-derived organic phosphorus compounds, such as phytate, which are less mobile and prone to be sorbed to mineral surfaces, whereas microbial-derived compounds like nucleic acids and simple phospho-monoester may represent more mobile forms of soil phosphorus. In our study, we estimated fluxes, composition, and bioavailability of DOP along a gradient in phosphorus availability at five sites on silicate bedrock across Germany (Bad Brückenau, Conventwald, Vessertal, Mitterfels and Lüss) and at a calcareous site in Switzerland (Schänis). Soil solution was collected at 0 down to 60 to 150 cm soil depth at different intervals. Since most solutions had very low P concentrations (<0.05 mg total dissolved P/L), soil solutions had to be concentrated by freeze-drying for the enzymatic characterization of DOP. In order to test the potential bioavailability, we used an enzyme assay distinguishing between phytate-like P (phytate), diester-like P (nucleic acids), monoester-like P (glucose-6-phosphate), and pyrophosphate of bulk molybdate unreactive phosphorus (MUP). First results from the enzymatic assay indicated that monoester-like P and diester-like P were the most prominent form of the hydrolysable DOP constituents. In leachates from the organic layer, there was a high enzymatic activity for monoester-like P, indicating high recycling efficiency and rapid hydrolysis of labile DOP constituents. DOP was the dominating P form in soil solution at some of the sites, with a greater contribution to total dissolved P in winter than in summer. Concentrations of DOP decreased along the phosphorus availability gradient from less to the more developed forest ecosystems.

The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

NASA Astrophysics Data System (ADS)

von Sperber, C.; Tamburini, F.; Brunner, B.; Bernasconi, S. M.; Frossard, E.

2015-07-01

Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (Pi) from organic phosphorus compounds (Porg). Phytic acid (myo-inositol hexakisphosphate, IP6) is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields available Pi and less phosphorylated inositol derivates as products. The hydrolysis of organic P compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ18O) of released Pi, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released Pi. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP6, adenosine 5'-monophosphate (AMP) and glycerophosphate (GPO4) as substrates. For a comparison to the δ18O of Pi released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP6 as a substrate were prepared. During the hydrolysis of IP6 by phytase, four of the six Pi were released, and one oxygen atom from water was incorporated into each Pi. This incorporation of oxygen from water into Pi was subject to an apparent inverse isotopic fractionation (ϵ ~ 6 to 10 ‰), which was similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ~ 7 ‰), where less than three Pi were released. The incorporation of oxygen from water into Pi during the hydrolysis of AMP and GPO4 by phytase yielded a normal isotopic fractionation (ϵ ~ -12 ‰), similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ϵ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking substrate dependency of the isotopic fractionation could be attributed to a difference in the δ18O values of the C-O-P bridging and non-bridging oxygen atoms in organic phosphate compounds.

The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

NASA Astrophysics Data System (ADS)

Sperber, C. v.; Tamburini, F.; Brunner, B.; Bernasconi, S. M.; Frossard, E.

2015-03-01

Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (Pi) from organic phosphorus compounds (Porg). Phytic acid (IP6) is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields plant available inorganic phosphate (Pi) and less phosphorylated inositol derivates as products. The hydrolysis of organic P-compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ18O) of released Pi, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released Pi. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP6, adenosine 5'monophosphate (AMP) and glycerophosphate (GPO4) as substrates. For a comparison to the δ18O of Pi released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP6 as substrate were prepared. During the hydrolysis of IP6 by phytase, four Pi are released, and one oxygen atom from water is incorporated into each Pi. This incorporation of oxygen from water into Pi is subject to an apparent inverse isotopic fractionation (ϵ ∼ 6 to 10‰), which is similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ∼ 7‰) where less than three Pi are released. The incorporation of oxygen from water into Pi during the hydrolysis of AMP and GPO4 by phytase yielded a normal isotopic fractionation (ϵ ∼ -12‰), again similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ɛ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking substrate-dependency of the isotopic fractionation could be attributed to a difference in the δ18O-values of the C-O-P bridging and non-bridging oxygen atoms in organic phosphate compounds.

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

NASA Astrophysics Data System (ADS)

Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

2011-05-01

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate this potential, on-chip adhesion islands are fabricated to immobilize MCF-7 human breast cancer cells. Viability studies are performed to assess the functionalization efficiency.

Imaging Primary Prostate Cancer and Bone Metastasis

DTIC Science & Technology

2006-04-01

BBN peptide has a pyroglutamic acid at the N-terminus and an amidated methionine at the C-termi- nus, further modification and radiolabeling of this...14), where Aca refers to ε-aminocaproic acid . For both compounds, in vitro assays, metabolic stability, and microPET studies were performed to...BBN(7- 14) (■) by PC-3 cells. Data are percentage of acid - resistant (internalized) radio- activity in cells for internalization, and percentage of

Bio-prospecting of distillery yeasts as bio-control and bio-remediation agents.

PubMed

Ubeda, Juan F; Maldonado, María; Briones, Ana I; Francisco, J Fernández; González, Francisco J

2014-05-01

This work constitutes a preliminary study in which the capacity of non-Saccharomyces yeasts isolated from ancient distilleries as bio-control agents against moulds and in the treatment of waste waters contaminated by heavy metals-i.e. bio-remediation-is shown. In the first control assays, antagonist effect between non-Saccharomyces yeasts, their extracts and supernatants against some moulds, analysing the plausible (not exhaustive) involved factors were qualitatively verified. In addition, two enzymatic degrading properties of cell wall plant polymers, quitinolitic and pectinolitic, were screened. Finally, their use as agents of bio-remediation of three heavy metals (cadmium, chromium and lead) was analysed semi-quantitatively. The results showed that all isolates belonging to Pichia species effectively inhibited all moulds assayed. Moreover, P. kudriavzevii is a good candidate for both bio-control and bio-remediation because it inhibited moulds and accumulated the major proportion of the three tested metals.

Somatic mutation detection in human biomonitoring.

PubMed

Olsen, L S; Nielsen, L R; Nexø, B A; Wassermann, K

1996-06-01

Somatic cell gene mutation arising in vivo may be considered to be a biomarker for genotoxicity. Assays detecting mutations of the haemoglobin and glycophorin A genes in red blood cells and of the hypoxanthine-guanine phosphoribosyltransferase and human leucocyte antigenes in T-lymphocytes are available in humans. This MiniReview describes these assays and their application to studies of individuals exposed to genotoxic agents. Moreover, with the implementation of techniques of molecular biology mutation spectra can now be defined in addition to the quantitation of in vivo mutant frequencies. We describe current screening methods for unknown mutations, including the denaturing gradient gel electrophoresis, single strand conformation polymorphism analysis, heteroduplex analysis, chemical modification techniques and enzymatic cleavage methods. The advantage of mutation detection as a biomarker is that it integrates exposure and sensitivity in one measurement. With the analysis of mutation spectra it may thus be possible to identify the causative genotoxic agent.

Investigation of the anticoagulant and antithrombotic effects of chlorogenic acid.

PubMed

Choi, Jun-Hui; Kim, Seung

2017-03-01

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. Fibrin clot and turbidity assays revealed that it was able to inhibit the formation of fibrin clot. Chlorogenic acid degraded blood clot and inhibited the enzymatic activity of procoagulant proteases, thrombin, activated factor X (FXa), and activated factor XIII (FXIIIa). Chlorogenic acid was found to delay activated partial thromboplastin time, prothrombin time, and thrombin time. PFA-100 assays showed that it prolonged the closure time of citrated whole human blood. It demonstrated the antithrombotic effect in collagen and epinephrine-induced acute thromboembolism mice model. These antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to NIH-3T3 and 3T3-L1 cells, make it a potential agent for thrombotic treatment and prevention. © 2016 Wiley Periodicals, Inc.

[Diagnosis of primary hyperlipoproteinemia in umbilical cord blood (author's transl)].

PubMed

Parwaresch, M R; Radzun, H J; Mäder, C

1977-10-01

The aim of the present investigation was to assay the frequency of primary dyslipoproteinemia in a random sample of one hundred newborns and to describe the minimal methodical requirements for sound diagnosis. After comparison of different methods total lipids were determined by gravimetry, cholesterol and triglycerides by enzymatic methods, nonesterified fatty acids by direct colorimetry; phospholipids were estimated indirectly. All measurements were applied to umbilical cord sera and to lipoprotein fractions separated by selective precipitation. The diagnosis of hyperlipoproteinemia type IV, which is the most frequent one in adults, is highly afflicted with pitfalls in the postnatal period. A primary hyper-alpha-liproteinemia occured in one case and type II-hyperlipoproteinemia in two cases, one of the parents being involved in each case. For mass screening triglycerides should be assayed in serum and cholesterol in precipitated and resolubilized LDL-fraction, for which the minimal requirements are described.

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

PubMed Central

Kuhl, Alexander; Svenstrup, Niels; Ladel, Christoph; Otteneder, Michael; Binas, Annegret; Schiffer, Guido; Brands, Michael; Lampe, Thomas; Ziegelbauer, Karl; Rübsamen-Waigmann, Helga; Haebich, Dieter; Ehlert, Kerstin

2005-01-01

Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 μg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired. PMID:15728893

Relation Between Deoxyribonucleic Acid and Intracytoplasmic Membranes in Escherichia coli O111a11

PubMed Central

Altenburg, Betty C.; Suit, Joan C.

1970-01-01

The possibility of a relationship between intracytoplasmic membranes and deoxyribonucleic acid (DNA) in Escherichia coli O111a1 has been investigated. To facilitate this investigation, a simple enzymatic assay for the amount of internal membrane present in a culture was developed. This assay was then used to show that the appearance of intracytoplasmic membranes is accompanied by an increase in the DNA content of the cells. Electron micrographs have confirmed this observation and have shown DNA to be in contact with the intracytoplasmic membranes. Extensive membranes were observed at sites of apparently unsuccessful attempts at cell division. These observations led to the conclusion that the internal membrane formed by strain O111a1 represents “extra” membrane, which is functional in that it contains sites for DNA replication, but is produced in excess because the organism is somehow defective in its regulation of membrane synthesis. Images PMID:4192984

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. Electronic supplementary information (ESI) available: Additional circular dichroism data and nanoparticle tracking analysis trace. See DOI: 10.1039/c6nr02009g

An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites.

PubMed

Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui

2016-03-01

Protein-inorganic nanoflowers, composed of protein and copper(II) phosphate (Cu3(PO4)2), have recently grabbed people's attention. Because the synthetic method requires no organic solvent and because of the distinct hierarchical nanostructure, protein-inorganic nanoflowers display enhanced catalytic activity and stability and would be a promising tool in biocatalytical processes and biological and biomedical fields. In this work, we first coimmobilized the enzyme, antibody, and Cu3(PO4)2 into a three-in-one hybrid protein-inorganic nanoflower to enable it to possess dual functions: (1) the antibody portion retains the ability to specifically capture the corresponding antigen; (2) the nanoflower has enhanced enzymatic activity and stability to produce an amplified signal. The prepared antibody-enzyme-inorganic nanoflower was first applied in an enzyme-linked immunosorbent assay to serve as a novel enzyme-labeled antibody for Escherichia coli O157:H7 (E. coli O157:H7) determination. The detection limit is 60 CFU L(-1), which is far superior to commercial ELISA systems. The three-in-one antibody (anti-E. coli O157:H7 antibody)-enzyme (horseradish peroxidase)-inorganic (Cu3(PO4)2) nanoflower has some advantages over commercial enzyme-antibody conjugates. First, it is much easier to prepare and does not need any complex covalent modification. Second, it has fairly high capture capability and catalytic activity because it is presented as aggregates of abundant antibodies and enzymes. Third, it has enhanced enzymatic stability compared to the free form of enzyme due to the unique hierarchical nanostructure.

Organic amendments to avocado crops induce suppressiveness and influence the composition and activity of soil microbial communities.

PubMed

Bonilla, Nuria; Vida, Carmen; Martínez-Alonso, Maira; Landa, Blanca B; Gaju, Nuria; Cazorla, Francisco M; de Vicente, Antonio

2015-05-15

One of the main avocado diseases in southern Spain is white root rot caused by the fungus Rosellinia necatrix Prill. The use of organic soil amendments to enhance the suppressiveness of natural soil is an inviting approach that has successfully controlled other soilborne pathogens. This study tested the suppressive capacity of different organic amendments against R. necatrix and analyzed their effects on soil microbial communities and enzymatic activities. Two-year-old avocado trees were grown in soil treated with composted organic amendments and then used for inoculation assays. All of the organic treatments reduced disease development in comparison to unamended control soil, especially yard waste (YW) and almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization. All of the organic amendments assayed in this study were able to suppress white root rot, although their suppressiveness appears to be mediated differentially. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Photoinitiator-Free Synthesis of Endothelial Cell Adhesive and Enzymatically Degradable Hydrogels

PubMed Central

Jones, Derek R.; Marchant, Roger E.; von Recum, Horst; Gupta, Anirban Sen; Kottke-Marchant, Kandice

2015-01-01

We report on a photoinitiator-free synthetic method of incorporating bioactivity into poly(ethylene glycol) (PEG) hydrogels in order to control physical properties, enzymatic biodegradability and cell-specific adhesiveness of the polymer network, while eliminating the need for UV-mediated photopolymerization. To accomplish this, hydrogel networks were polymerized using Michael addition with four-arm PEG acrylate (10 kDa), using a collagenase sensitive peptide (CSP) as a crosslinker, and introducing an endothelial cell adhesive peptide either terminally (RGD) or attached to the crosslinking peptide sequence (CSP-RGD). The efficiency of the Michael addition reactions were determined by NMR and Ellman’s assay. Successful decoupling of cell adhesivity and physical properties was demonstrated by quantifying and comparing the swelling ratios and Young’s Moduli of various hydrogel formulations. Degradation profiles were established by incubating functionalized hydrogels in collagenase solutions (0.0 – 1.0 µg/mL), demonstrating that functionalized hydrogels degraded at a rate dependent upon collagenase concentration. Moreover, it was shown that the degradation rate was independent of CSP-RGD concentration. Cell attachment and proliferation on functionalized hydrogels were compared for various RGD concentrations, providing evidence that cell attachment and proliferation were directly related to relative amounts of the CSP-RGD combination peptide. An increase in cell viability was achieved using Michael addition techniques when compared to UV-polymerization, and was assessed by a LIVE/DEAD fluorescence assay. This photoinitiator-free method shows promise in creating hydrogel-based tissue engineering scaffolds allow for decoupled cell adhesivity and physical properties and that render greater cell viability. PMID:25462848

Heparanase induced by advanced glycation end products (AGEs) promotes macrophage migration involving RAGE and PI3K/AKT pathway

PubMed Central

2013-01-01

Background Advanced glycation end products (AGEs), inflammatory-associated macrophage migration and accumulation are crucial for initiation and progression of diabetic vascular complication. Enzymatic activity of heparanase (HPA) is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, HPA enhances the phosphorylation of selected signaling molecules including AKT pathway independent of enzymatic activity. However, virtually nothing is presently known the role of HPA during macrophage migration exposed to AGEs involving signal pathway. Methods These studies were carried out in Ana-1 macrophages. Macrophage viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. HPA and AKT protein expression in macrophages are analysed by Western blotting and HPA mRNA expression by real time quantitative RT-PCR. Release of HPA was determined by ELISA. Macrophage migration was assessed by Transwell assays. Results HPA protein and mRNA were found to be increased significantly in AGEs-treated macrophages. Pretreatment with anti-HPA antibody which recognizes the nonenzymatic terminal of HPA prevented AGEs-induced AKT phosphorylation and macrophage migration. LY294002 (PI3k/AKT inhibitor) inhibited AGEs-induced macrophage migration. Furthermore, pretreatment with anti-receptor for advanced glycation end products (RAGE) antibody attenuated AGEs-induced HPA expression, AKT phosphorylation and macrophage migration. Conclusions These data indicate that AGEs-induced macrophage migration is dependent on HPA involving RAGE-HPA-PI3K/AKT pathway. The nonenzymatic activity of HPA may play a key role in AGEs-induced macrophage migration associated with inflammation in diabetic vascular complication. PMID:23442498

Headache and neuropsychic disorders in the puerperium: a case report with suspected deficiency of urea cycle enzymes.

PubMed

Tonini, Maria Clara; Bignamini, V; Mattioli, M

2011-05-01

An enzymatic abnormality of the urea cycle is a metabolic disorder occasionally seen in adults, but particularly in the puerperium. The main risk is acute hyperammoniemic encephalopathy, leading to psychosis, coma and even death if not diagnosed promptly and treated appropriately. Headache is frequent in the puerperium normally manifesting between 3 and 6 days after delivery. We describe here a 39-year-old woman, who 3 days after delivery presented diffuse tension-type headache and depression, followed by behavioral disorders, psychomotor agitation, epileptic seizures, and finally coma 2 days later. Pregnancy and normal delivery: routine blood chemistry findings, CT scan, MR imaging, angio-MR of the brain, and lumbar puncture were normal. EEG when seizures started, it showed diffuse slowing, as in the case of metabolic encephalopathy. This led us to assay blood ammonia, which was high at >400 mmol. Liver function and abdominal US were normal; hence, we suspected a urea cycle enzymatic abnormality, and requested for genetic tests. These confirmed a congenital primary metabolic deficiency of arginine succinate synthetase, with high citrullinemia (type II, adult form). Dialysis was started promptly, with initially iv arginine, then orally, plus medical therapy for the hyperammoniemia and a low protein diet; plasma ammonia dropped swiftly to normal, and her state of consciousness gradually improved until all the clinical symptoms had resolved. Ammonia assay should always be considered in the first few days of the puerperium in women with headache and behavioral disorders, to exclude an inborn deficiency of the urea cycle, which may have gone unnoticed until then.

Do alterations in follicular fluid proteases contribute to human infertility?

PubMed

Cookingham, Lisa Marii; Van Voorhis, Bradley J; Ascoli, Mario

2015-05-01

Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.

PubMed

Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni

2014-03-01

Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.

Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

PubMed

Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

2018-05-07

Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

NASA Astrophysics Data System (ADS)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

A smartphone-readable barcode assay for the detection and quantitation of pesticide residues.

PubMed

Guo, Juan; Wong, Jessica X H; Cui, Caie; Li, Xiaochun; Yu, Hua-Zhong

2015-08-21

In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method.

Real-time monitoring of lactate extrusion and glucose consumption of cultured cells using a lab-on-valve system.

PubMed

Schulz, Craig M; Scampavia, Louis; Ruzicka, Jaromir

2002-12-01

Microsequential injection (microST) provides microfluidic operations that are ideally suited for cellular function studies and as a means of validating targets for drug discovery. MicroSI carried out within the lab-on-valve (LOV) manifold, is an ideal platform for spectroscopic studies on living cells that are grown on microcarrier beads and kept thermostated while their metabolism is probed in real-time. In this paper a microbioreactor is integrated into the LOV manifold allowing measurement of cellular lactate extrusion and glucose consumption rates of a cell culture that is automatically renewed prior to each measurement. Glucose consumption and lactate extrusion are monitored using NAD-linked enzymatic assays. The microSI-LOV setup has demonstrated a linear analysis range of 0.05-1.00 mM for lactate and 0.1-5.6 mM for glucose. These assays were conducted in a serial fashion requiring 3 microL of cellular perfusate and 10 s for glucose determination and 30 s for the lactate assay. Overall waste generated per lactate/glucose assay is < 200 microL. This work was performed using two different transfected hepatocyte cell lines, which adhere to Cytopore microcarrier beads. This novel approach to metabolic screening allows for the rapid evaluation of the effects of dosing cells with chemical agents.