Science.gov

Sample records for activity assays demonstrated

  1. Rubisco Activase Activity Assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, Rubisco. This action modulates the activation state of Rubisco by removing tightly-bound inhibitory s...

  2. Performance in the WIPP nondestructive assay performance demonstration program

    SciTech Connect

    Marcinkiewicz, C.J.; Connolly, M.J.; Becker, G.K.

    1997-11-01

    Measurement facilities performing nondestructive assay (NDA) of wastes intended for disposal at the United States Department of Energy (DOE) Waste Isolation Pilot Plant (WIPP) are required to demonstrate their ability to meet specific Quality Assurance Objectives (QAOs). This demonstration is performed, in part, by participation in the NDA Performance Demonstration Program (PDP). The PDP is funded and managed by the Carlsbad Area Office (CAO) of DOE and is conducted by the Idaho National Engineering Laboratory. It tests the characteristics of precision, system bias and/or total uncertainty through the measurement of variable, blind combinations of simulated waste drums and certified radioactive standards. Each facility must successfully participate in the PDP using each different type of measurement system planned for use in waste characterization. The first cycle of the PDP using each different type of measurement system planned for use in waste characterization. The first cycle of the PDP was completed in July 1996 and the second is scheduled for completion by December 1996. Seven sites reported data in cycle 1 for 11 different measurement systems. This paper describes the design and operation of the PDP and provides the performance data from cycle 1. It also describes the preliminary results from cycle 2 and updates the status and future plans for the NDA PDP. 4 refs., 9 figs., 11 tabs.

  3. Demonstration of Active Combustion Control

    NASA Technical Reports Server (NTRS)

    Lovett, Jeffrey A.; Teerlinck, Karen A.; Cohen, Jeffrey M.

    2008-01-01

    The primary objective of this effort was to demonstrate active control of combustion instabilities in a direct-injection gas turbine combustor that accurately simulates engine operating conditions and reproduces an engine-type instability. This report documents the second phase of a two-phase effort. The first phase involved the analysis of an instability observed in a developmental aeroengine and the design of a single-nozzle test rig to replicate that phenomenon. This was successfully completed in 2001 and is documented in the Phase I report. This second phase was directed toward demonstration of active control strategies to mitigate this instability and thereby demonstrate the viability of active control for aircraft engine combustors. This involved development of high-speed actuator technology, testing and analysis of how the actuation system was integrated with the combustion system, control algorithm development, and demonstration testing in the single-nozzle test rig. A 30 percent reduction in the amplitude of the high-frequency (570 Hz) instability was achieved using actuation systems and control algorithms developed within this effort. Even larger reductions were shown with a low-frequency (270 Hz) instability. This represents a unique achievement in the development and practical demonstration of active combustion control systems for gas turbine applications.

  4. Indirect conductimetric assay of antibacterial activities.

    PubMed

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  5. An Undergraduate Laboratory Activity Demonstrating Bacteriophage Specificity†

    PubMed Central

    Allen, Mary E.; Gyure, Ruth A.

    2013-01-01

    Bacteriophage are among the most diverse and numerous microbes inhabiting our planet. Yet many laboratory activities fail to engage students in meaningful exploration of their diversity, unique characteristics, and abundance. In this curriculum activity students use a standard plaque assay to enumerate bacteriophage particles from a natural sample and use the scientific method to address questions about host specificity and diversity. A raw primary sewage sample is enriched for bacteriophage using hosts in the family Enterobacteriaceae. Students hypothesize about host specificity and use quantitative data (serial dilution and plaque assay) to test their hypotheses. Combined class data also help them answer questions about phage diversity. The exercise was field tested with a class of 47 students using pre- and posttests. For all learning outcomes posttest scores were higher than pretest scores at or below p = 0.01. Average individualized learning gain (G) was also calculated for each learning outcome. Students’ use of scientific language in reference to bacteriophage and host interaction significantly improved (p = 0.002; G = 0.50). Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, p = 0.01) and to explain the plaque assay method (G = 0.33, p = 0.002). At the end of the exercise students also demonstrated improved knowledge and understanding of phage specificity as related to phage therapy in humans (p < 0.001; G = 51). PMID:23858357

  6. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  7. Assaying nonspecific phospholipase C activity.

    PubMed

    Pejchar, Přemysl; Scherer, Günther F E; Martinec, Jan

    2013-01-01

    Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC activity, to phosphatidic acid (PA) that participates in ABA signaling pathways. Here we describe a step-by-step protocol, which can be used to determine in situ or in vitro NPC activity. Determination is based on quantification of fluorescently labeled DAG as a product of cleavage of the fluorescently labeled substrate lipid, phosphatidylcholine. High-performance thin-layer chromatography is used for separation of fluorescent DAG. The spot is visualized with a laser scanner and the relative amounts of fluorescent DAG are quantified using imaging software. PMID:23681535

  8. Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories

    PubMed Central

    Levine, Gail L.; Sambor, Anna; Carter, Donald K.; Sato, Alicia; Kopycinski, Jakub; Hayes, Peter; Hahn, Bridget; Birungi, Josephine; Tarragona-Fiol, Tony; Wan, Hong; Randles, Mark; Cooper, Andrew Raxworthy; Ssemaganda, Aloysius; Clark, Lorna; Kaleebu, Pontiano; Self, Steven G.; Koup, Richard; Wood, Blake; McElrath, M. Juliana; Cox, Josephine H.; Hural, John; Gilmour, Jill

    2010-01-01

    Background The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates. Methods We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study. Findings Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study. Interpretation The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of

  9. Performance Demonstration Program Plan for Nondestructive Assay of Boxed Wastes for the TRU Waste Characterization Program

    SciTech Connect

    None, None

    2009-10-01

    Each testing and analytical facility performing waste characterization activities for the Waste Isolation Pilot Plant (WIPP) participates in the Performance Demonstration Program (PDP) to comply with the Transuranic Waste Acceptance Criteria for the Waste Isolation Pilot Plant (WAC) (DOE/WIPP-02-3122) and the Quality Assurance Program Document (QAPD) (CBFO-94-1012). The PDP serves as a quality control check for data generated in the characterization of waste destined for WIPP. Single-blind audit samples are prepared and distributed to each of the facilities participating in the PDP. Different PDPs evaluate the analyses of simulated headspace gases (HSGs), constituents of the Resource Conservation and Recovery Act (RCRA), and transuranic (TRU) radionuclides using nondestructive assay (NDA) techniques.

  10. Performance Demonstration Program Plan for Nondestructive Assay of Drummed Wastes for the TRU Waste Characterization Program

    SciTech Connect

    N /A

    2009-04-01

    Each testing and analytical facility performing waste characterization activities for the Waste Isolation Pilot Plant (WIPP) participates in the Performance Demonstration Program (PDP) to comply with the Transuranic Waste Acceptance Criteria for the Waste Isolation Pilot Plant (WAC) (DOE/WIPP-02-3122) and the Quality Assurance Program Document (QAPD) (CBFO-94-1012). The PDP serves as a quality control check for data generated in the characterization of waste destined for WIPP. Single blind audit samples are prepared and distributed to each of the facilities participating in the PDP. The PDP evaluates analyses of simulated headspace gases, constituents of the Resource Conservation and Recovery Act (RCRA), and transuranic (TRU) radionuclides using nondestructive assay (NDA) techniques.

  11. Assay of DAGLα/β Activity.

    PubMed

    Bisogno, Tiziana

    2016-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail. PMID:27245901

  12. A miniaturized fibrinolytic assay for plasminogen activators

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

    1991-01-01

    This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

  13. Use of a Simple, Colorimetric Assay to Demonstrate Conditions for Induction of Nitrate Reductase in Plants.

    ERIC Educational Resources Information Center

    Harley, Suzanne M.

    1993-01-01

    Nitrate assimilation by plants provides an excellent system for demonstrating control of gene expression in a eukaryotic organism. Describes an assay method that allows students to complete experiments designed around the measurement of nitrate reductase within a three-hour laboratory experiment. (PR)

  14. Analyte detection using an active assay

    DOEpatents

    Morozov, Victor; Bailey, Charles L.; Evanskey, Melissa R.

    2010-11-02

    Analytes using an active assay may be detected by introducing an analyte solution containing a plurality of analytes to a lacquered membrane. The lacquered membrane may be a membrane having at least one surface treated with a layer of polymers. The lacquered membrane may be semi-permeable to nonanalytes. The layer of polymers may include cross-linked polymers. A plurality of probe molecules may be arrayed and immobilized on the lacquered membrane. An external force may be applied to the analyte solution to move the analytes towards the lacquered membrane. Movement may cause some or all of the analytes to bind to the lacquered membrane. In cases where probe molecules are presented, some or all of the analytes may bind to probe molecules. The direction of the external force may be reversed to remove unbound or weakly bound analytes. Bound analytes may be detected using known detection types.

  15. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-08-01

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design.

  16. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-09-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. They are using a Schlumberger neutron generator for the direct measurement of the fissile material content in spent fuel, in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics for the detection of very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. They have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The results to data are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference.

  17. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  18. Mining Chemical Activity Status from High-Throughput Screening Assays

    PubMed Central

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare. PMID:26658480

  19. An Overhead Projection Demonstration of Optical Activity

    ERIC Educational Resources Information Center

    Hill, John W.

    1973-01-01

    Describes the use of two polarizing lenses, a yellow filter, an oatmeal bos, a piece of cardboard, a 1,000 ml beaker, and an overhead projector to demonstrate compound optical activity to large classes. Indicates the presence of an accuracy within 1-2 degrees of usually acceptable data. (CC)

  20. Quantitative comparisons of in vitro assays for estrogenic activities.

    PubMed Central

    Fang, H; Tong, W; Perkins, R; Soto, A M; Prechtl, N V; Sheehan, D M

    2000-01-01

    Substances that may act as estrogens show a broad chemical structural diversity. To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes. We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens. We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities. In this examination, we combined individual data sets in a specific, quantitative data mining exercise. Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots. The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded (r(2) is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays). Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays. Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays. Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques. Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined. The results should provide guidance for expanded data mining examinations and the selection of appropriate

  1. Performance Demonstration Program Plan for Nondestructive Assay of Drummed Wastes for the TRU Waste Characterization Program

    SciTech Connect

    Carlsbad Field Office

    2005-08-03

    The Performance Demonstration Program (PDP) for Nondestructive Assay (NDA) is a test program designed to yield data on measurement system capability to characterize drummed transuranic (TRU) waste generated throughout the Department of Energy (DOE) complex. The tests are conducted periodically and provide a mechanism for the independent and objective assessment of NDA system performance and capability relative to the radiological characterization objectives and criteria of the Office of Characterization and Transportation (OCT). The primary documents requiring an NDA PDP are the Waste Acceptance Criteria for the Waste Isolation Pilot Plant (WAC), which requires annual characterization facility participation in the PDP, and the Quality Assurance Program Document (QAPD). This NDA PDP implements the general requirements of the QAPD and applicable requirements of the WAC. Measurement facilities must demonstrate acceptable radiological characterization performance through measurement of test samples comprised of pre-specified PDP matrix drum/radioactive source configurations. Measurement facilities are required to analyze the NDA PDP drum samples using the same procedures approved and implemented for routine operational waste characterization activities. The test samples provide an independent means to assess NDA measurement system performance and compliance per criteria delineated in the NDA PDP Plan. General inter-comparison of NDA measurement system performance among DOE measurement facilities and commercial NDA services can also be evaluated using measurement results on similar NDA PDP test samples. A PDP test sample consists of a 55-gallon matrix drum containing a waste matrix type representative of a particular category of the DOE waste inventory and nuclear material standards of known radionuclide and isotopic composition typical of DOE radioactive material. The PDP sample components are made available to participating measurement facilities as designated by the

  2. Guidance manual for conducting technology demonstration activities

    SciTech Connect

    Jolley, Robert L.; Morris, Michael I.; Singh, Suman P.N.

    1991-12-01

    This demonstration guidance manual has been prepared to assist Martin Marietta Energy Systems, Inc. (Energy Systems), staff in conducting demonstrations. It is prepared in checklist style to facilitate its use and assumes that Energy Systems personnel have project management responsibility. In addition to a detailed step-by-step listing of procedural considerations, a general checklist, logic flow diagram, and several examples of necessary plans are included to assist the user in developing an understanding of the many complex activities required to manage technology demonstrations. Demonstrations are pilot-scale applications of often innovative technologies to determine the commercial viability of the technologies to perform their designed function. Demonstrations are generally conducted on well-defined problems for which existing technologies or processes are less than satisfactory in terms of effectiveness, cost, and/or regulatory compliance. Critically important issues in demonstration management include, but are not limited to, such factors as communications with line and matrix management and with the US Department of Energy (DOE) and Energy Systems staff responsible for management oversight, budgetary and schedule requirements, regulatory compliance, and safety.

  3. Active and passive computed tomography for nondestructive assay

    SciTech Connect

    Bernardi, R T; Camp, D E; Clard, D; Jackson, J A; Martz, H E, Decman, D J; Roberson, G P

    1998-10-28

    Traditional gamma-ray methods used to characterize nuclear waste introduce errors that are related to non-uniform measurement responses associated with unknown radioactive source and matrix material distributions. These errors can be reduced by applying an active and passive tomographic technique (A&PCT) developed at the Lawrence Livermore National Laboratory (LLNL). The technique uses an external radioactive source and active tomography to map the attenuation within a waste barrel as a function of mono-energetic gamma-ray energy. Passive tomography is used to localize and identify specific radioactive waste within the same container. Reconstruction of the passive data using the attenuation maps at specific energies allows internal waste radioactivity to be corrected for any overlying heterogeneous materials, thus yielding an absolute assay of the waste activity. LLNL and Bio-Imaging Research, Inc. have collaborated in a technology transfer effort to integrate an A&PCT assay system into a mobile waste characterization trailer. This mobile system has participated in and passed several formal DOE-sponsored performance demonstrations, tests and evaluations. The system is currently being upgraded with multiple detectors to improve throughput, automated gamma-ray analysis code to simplify the assay, and a new emission reconstruction code to improve accuracy

  4. Biochemical assays on plasminogen activators and hormones from kidney sources

    NASA Technical Reports Server (NTRS)

    Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

    1988-01-01

    Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

  5. Experimental Demonstration of Active Electromagnetic Cloaking

    NASA Astrophysics Data System (ADS)

    Selvanayagam, Michael; Eleftheriades, George V.

    2013-10-01

    Active electromagnetic cloaking uses an array of elementary sources to cancel the scattered fields created by an object. An active interior cloak does this by placing the sources along the boundary of the object. This process can be thought of as introducing a discontinuity in the field to cancel out the scattered field by the object. Here, an experimental version of a thin active cloak at microwave frequencies is demonstrated for an aluminum cylinder with a radius of 0.56λ. The cloak consists of a 12-element magnetic-dipole array. By controlling the weights of the current on each element of the array, the scattering off of the cylinder is reduced in the backward and forward directions. The ability to disguise the aluminum cylinder as another object by varying the weights of the dipole array is also demonstrated. Finally, potential ways of overcoming the constraint of requiring a priori knowledge of the incident field leading to camouflaging-type behavior are discussed.

  6. How-to-do-it: Immunological Assays for the Classroom 1. Enzyme Linked Immunosorbent Assay (ELISA): A Laboratory Tool for Demonstration of Antibody-Antigen Interaction.

    ERIC Educational Resources Information Center

    Russo, A. J.; And Others

    1984-01-01

    Background information, list of required materials, and procedures are provided for an immunological assay which has been modified for use as a classroom/laboratory demonstration of antigen-antibody reaction. The assay is designed for a two and one-half hour laboratory period but may be modified for one hour laboratories. (JN)

  7. Recent Applications for in Vitro Antioxidant Activity Assay.

    PubMed

    Bunaciu, Andrei A; Danet, Andrei Florin; Fleschin, Şerban; Aboul-Enein, Hassan Y

    2016-09-01

    This review presents some of the most recent aspects related to antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Taking into account the reactions involved, in the antioxidant activity determinations, the assays can be classified into two main types: hydrogen atom transfer reactions and electron transfer. This review focuses on analytical methods used for antioxidant activity assay, published in the period 2009-2014. PMID:26575594

  8. A Rapid and Quantitative Recombinase Activity Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  9. Novel assay for direct fluorescent imaging of sialidase activity

    NASA Astrophysics Data System (ADS)

    Tomin, A.; Shkandina, T.; Bilyy, R.

    2011-07-01

    Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 3.2.1.18, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.

  10. Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays

    PubMed Central

    Spence, Janice M.; Rothberg, Paul G.; Wang, Nancy; Burack, W. Richard

    2011-01-01

    We demonstrate an approach that allowed rapid development of a robust assay for the detection of chromosomal translocations. The method includes highly multiplexed PCR with analysis of the PCR products performed by array detection. As proof of principle, we applied this approach to the detection of IGH@-BCL2 translocations in DNA prepared from FFPE specimens. This translocation and specimen type were chosen because of the known difficulties associated with PCR-based detection of this lesion and the additional loss of sensitivity associated with FFPE samples. The multiplex PCR with array detection method detected the IGH@-BCL2 translocation in 26 of 36 FFPE follicular lymphoma specimens, whereas the BIOMED-2 assay detected 13 of 36 specimens. This increased sensitivity was the result of both the increased density of BCL2 primers and identification of PCR products by low-density array. The method was specific and allowed mapping of the BCL2 break point in all cases. The method detected the IGH@-BCL2 lesion when the tumor DNA was diluted more than 1:20 in normal DNA but not when it was diluted more than 1:100. This sensitivity allows detection of diagnostically relevant levels of IGH@-BCL2 but will not detect the rare cells with IGH@-BCL2 translocations in healthy individuals. PMID:21497287

  11. A DIRECT METHOD TO ASSAY NEUROTOXIC ESTERASE ACTIVITY

    EPA Science Inventory

    A direct photometric method for assaying neurotoxic esterase (NTE) activity of chicken brain microsomal preparation has been developed using 4-nitrophenyl esters as substrates. Paired samples of the microsomal preparation were preincubated for 20 min. with paraoxon plus either (a...

  12. Demonstration of Zoospore Activities by Fungi.

    ERIC Educational Resources Information Center

    McCarter, States M.

    1978-01-01

    The demonstrations and experiments described in this article are appropriate for junior high school and older students, including beginning students in college biology or botany. Included are culture and observation of zoospores, zoospore attraction to plant roots, and other topics. (BB)

  13. Simple Activity Demonstrates Wind Energy Principles

    ERIC Educational Resources Information Center

    Roman, Harry T.

    2012-01-01

    Wind energy is an exciting and clean energy option often described as the fastest-growing energy system on the planet. With some simple materials, teachers can easily demonstrate its key principles in their classroom. (Contains 1 figure and 2 tables.)

  14. Nondestructive assay using active and passive computed tomography

    SciTech Connect

    Roberson, G. P. ,LLNL

    1998-07-01

    The United States Department of Energy (DOE) has over 600,000 transuranic (TRU) waste drums temporarily stored at nearly 40 sites within the United States. Contents of these drums must be characterized before they are transported for permanent disposal. Traditional gamma-ray methods used to characterize nuclear waste introduce errors that are related to nonuniform measurement responses associated with unknown radioactive source and matrix material distributions. These errors can be reduced by application of tomographic techniques, that measure these distributions. The Lawrence Livermore National Laboratory (LLNL) has developed two tomographic-based waste assay systems. They use external radioactive sources and tomography-protocol to map the attenuation within a waste drum as a function of mono-energetic gamma-ray energy in waste containers. Passive tomography is used to localize and identify specific radioactive waste contents within the same waste containers. Reconstruction of the passive data via the active images allows internal waste radioactivities in a drum to be corrected for any overlying heterogeneous materials, thus yielding an absolute assay of the waste radioactivities. Calibration of both systems requires only point source measurements and are independent of matrix materials. The first system is housed at LLNL and was developed to study and validate research concepts. The second system is being developed with Bioimaging Research, Inc. (BIR) and is housed within a mobile waste characterization trailer. This system has traveled to three DOE facilities to demonstrate the active and passive computed tomography capability. Both systems have participated in and successfully passed the requirements of formal DOE-sponsored intercomparison studies. The systems have measured approximately 1 to 100 grains of plutonium within a variety of waste matrix materials. Laboratory and field results from these two systems over the past several years show that both systems

  15. An in vivo assay for chemoattractant activity.

    PubMed

    Zetter, B R; Rasmussen, N; Brown, L

    1985-09-01

    We have devised an implantable device for the study of leukocyte chemoattraction. The device consists of a 0.25-mm thick patch of Dacron fabric coupled to a disc of ethylene vinyl acetate copolymer. Such polymers can release biologically active molecules at a constant rate for at least 18 days. Attracted cells invade and are trapped within the Dacron fabric. Upon removal from the host, the fabric patches are sectioned and stained to reveal the distribution of attracted cells. Distinct patterns of cellular accumulation can be seen for different chemoattractant molecules. These include the attraction of eosinophils by histamine, monocytes by tuftsin, and mast cells by glycyl-histidyl-lysine. Maximal accumulation of specific cell types occurs at postimplantation days 1 to 2 for neutrophils, days 3 to 5 for monocytes, and days 5 to 6 for macrophages and eosinophils. Control polymers fail to cause significant leukocyte accumulation, indicating that neither the polymer nor the Dacron fabric provokes an inflammatory response. PMID:3162062

  16. Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

    PubMed Central

    Schwartz, David Eric; Gong, Ping; Shepard, Kenneth L.

    2008-01-01

    We present an active oligonucleotide microarray platform for time-resolved Förster resonance energy transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors. PMID:18515059

  17. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Veyo, S.E.

    1995-08-01

    The development of a viable fuel cell driven electrical power generation system involves not only the development of cell and stack technology, but also the development of the overall system concept, the strategy for control, and the ancillary subsystems. The design requirements used to guide system development must reflect a customer focus in order to evolve a commercial product. In order to obtain useful customer feedback, Westinghouse has practiced the deployment with customers of fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units have served to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  18. Selected demonstration and educational products/activities

    SciTech Connect

    Williams, R.J.; Mann, H.C.

    1992-07-01

    The information in this paper was assembled for several informal presentations to a variety of visitor groups during the summer of 1992. A number of staff members at TVA`s National Fertilizer and Environmental Research Center (NFERC) found it useful as a quick overview for their use and for their sharing with external colleagues and customers. The paper is not meant to be an exhaustive list or explanation of all products and services available from NFERC. However, the authors believe it will give a flavor and tenor of some of the ongoing activities of the Center, especially those activities relating to the retail fertilizer dealer. Programs over the years have focused on key aspects of nutrient efficiency and management. TVA is uniquely positioned to assist the fertilizer industry and US agriculture in protecting the environment from potential adverse environmental impacts of agriculture, especially for fertilizer and the attendant agrichemicals. TVA has the technical base and an ongoing working relationship with the fertilizer industry in technology development and introduction. Dealer education is very important in TVA programs in two aspects: (1) education for the dealer in meeting new environmental stewardship challenges from an operational perspective; and (2) education for the dealer in meeting the site-specific information needs of the farmer.

  19. Improved assays for xenosensor activation based on reverse transfection.

    PubMed

    Küblbeck, Jenni; Anttila, Teemu; Pulkkinen, Juha T; Honkakoski, Paavo

    2015-10-01

    Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies. PMID:26187274

  20. A supplemented soft agar chemotaxis assay demonstrates the Helicobacter pylori chemotactic response to zinc and nickel

    PubMed Central

    Sanders, Lisa; Andermann, Tessa M.

    2013-01-01

    Directed motility, or chemotaxis, is required for Helicobacter pylori to establish infection in the stomach, although the full repertoire of this bacterium’s chemotactic responses is not yet known. Here we report that H. pylori responds to zinc as an attractant and nickel as a repellent. To reach this conclusion, we employed both a temporal chemotaxis assay based on bacterial reversals and a supplemented soft agar spatial assay. We refined the temporal assay using a previously described chemorepellent, acid, and found that H. pylori requires rich media with serum to maintain optimal swimming motility. Surprisingly, we found that some strains respond to acid as an attractant, and that the TlpC chemoreceptor correlated with whether acid was sensed as an attractant or repellent. Using this same assay, we detected weak repellent responses to nickel and copper, and a varied response to zinc. We thus developed an alternative spatial chemotactic assay called the supplemented soft agar assay, which utilizes soft agar medium supplemented with the test compound. With Escherichia coli, the attractant serine slowed overall bacterial migration, while the repellent nickel increased the speed of overall migration. In H. pylori we detected slowed migration with doubled tryptone media, as well as zinc, consistent with an attractant response. In contrast, nickel increased migration, consistent with repulsion. PMID:23139399

  1. Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

    PubMed Central

    Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

    2014-01-01

    The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

  2. Polarographic assay based on hydrogen peroxide scavenging in determination of antioxidant activity of strong alcohol beverages.

    PubMed

    Gorjanović, Stanislava Z; Novaković, Miroslav M; Vukosavljević, Predrag V; Pastor, Ferenc T; Tesević, Vele V; Suznjević, Desanka Z

    2010-07-28

    Total antioxidant (AO) activity of strong alcohol beverages such as wine and plum brandies, whiskeys, herbal and sweet fruit liqueurs have been assessed using a polarographic assay based on hydrogen peroxide scavenging (HPS). Rank of order of total AO activity, expressed as percentage of decrease of anodic oxidation current of hydrogen peroxide, was found analogous with total phenolic content estimated by Folin-Ciocalteau (FC) assay and radical scavenging capacity against the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). Application of the assay for surveying of a quarter century long maturation of plum brandy in oak barrel was demonstrated. In addition, influence of different storage conditions on preservation of AO activity of some herbal liqueurs was surveyed. Wide area of application of this simple, fast, low cost and reliable assay in analysis and quality monitoring of various strong alcohol beverages was confirmed. PMID:20604507

  3. Development of a new colorimetric assay for lipoxygenase activity.

    PubMed

    Lu, Weiqiang; Zhao, Xue; Xu, Zhongyu; Dong, Ningning; Zou, Shien; Shen, Xu; Huang, Jin

    2013-10-15

    Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases that catalyze the hydroperoxidation of lipids, containing a cis,cis-1,4-pentadiene structure. A rapid and reliable colorimetric assay for determination of the activity of three human functional lipoxygenase isoforms (5-lipoxygenase, platelet 12-lipoxygenase, and 15-lipoxygenase-1) is developed in this article. In the new assay, LOX-derived lipid hydroperoxides oxidize the ferrous ion (Fe²⁺) to the ferric ion (Fe³⁺), the latter of which binds with thiocyanate (SCN⁻) to generate a red ferrithiocyanate (FTC) complex. The absorbance of the FTC complex can be easily measured at 480 nm. Because 5-LOX can be stimulated by many cofactors, the effects of its cofactors (Ca²⁺, ATP, dithiothreitol, glutathione, L-α-phosphatidylcholine, and ethylenediaminetetraacetic acid) on the color development of the FTC complex are also determined. The assay is adaptive for purified LOXs and cell lysates containing active LOXs. We use the new colorimetric assay in a 96-well format to evaluate several well-known LOX inhibitors, the IC₅₀ values of which are in good agreement with previously reported data. The reliability and reproducibility of the assay make it useful for in vitro screening for inhibitors of LOXs and, therefore, should accelerate drug discovery for clinical application. PMID:23811155

  4. Toxin activity assays, devices, methods and systems therefor

    DOEpatents

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2016-04-05

    Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  5. A new robust kinetic assay for DAP epimerase activity.

    PubMed

    Hor, Lilian; Peverelli, Martin G; Perugini, Matthew A; Hutton, Craig A

    2013-10-01

    DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics. PMID:23838343

  6. A chromogenic assay for limit dextrinase and pullulanase activity.

    PubMed

    Bøjstrup, Marie; Christensen, Caspar Elo; Windahl, Michael Skovbo; Henriksen, Anette; Hindsgaul, Ole

    2014-03-15

    A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide (Glc-maltotriosyl-maltotriose) was found to be a suitable specific substrate for starch debranching enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined, and the selectivity of the substrate in a complex cereal-based extract is established. PMID:24333247

  7. Assay for Lipolytic and Proteolytic Activity Using Marine Substrates

    PubMed Central

    Tom, Raymond A.; Crisan, Eli V.

    1975-01-01

    Nondestructive assay procedures for determining microbial lipolytic and proteolytic activity on marine substrates were developed and tested with 287 isolates of bacteria, filamentous fungi, and yeasts. A definite substrate specificity was noted when the enzymatic activities on marine and nonmarine substrates was compared. Of 170 lipolytic isolates, 14 were only active on menhaden oil, 11 could hydrolyze menhaden oil and Tween 80 and/or tributyrin, and 145 isolates could only hydrolyze one or both of the nonmarine lipids. Of the 198 proteolytic isolates, 10 were specific for codfish extract, 152 were active against the marine substrate plus casein and/or gelatin, and 36 were specific for nonmarine substrates. PMID:1167775

  8. More insights into a human adipose tissue GPAT activity assay

    PubMed Central

    Morgan-Bathke, Maria; Chen, Liang; Oberschneider, Elisabeth; Harteneck, Debra; Jensen, Michael D

    2016-01-01

    ABSTRACT Adipose tissue fatty acid storage varies according to sex, adipose tissue depot and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We recently published findings based on the glycerol 3-phosphate acyltransferase (GPAT) enzyme activity assay we optimized for use with human adipose tissue. These findings include a decrease in total GPAT and GPAT1 as a function of adipocyte size in both omental and subcutaneous adipose tissue and a strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots and between these storage factors and palmitate storage rates into TAG. The aim of this commentary is to expand upon the data from our recent publication. We describe here additional details on the optimization of the GPAT enzyme activity assay, a correlation between DGAT and percentage palmitate in the diacylglycerol fraction, and sex differences in fatty acid storage factors and storage rates into TAG at high palmitate concentrations. PMID:27144101

  9. Novel yeast cell dehydrogenase activity assay in situ.

    PubMed

    Berłowska, Joanna; Kregiel, Dorota; Klimek, Leszek; Orzeszyna, Bartosz; Ambroziak, Wojciech

    2006-01-01

    The aim of this research was to develop a suitable method of succinate dehydrogenase activity assay in situ for different industrial yeast strains. For this purpose different compounds: EDTA, Triton X-100, sodium deoxycholate, digitonin, nystatin and beta-mercaptoethanol were used. The permeabilization process was controlled microscopically by primuline staining. Enzyme assay was conducted in whole yeast cells with Na-succinate as substrate, phenazine methosulfate (PMS) as electron carrier and in the presence one of two different tetrazolium salts: tetrazolium blue chloride (BT) or cyanoditolyl tetrazolium chloride (CTC) reduced during the assay. In comparabile studies of yeast vitality the amount of intracellular ATP was determined according to luciferin/luciferase method. During the succinate dehydrogenase assay in intact yeast cells without permeabilization, BT formazans were partially visualized in the cells, but CTC formazans appeared to be totally extracellular or associated with the plasma membrane. Under these conditions there was no linear relationship between formazan color intensity signal and yeast cell density. From all chemical compounds tested, only digitonin was effective in membrane permeabilization without negative influence on cell morphology. Furthermore, with digitonin-treated cells a linear relationship between formazan color intensity signal and yeast cell number was noticed. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of the tested yeast strains. PMID:17419290

  10. An Anesthetic Drug Demonstration and an Introductory Antioxidant Activity Experiment with "Eugene, the Sleepy Fish"

    ERIC Educational Resources Information Center

    Barcena, Homar; Chen, Peishan

    2016-01-01

    Students are introduced to spectrophotometry in comparing the antioxidant activity of pure eugenol and oil of cloves from a commercial source using a modified ferric reducing antioxidant power (FRAP) assay. The extraction of the essential oil from dried cloves is demonstrated to facilitate discussions on green chemistry. The anesthetic properties…

  11. The uronic acids assay: a method for the determination of chemical activity on biofilm EPS.

    PubMed

    Mojica, Kristina D A; Cooney, Michael J

    2010-01-01

    In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS. PMID:20087802

  12. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  13. Demonstrating Enzyme Activation: Calcium/Calmodulin Activation of Phosphodiesterase

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2004-01-01

    Demonstrating the steps of a signal transduction cascade usually involves radioactive materials and thus precludes its use in undergraduate teaching labs. Developing labs that allow the visual demonstration of these steps without the use of radioactivity is important for allowing students hands-on methods of illustrating each step of a signal…

  14. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  15. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  16. Lack of activity of cadmium in in vitro estrogenicity assays

    SciTech Connect

    Silva, Elisabete . E-mail: elisabete.silva@pharmacy.ac.uk; Lopez-Espinosa, Maria Jose; Molina-Molina, Jose-Manuel; Fernandez, Marieta; Olea, Nicolas; Kortenkamp, Andreas

    2006-10-01

    Prompted by reports about strong estrogenic effects of cadmium, attempts were made to reproduce these observations using the yeast estrogen screen (YES) and the E-Screen assays. For the first time, possible activation of the Src/MAPK pathway was also investigated. In the YES, only a slight activation (10% of a maximal effect) of the estrogen receptor alpha (ER{alpha}) was observed at cadmium concentrations between 5 x 10{sup -7} M and 5 x 10{sup -6} M. In the E-Screen assay, carried out by two laboratories, the heavy metal was without observable cell proliferative effects when tested in the range between 6 x 10{sup -11} M and 1 x 10{sup -5} M. However, in both assays, cadmium led to a reduction of the effects of 17{beta}-estradiol (E2). Treatment of MCF-7 human breast cancer cells with 1 x 10{sup -7} M cadmium failed to induce phosphorylation of Src and the MAP kinases Erk1 and Erk2-effects shown to occur with E2 and epidermal growth factor (EGF). In summary, we were unable to confirm the strong estrogenicity of cadmium reported recently by a number of laboratories. This apparent absence of effects in our hands is not due to a lack of uptake of the metal or to effective protection against cadmium by high levels of glutathione or metallothionein, since toxicity and an antagonism of E2 responses were observed both in the YES and the E-Screen.

  17. Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay.

    PubMed

    Yavo, B; Brunetti, I L; da Fonseca, L M; Catalani, L H; Campa, A

    2001-01-01

    In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme. PMID:11590700

  18. A Rapid Method for Assaying Thiaminase I Activity in Diverse Biological Samples

    PubMed Central

    Kraft, Clifford E.; Gordon, Eric R. L.; Angert, Esther R.

    2014-01-01

    Vitamin B1 (thiamine) deficiencies can lead to neurological disorders, reproductive failure and death in wild and domestic animal populations. In some cases, disease is brought about by the consumption of foods high in thiaminase I activity. Levels of thiaminase activity in these foods are highly variable and the factors leading to production of this enzyme are poorly understood. Here we describe improvements in a spectrophotometric thiaminase I activity assay that measures the disappearance of 4-nitrothiophenol, a favored nucleophile co-substrate that replaces the thiazole portion of thiamine during the inactivation of thiamine by the enzyme. Scalable sample processing protocols and a 96-well microtiter plate format are presented that allow the rapid evaluation of multiple, replicated samples in the course of only a few hours. Observed levels of activity in bacterial culture supernatant, fish, ferns and molluscs using this colorimetric assay were similar to previously published reports that employed a radiometric method. Organisms devoid of thiaminase I, based upon previous work, showed no activity with this assay. In addition, activity was found in a variety of fishes and one fern species from which this enzyme had not previously been reported. Overall, we demonstrate the suitability of this technique for measuring thiaminase I activity within small amounts of tissue and environmental samples with replication levels that were heretofore prohibitive. The assay provides a considerable improvement in the ability to examine and understand the properties of an enzyme that has a substantial influence on organism and ecosystem health. PMID:24675843

  19. Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

    PubMed Central

    Lopez-Gigosos, Rosa M.; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin

    2015-01-01

    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R2 = 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques. PMID:26276114

  20. Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays.

    PubMed

    Newberry, Kim; Wang, Shuya; Hoque, Nina; Kiss, Laszlo; Ahlijanian, Michael K; Herrington, James; Graef, John D

    2016-06-01

    In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na(+) and Ca(2+) channel blockers, as well as enhanced with K(+) channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain. PMID:27052585

  1. Classroom Activities and Demonstrations for Use in Behavioral Science Courses.

    ERIC Educational Resources Information Center

    Cology, Lorry J.

    This compilation provides descriptions of and resource materials for 25 classroom activities or demonstrations for behavioral science courses. For each activity, the following information is provided: subject area, source, time required and materials needed. In addition, discussion questions and comments on the value and use of the activities are…

  2. 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. PMID:27245904

  3. Application of the E-screen assay to test for oestrogenically active substances in swine feed.

    PubMed

    Bitsch, N; Körner, W; Postupka, S; Brunn, H

    2001-12-01

    A pig breeder in central Hesse (Germany) noticed the occurrence of enlarged vulvae in female piglets. Intoxication with oestrogenically active substances by contamination of two feed mixes ingested by the mother sows appeared to be a possible cause. Using a combined technique of the DFG analytical method S19 and the E-screen assay, two feed samples were found to contain powerful oestrogenically active compounds. By co-incubation with the anti-oestrogen tamoxifen it could be clearly demonstrated that the oestrogenic activity was mediated by the oestrogen receptor. These results demonstrate that use of the E-screen assay in combination with the DFG analytical method S19 provides a simple and readily usable prescreening method for the routine detection of oestrogenically active compounds in animal feed. The results from the E-screen assay show that the sows ingested 10-80 microg oestradiol equivalents per day in their feed. Because of the bioavailability of these substances, the oestrogenic active compounds seem to be transferred into the milk and passed to the piglets via suckling. The milk of the dam appears to contain this substance in biologically active form and at such high concentrations that the female piglets had enlarged vulvae. PMID:11906561

  4. The Peptide Microarray-Based Resonance Light Scattering Assay for Sensitively Detecting Intracellular Kinase Activity.

    PubMed

    Li, Tao; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-01-01

    The peptide microarray technology is a robust, reliable, and efficient technique for large-scale determination of enzyme activities, and high-throughput profiling of substrate/inhibitor specificities of enzymes. Here, the activities of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) in different cell lysates have been detected by a peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle (GNP) probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 (human glioma cell) cell lysate (corresponding to 200 cells) is achieved by a selected peptide substrate. The experimental results also demonstrate that the RLS assay can be employed to evaluate the chemical regulation of intracellular kinase activity. PMID:26490469

  5. Demonstrating Electrical Activity in Nerve and Muscle. Part II

    ERIC Educational Resources Information Center

    Robinson, D. J.

    1976-01-01

    Describes the construction of an amplifier and force transducer that can be used to demonstrate electrical activity in nerve and muscle using the gastrocnemius muscle and sciatic nerve of the frog. (MLH)

  6. Demonstrating Electrical Activity in Nerve and Muscle. Part I

    ERIC Educational Resources Information Center

    Robinson, D. J.

    1975-01-01

    Describes a demonstration for showing the electrical activity in nerve and muscle including action potentials, refractory period of a nerve, and fatigue. Presents instructions for constructing an amplifier, electronic stimulator, and force transducer. (GS)

  7. Neutron coincidence imaging for active and passive neutron assays

    SciTech Connect

    Estep, R. J.; Brunson, G. S.; Melton, S. G.

    2001-01-01

    Neutron multiplicity assay algorithms for {sup 240}Pu assume a point source of fission neutrons that are detected in a single detector channel. The {sup 240}Pu in real waste, however, is more likely to be distributed throughout the container in some random way. For different reasons, this leads to significant errors when using either multiplicity or simpler coincidence analyses. Reduction of these errors can be achieved using tomographic imaging. In this talk we report on our results from using neutron singles and coincidence data between tagged detector pairs to provide enhanced tomographic imaging capabilities to a crate nondestructive assay system. Only simulated passive coincidence data is examined here, although the higher signal rates from active coincidence counting hold more promise for waste management. The active coincidence approach has significantly better sensitivity than the passive and is not significantly perturbed by (alpha,n) contributions. Our study was based primarily on simulated neutron pulse trains derived from the Los Alamos SIM3D software, which were subjected to analysis using the Los Alamos CTEN-FIT and TGS-FIT software. We found significantly improved imaging capability using the coincidence and singles rate data than could be obtained using the singles rate alone.

  8. The Importance of Engaging Pupils Actively in Demonstrations

    ERIC Educational Resources Information Center

    Suomela, Liisa; Juuti, Kalle; Ahtee, Maija

    2013-01-01

    Demonstrating is a traditional method in teaching science that can raise interest and encourage pupils to think about a topic. While demonstrating, the teacher can focus the pupils' attention on the relevant facts and introduce scientific principles and concepts. Through discussion and actively making observations and inferences, rather than…

  9. Demonstration of nitric oxide on asbestos and silicon carbide fibers with a new ultraviolet spectrophotometric assay.

    PubMed Central

    Leanderson, P; Lagesson, V; Tagesson, C

    1997-01-01

    Nitric oxide (NO) has a number of important functions in biological systems and may play a role in the toxicity of mineral fibers. We investigated whether NO might be present on the surface of mineral fibers and if crocidolite could adsorb NO from NO gas or cigarette smoke. NO was determined with a new gas chromatography-ultraviolet spectrophotometric technique after thermal desorption from the fiber surface and injection in a gas flow cell. NO was found in different amounts on chrysotile B, crocidolite, amosite, and silicon carbide whiskers. There was a strong correlation between the amount of NO and the specific surface area of these fibers (r = 0.98). NO could not be demonstrated on rockwool fibers [man-made vitreous fiber(s) (MMVF)21 and MMVF22] or silicon nitride whiskers. NO on crocidolite, amosite, and silicon carbide whiskers was readily desorbed from the fibers at increased temperature, while NO on chrysotile B seemed to be more firmly adsorbed to the fiber and required a longer period of time to be desorbed. The amount of NO bound to crocidolite increased from 34 micrograms/g fiber to 85 and 474 micrograms/g after exposing the fibers to cigarette smoke and NO gas, respectively. These findings indicate that a) NO adsorbs to fiber surfaces, b) some fibers adsorb more NO than others, c) some fibers adsorb NO more strongly than others, and d) the amounts of NO on fibers may be increased after exposure of the fiber to cigarette smoke or other sources of NO. The biological significance of NO on mineral fibers remains to be investigated. Images Figure 3. PMID:9400696

  10. Activity-based assay for ricin-like toxins

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2007-02-06

    A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

  11. Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion.

    PubMed

    Vuojola, Johanna; Riuttamäki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero

    2012-05-01

    Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC(50) values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an internally quenched substrate molecule diminishes the background resulting from radiatively excited acceptor molecules. The reduction of autofluorescence and radiative background result in high signal-to-background ratios (ratios of approximately 100 were obtained). By further utilizing assay miniaturization and signal enhancement in a white microtitration plate, a significant reduction in the reagent consumption can be achieved rendering the assay applicable for high-throughput screening. PMID:22502613

  12. An Activity for Demonstrating the Concept of a Neural Circuit

    ERIC Educational Resources Information Center

    Kreiner, David S.

    2012-01-01

    College students in two sections of a general psychology course participated in a demonstration of a simple neural circuit. The activity was based on a neural circuit that Jeffress proposed for localizing sounds. Students in one section responded to a questionnaire prior to participating in the activity, while students in the other section…

  13. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  14. Development and utilization of activated STAT3 detection assays for screening a library of secreted proteins.

    PubMed

    Fursov, Natalie; Gates, Irina V; Panavas, Tadas; Giles-Komar, Jill; Powers, Gordon

    2011-08-01

    Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications. PMID:21294636

  15. A simple liposome assay for the screening of zinc ionophore activity of polyphenols.

    PubMed

    Clergeaud, Gael; Dabbagh-Bazarbachi, Husam; Ortiz, Mayreli; Fernández-Larrea, Juan B; O'Sullivan, Ciara K

    2016-04-15

    An efficient liposomal system for screening the zinc ionophore activity of a selected library consisting of the most relevant dietary polyphenols is presented. The zinc ionophore activity was demonstrated by exploring the use of zinc-specific fluorophore FluoZin-3 loaded liposomes as simple membrane tools that mimic the cell membrane. The zinc ionophore activity was demonstrated as the capacity of polyphenols to transport zinc cations across the liposome membrane and increase the zinc-specific fluorescence of the encapsulated fluorophore FluoZin-3. In addition, the zinc chelation strength of the polyphenols was also tested in a competition assay based on the fluorescence quenching of zinc-dependent fluorescence emitted by zinc-FluoZin-3 complex. Finally, the correlation between the chelation capacity and ionophore activity is demonstrated, thus underlining the sequestering or ionophoric activity that the phenolic compounds can display, thus, providing better knowledge of the importance of the structural conformation versus their biological activity. Furthermore, the assays developed can be used as tools for rapid, high-throughput screening of families of polyphenols towards different biometals. PMID:26617034

  16. A Transgenic Mouse Assay for Agouti Protein Activity

    PubMed Central

    Perry, W. L.; Hustad, C. M.; Swing, D. A.; Jenkins, N. A.; Copeland, N. G.

    1995-01-01

    The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein. PMID:7635291

  17. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  18. Integrated gasification combined-cycle research development and demonstration activities

    SciTech Connect

    Ness, H.M.; Reuther, R.B.

    1995-12-01

    The United States Department of Energy (DOE) has selected six integrated gasification combined-cycle (IGCC) advanced power systems for demonstration in the Clean Coal Technology (CCT) Program. DOE`s Office of Fossil Energy, Morgantown Energy Technology Center, is managing a research development and demonstration (RD&D) program that supports the CCT program, and addresses long-term improvements in support of IGCC technology. This overview briefly describes the CCT projects and the supporting RD&D activities.

  19. The synchronous active neutron detection system for spent fuel assay

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-10-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit the unique operating features of a 14-MeV neutron generator developed by Schlumberger. This generator and a novel detection system will be applied to the direct measurement of the fissile material content in spent fuel in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics to detect very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed {open_quotes}lock-in{close_quotes} amplifiers. The authors have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. This approach is possible because the Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. The results to date are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference. The interrogating neutrons appear to be nonthermal and penetrating. Although a significant amount of work remains to fully explore the relevant physics and optimize the instrument design, the underlying concept appears sound.

  20. EVALUATION AND DEMONSTRATION OF THE CHEMICALLY ACTIVE FLUID BED

    EPA Science Inventory

    The report gives results of the operation of a 17-MW Chemically Active Fluid Bed (CAFB) demonstration unit, retrofitted to a natural gas boiler. The CAFB process gasifies high-sulfur, high-metals-content liquid and solid fuels. Residual oil, lignite, and bituminous coal were gasi...

  1. Chemistry: Experiments, Demonstrations and Other Activities Suggested for Chemistry.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Bureau of Secondary Curriculum Development.

    This publication is a handbook used in conjunction with the course of study in chemistry developed through the New York State Education Department and The University of the State of New York. It contains experiments, demonstrations, and other activities for a chemistry course. Areas covered include the science of chemistry, the atomic structure of…

  2. A high-content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling.

    PubMed

    Saydmohammed, Manush; Vollmer, Laura L; Onuoha, Ezenwa Obi; Vogt, Andreas; Tsang, Michael

    2011-09-01

    Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF. PMID:21932436

  3. Demonstration of immunoglobulin M class antibodies to Toxoplasma gondii antigenic component p35000 by enzyme-linked antigen immunosorbent assay.

    PubMed Central

    Lindenschmidt, E G

    1986-01-01

    On the basis that 89% of 48 acute-phase toxoplasmosis patients showed immunoglobulin M (IgM) class antibodies to the 35,000-molecular-weight antigenic component (p35000) of Toxoplasma gondii, as demonstrated by IgM immunoblotting, the antigen was purified by sucrose gradient centrifugation and enzyme labeled for use in an enzyme-linked antigen immunosorbent assay (ELA) for the demonstration of IgM class antibodies to the p35000 component. The ELA showed a specificity of 96% with 139 serum specimens at a serum dilution of only 1:5. The test serologically detected 73 symptomatic acute-phase toxoplasmosis patients; 64 were positive in the 19S IgM indirect immunofluorescent-antibody test, and 9 were negative, although they showed IgM antibodies to p35000, as demonstrated by IgM immunoblotting. Also, the ELA turned out to be independent of IgM rheumatoid factors in six acute-phase toxoplasmosis serum specimens. PMID:3536996

  4. Central Limit Theorem: New SOCR Applet and Demonstration Activity

    PubMed Central

    Dinov, Ivo D.; Christou, Nicolas; Sanchez, Juana

    2011-01-01

    Modern approaches for information technology based blended education utilize a variety of novel instructional, computational and network resources. Such attempts employ technology to deliver integrated, dynamically linked, interactive content and multifaceted learning environments, which may facilitate student comprehension and information retention. In this manuscript, we describe one such innovative effort of using technological tools for improving student motivation and learning of the theory, practice and usability of the Central Limit Theorem (CLT) in probability and statistics courses. Our approach is based on harnessing the computational libraries developed by the Statistics Online Computational Resource (SOCR) to design a new interactive Java applet and a corresponding demonstration activity that illustrate the meaning and the power of the CLT. The CLT applet and activity have clear common goals; to provide graphical representation of the CLT, to improve student intuition, and to empirically validate and establish the limits of the CLT. The SOCR CLT activity consists of four experiments that demonstrate the assumptions, meaning and implications of the CLT and ties these to specific hands-on simulations. We include a number of examples illustrating the theory and applications of the CLT. Both the SOCR CLT applet and activity are freely available online to the community to test, validate and extend (Applet: http://www.socr.ucla.edu/htmls/SOCR_Experiments.html and Activity: http://wiki.stat.ucla.edu/socr/index.php/SOCR_EduMaterials_Activities_GeneralCentralLimitTheorem). PMID:21833159

  5. Central Limit Theorem: New SOCR Applet and Demonstration Activity.

    PubMed

    Dinov, Ivo D; Christou, Nicolas; Sanchez, Juana

    2008-07-01

    Modern approaches for information technology based blended education utilize a variety of novel instructional, computational and network resources. Such attempts employ technology to deliver integrated, dynamically linked, interactive content and multifaceted learning environments, which may facilitate student comprehension and information retention. In this manuscript, we describe one such innovative effort of using technological tools for improving student motivation and learning of the theory, practice and usability of the Central Limit Theorem (CLT) in probability and statistics courses. Our approach is based on harnessing the computational libraries developed by the Statistics Online Computational Resource (SOCR) to design a new interactive Java applet and a corresponding demonstration activity that illustrate the meaning and the power of the CLT. The CLT applet and activity have clear common goals; to provide graphical representation of the CLT, to improve student intuition, and to empirically validate and establish the limits of the CLT. The SOCR CLT activity consists of four experiments that demonstrate the assumptions, meaning and implications of the CLT and ties these to specific hands-on simulations. We include a number of examples illustrating the theory and applications of the CLT. Both the SOCR CLT applet and activity are freely available online to the community to test, validate and extend (Applet: http://www.socr.ucla.edu/htmls/SOCR_Experiments.html and Activity: http://wiki.stat.ucla.edu/socr/index.php/SOCR_EduMaterials_Activities_GeneralCentralLimitTheorem). PMID:21833159

  6. Comparison of two methods for assaying reducing sugars in the determination of carbohydrase activities.

    PubMed

    Gusakov, Alexander V; Kondratyeva, Elena G; Sinitsyn, Arkady P

    2011-01-01

    The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides. Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, β-glucanase, xylanase, and β-mannanase activities was carried out. When cellulase activities against CMC were measured, the DNS assay gave activity values, which were typically 40-50% higher than those obtained with the NS assay. In the analysis of the xylanase, β-mannanase, and β-glucanase activities, the overestimations by the DNS assay were much more pronounced (the observed differences in the activities were 3- to 13-fold). Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed. PMID:21647284

  7. Demonstrating Optical Activity Using an iPad

    ERIC Educational Resources Information Center

    Schwartz, Pauline M.; Lepore, Dante M.; Morneau, Brandy N.; Barratt, Carl

    2011-01-01

    Optical activity using an iPad as a source of polarized light is demonstrated. A sample crystal or solution can be placed on the iPad running a white screen app. The sample is viewed through a polarized filter that can be rotated. This setup can be used in the laboratory or with a document camera to easily project in a large lecture hall.…

  8. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  9. A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB

    PubMed Central

    Ferrari, Annette; Rijnbrand, R.; Erwin, Alice L.

    2015-01-01

    A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux. PMID:25645845

  10. Activation of chemical promutagens by Selenastrum capricornutum in the plant cell/microbe coincubation assay

    SciTech Connect

    Gentile, J.M.; Lippert, M.; Johnson, P.; Shafer, T. )

    1990-05-01

    The critical balance of organisms living in aquatic environments is influenced by the presence and relationship of plants to those environments. However, even though plants occupy a fundamental trophic level within aquatic ecosystems, few studies have focused upon the effect of xenobiotics on aquatic plants, and even fewer studies have dealt with xenobiotic metabolism by aquatic plants. It is well established that plants can metabolize chemicals into mutagens. The impact of these unique plant-activated chemical mutagens on ecosystems, food chains and, ultimately, human health is an important question that will require intensive and integrative investigation. The plant cell/microbe coincubation assay is particularly advantageous for use with unicellular algae. The conditions of this assay are such that chemical metabolism and subsequent mutagen detection can be followed in intact algal cells under simulated field conditions. The purpose of this research was to demonstrate that a unicellular algal species could be used effectively in the plant cell/microbe coincubation assay to activate model chemical mutagens.

  11. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  12. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    PubMed

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts. PMID:24134852

  13. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    SciTech Connect

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C.; Miller, R.T.

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  14. Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates.

    PubMed

    Spinelli, Laura; Leslie, Nicholas R

    2016-01-01

    PTEN is a one of the most frequently mutated tumor suppressors in human cancers. It is essential for regulating diverse biological processes and through its lipid phosphatase activity regulates the PI 3-Kinase signaling pathway. Sensitive phosphatase assays are employed to study the catalytic activity of PTEN against phospholipid substrates. Here we describe protocols to assay PTEN lipid phosphatase activity using either purified enzyme (purified PTEN lipid phosphatase assay) or PTEN immunopurified from tissues or cultured cells (cellular IP PTEN lipid phosphatase assay) against vesicles containing radiolabeled PIP3 substrate. PMID:27514802

  15. A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

    PubMed Central

    Lloyd, A J; Thomann, H U; Ibba, M; Söll, D

    1995-01-01

    We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS. PMID:7659511

  16. Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

    PubMed Central

    Grin, Yossi; Admoni, Tamar; Priel, Esther

    2014-01-01

    Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay. In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain. Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP's for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region. PMID:25225832

  17. Telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (TRAP) assay.

    PubMed

    Grin, Yossi; Admoni, Tamar; Priel, Esther

    2014-01-01

    Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay. In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain. Compared to the traditional TRAP assay that utilize (32)P labeled radioactive dCTP's for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region. PMID:25225832

  18. A direct fluorescence-based assay for RGS domain GTPase accelerating activity.

    PubMed

    Willard, Francis S; Kimple, Adam J; Johnston, Christopher A; Siderovski, David P

    2005-05-15

    Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Galpha subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Galpha subunits. In this article, we describe the use of the ribose-conjugated fluorescent guanine nucleotide analog BODIPYFL-GTP as a spectroscopic probe to measure intrinsic and RGS protein-catalyzed nucleotide hydrolysis by Galphao. BODIPYFL-GTP bound to Galphao exhibits a 200% increase in fluorescence quantum yield. Hydrolysis of BODIPYFL-GTP to BODIPYFL-GDP reduces the quantum yield to 27% above its unbound value. We demonstrate that BODIPYFL-GTP can be used as a rapid real-time probe for measuring RGS domain-catalyzed GTP hydrolysis by Galphao. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Galphao and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds. PMID:15840508

  19. High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species.

    PubMed

    Inglin, Raffael C; Stevens, Marc J A; Meile, Lukas; Lacroix, Christophe; Meile, Leo

    2015-07-01

    We describe high-throughput screening techniques to rapidly detect either antimicrobial activity, using an agar-well diffusion assay in microtiter plates, or antifungal activity using an agar-spot assay in 24-well plates. 504 Lactobacillus isolates were screened with minimal laboratory equipment and screening rates of 2000-5000 individual antimicrobial interactions. PMID:25937247

  20. Sensitive, coupled assay for plasminogen activator using a thiol ester substrate for plasmin

    SciTech Connect

    Coleman, P L; Green, G D.J.

    1980-01-01

    Several assays for plasminogen activator employ a direct assay method. These are remarkably sensitive methods, yet they suffer in comparison to the sensitivity of coupled methods. Coupling the assay with plasminogen not only amplifies the sensitivity by the multiplicative effect of plasmin, but insures that only those proteases specific for plasminogen are assayed. The choice of substrate for plasmin is critical. A thiol ester substrate, thiobenzyl benzyloxy-carbonyl-L-lysinate (Z-Lys-SBzl), has been synthesized which combines high k/sub cat/ with alkaline stability. In an effort to characterize the plasminogen activator from hepatoma tissue culture (HTC) and its hormonally-controlled inhibitor, Z-Lys-SBzl was used in a coupled approach providing an assay which is superior to the /sup 125/I-fibrinolytic assay. It is also extremely sensitive to plasminogen activator and can be used for routine analysis of purification as well as kinetic and binding studies. (ERB)

  1. Residential construction demonstration project, Cycle II: Active ventilation

    SciTech Connect

    Not Available

    1991-01-01

    This report documents the analysis of the performance of natural and mechanical ventilation in Pacific Northwest homes. The analysis was part of Cycle II of the Residential Construction Demonstration Project, sponsored by Bonneville Power Administration (BPA). Since 1986, the Residential Construction Demonstration Project (RCDP) has sponsored the collection of data on energy efficient homes in the Pacific Northwest that comply with these new standards and requirements. Cycle II of RCDP was conducted between September 1987 and April 1990. It concentrated on energy innovations in homes built to the Super Good Cents specification. All of the test homes have electric heat and mechanical ventilation systems. Seven different types of active ventilation systems are represented in the homes. Three of these system types are equipped with heat recovery devices, and are represented in approximately a quarter of the test homes. The potential for both natural and mechanical ventilation was measured. Potential structural leakage was measured by blower door testing. Flow rate and operating time of mechanical ventilation systems were measured with flow hoods and hour meters. Actual ventilation was measured by using a passive tracer gas technique for several weeks during the heating season and at times of normal occupancy.

  2. Residential construction demonstration project, Cycle II: Active ventilation

    SciTech Connect

    Not Available

    1991-12-31

    This report documents the analysis of the performance of natural and mechanical ventilation in Pacific Northwest homes. The analysis was part of Cycle II of the Residential Construction Demonstration Project, sponsored by Bonneville Power Administration (BPA). Since 1986, the Residential Construction Demonstration Project (RCDP) has sponsored the collection of data on energy efficient homes in the Pacific Northwest that comply with these new standards and requirements. Cycle II of RCDP was conducted between September 1987 and April 1990. It concentrated on energy innovations in homes built to the Super Good Cents specification. All of the test homes have electric heat and mechanical ventilation systems. Seven different types of active ventilation systems are represented in the homes. Three of these system types are equipped with heat recovery devices, and are represented in approximately a quarter of the test homes. The potential for both natural and mechanical ventilation was measured. Potential structural leakage was measured by blower door testing. Flow rate and operating time of mechanical ventilation systems were measured with flow hoods and hour meters. Actual ventilation was measured by using a passive tracer gas technique for several weeks during the heating season and at times of normal occupancy.

  3. In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture

    USGS Publications Warehouse

    Troyer, R.M.; Garver, K.A.; Ranson, J.C.; Wargo, A.R.; Kurath, G.

    2008-01-01

    A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72 h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested. ?? 2008.

  4. A MEMBRANE FILTER PROCEDURE FOR ASSAYING CYTOTOXIC ACTIVITY IN HETEROTROPHIC BACTERIA ISOLATED FROM DRINKING WATER

    EPA Science Inventory

    Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Wate...

  5. Herpes Murine Model as a Biological Assay to Test Dialyzable Leukocyte Extracts Activity

    PubMed Central

    Salinas-Jazmín, Nohemí; Estrada-Parra, Sergio; Becerril-García, Miguel Angel; Limón-Flores, Alberto Yairh; Vázquez-Leyva, Said; Pavón, Lenin; Velasco-Velázquez, Marco Antonio; Pérez-Tapia, Sonia Mayra

    2015-01-01

    Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines. PMID:25984538

  6. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

    SciTech Connect

    Ueda, Yoshihiro; Morigaki, Kenichi . E-mail: morigaki-kenichi@aist.go.jp; Tatsu, Yoshiro; Yumoto, Noboru; Imaishi, Hiromasa . E-mail: himaish@kobe-u.ac.jp

    2007-04-20

    We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.

  7. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes.

    PubMed

    Ueda, Yoshihiro; Morigaki, Kenichi; Tatsu, Yoshiro; Yumoto, Noboru; Imaishi, Hiromasa

    2007-04-20

    We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s. PMID:17335776

  8. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel. PMID:24571675

  9. Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity

    PubMed Central

    Muren, Natalie B.; Barton, Jacqueline K.

    2013-01-01

    Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by these enzymes is associated with cancer initiation and progression. Here we describe a non-radioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-modified electrodes confers protection from restriction for signal-on detection. We implement this assay with a multiplexed chip platform and show robust detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Essential to work with human methyltransferases, our unique assay design allows activity measurements on both unmethylated and hemimethylated DNA substrates. We validate this assay by comparison with a conventional radioactive method. The advantages of electrochemistry over radioactivity and fluorescence make this assay an accessible and promising new approach for the sensitive, label-free detection of human methyltransferase activity. PMID:24164112

  10. Freeze-dried activated substrate for factor VIII assays.

    PubMed

    Margolis, J

    1987-01-01

    Factor VIII-deficient plasma (natural or artificial) mixed with kaolin and phospholipid can be lyophilized to provide ready-to-use substrate which is stable for months at 4 degrees C and usable after many weeks at room temperature. Factor VIII assays are much simplified and more reproducible using this reagent and can be quantified with the aid of a programmable calculator according to the equation (formula; see text) as % of standard and X, S and B are clotting times of test, standard and blank samples respectively. The slope of the log/log function (k) is approximately--6.5. PMID:3111909

  11. In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity.

    PubMed

    Zufferey, Rachel

    2016-01-01

    Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-(3)H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-(3)H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme. PMID:26780155

  12. In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

    PubMed Central

    Zufferey, Rachel

    2016-01-01

    Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme. PMID:26780155

  13. Antiviral activity of a Rac GEF inhibitor characterized with a sensitive HIV/SIV fusion assay

    SciTech Connect

    Pontow, Suzanne; Harmon, Brooke; Campbell, Nancy; Ratner, Lee

    2007-11-10

    A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

  14. Serotonin (5-hydroxytryptamine) glucuronidation in vitro: assay development, human liver microsome activities and species differences.

    PubMed

    Krishnaswamy, S; Duan, S X; Von Moltke, L L; Greenblatt, D J; Sudmeier, J L; Bachovchin, W W; Court, M H

    2003-02-01

    1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5-O-glucuronide by (1)H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K(m) and V(max) of 8.8 +/- 0.3 mM and 43.4 +/- 0.4 nmoles min(-1) mg(-1) protein, respectively, for human liver microsomes, and 5.9 +/- 0.2 mM and 15.8 +/- 0.2 nmoles min(-1) mg(-1), respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro. PMID:12623759

  15. Development and Demonstration of Active Noise Control Concepts

    NASA Technical Reports Server (NTRS)

    Kraft, R.; Hu, Z.; Sommerfeldt, S.; Walker, B.; Hersh, A.; Luo, H.; Spencer, M.; Hallman, D.; Mitchell, C.; Sutliff, D.

    2000-01-01

    This report details design methods for and feasibility of an Active Noise Control (ANC) system using flush-wall-mounted sensors and actuators to reduce turbofan engine rotor-stator interaction noise. ANC concepts capable of suppressing discrete-tone spinning modes containing several cut-on radial mode were identified, developed analytically, and evaluated. Separate ANC systems that suppressed at least three radial modes in a cylindrical inlet duct and three radial modes in an exhaust annulus were developed. These designs resulted in inlet duct and exhaust duct tests that were performed at NASA on the 4-ft ANC Fan in the NASA Glenn AAPL facility. Effective suppression of 2-BPF spinning mode m = 2 tone noise was achieved over a range of fan speeds 1800 to 2450 rpm, where up to 4 radials were present. In the inlet duct, up to 12 dB reduction was obtained for 3 radial modes, and up to 4 dB was obtained with 4 radial modes. In the exhaust duct, up to 15 dB PWL reduction was obtained with either two or three radial modes present. Thus, the ability to suppress multiple radial modes for tones in both the inlet and exhaust ducts has been successfully demonstrated. Implications of ANC system design requirements on installation and system integration issues for ANC systems capable of suppressing higher order radial mode content when applied to a 767 using twin CF6 engines were evaluated analytically. The analytical results indicated an ANC system must be part of an integrated design to be effective.

  16. Photography by Cameras Integrated in Smartphones as a Tool for Analytical Chemistry Represented by an Butyrylcholinesterase Activity Assay

    PubMed Central

    Pohanka, Miroslav

    2015-01-01

    Smartphones are popular devices frequently equipped with sensitive sensors and great computational ability. Despite the widespread availability of smartphones, practical uses in analytical chemistry are limited, though some papers have proposed promising applications. In the present paper, a smartphone is used as a tool for the determination of cholinesterasemia i.e., the determination of a biochemical marker butyrylcholinesterase (BChE). The work should demonstrate suitability of a smartphone-integrated camera for analytical purposes. Paper strips soaked with indoxylacetate were used for the determination of BChE activity, while the standard Ellman’s assay was used as a reference measurement. In the smartphone-based assay, BChE converted indoxylacetate to indigo blue and coloration was photographed using the phone’s integrated camera. A RGB color model was analyzed and color values for the individual color channels were determined. The assay was verified using plasma samples and samples containing pure BChE, and validated using Ellmans’s assay. The smartphone assay was proved to be reliable and applicable for routine diagnoses where BChE serves as a marker (liver function tests; some poisonings, etc.). It can be concluded that the assay is expected to be of practical applicability because of the results’ relevance. PMID:26110404

  17. Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations.

    PubMed

    Settivari, Raja S; Gehen, Sean C; Amado, Ricardo Acosta; Visconti, Nicolo R; Boverhof, Darrell R; Carney, Edward W

    2015-07-01

    Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations. PMID:25981449

  18. Photography by Cameras Integrated in Smartphones as a Tool for Analytical Chemistry Represented by an Butyrylcholinesterase Activity Assay.

    PubMed

    Pohanka, Miroslav

    2015-01-01

    Smartphones are popular devices frequently equipped with sensitive sensors and great computational ability. Despite the widespread availability of smartphones, practical uses in analytical chemistry are limited, though some papers have proposed promising applications. In the present paper, a smartphone is used as a tool for the determination of cholinesterasemia i.e., the determination of a biochemical marker butyrylcholinesterase (BChE). The work should demonstrate suitability of a smartphone-integrated camera for analytical purposes. Paper strips soaked with indoxylacetate were used for the determination of BChE activity, while the standard Ellman's assay was used as a reference measurement. In the smartphone-based assay, BChE converted indoxylacetate to indigo blue and coloration was photographed using the phone's integrated camera. A RGB color model was analyzed and color values for the individual color channels were determined. The assay was verified using plasma samples and samples containing pure BChE, and validated using Ellmans's assay. The smartphone assay was proved to be reliable and applicable for routine diagnoses where BChE serves as a marker (liver function tests; some poisonings, etc.). It can be concluded that the assay is expected to be of practical applicability because of the results' relevance. PMID:26110404

  19. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

    PubMed Central

    Körner, Wolfgang; Vinggaard, Anne Marie; Térouanne, Béatrice; Ma, Risheng; Wieloch, Carise; Schlumpf, Margret; Sultan, Charles; Soto, Ana M

    2004-01-01

    We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%. PMID

  20. POSSIBLE ERRORS IN ASSAY FOR B-GLYCOSIDASE ACTIVITY

    EPA Science Inventory

    Because intestinal B-glycosidase enzymes activate the toxicity and/or carcinogenicity of many environmental chemicals, accurate analysis of their activities is toxicologically important. owever, previous work in this lab indicated that widespread use of the glycosides of p-nitrop...

  1. Disk Diffusion Assay to Assess the Antimicrobial Activity of Marine Algal Extracts.

    PubMed

    Desbois, Andrew P; Smith, Valerie J

    2015-01-01

    Marine algae are a relatively untapped source of bioactive natural products, including those with antimicrobial activities. The ability to assess the antimicrobial activity of cell extracts derived from algal cultures is vital to identifying species that may produce useful novel antibiotics. One assay that is used widely for this purpose is the disk diffusion assay due to its simplicity, rapidity, and low cost. Moreover, this assay gives output data that are easy to interpret and can be used to screen many samples at once irrespective of the solvent used during preparation. In this chapter, a step-by-step protocol for performing a disk diffusion assay is described. The assay is particularly well suited to testing algal cell extracts and fractions resulting from separation through bioassay-guided approaches. PMID:26108520

  2. Imaging techniques for assaying lymphocyte activation in action

    PubMed Central

    Balagopalan, Lakshmi; Sherman, Eilon; Barr, Valarie A.; Samelson, Lawrence E.

    2012-01-01

    Imaging techniques have greatly improved our understanding of lymphocyte activation. Technical advances in spatial and temporal resolution and new labelling tools have enabled researchers to directly observe the activation process. Consequently, research using imaging approaches to study lymphocyte activation has expanded, providing an unprecedented level of cellular and molecular detail in the field. As a result, certain models of lymphocyte activation have been verified, others have been revised and yet others have been replaced with new concepts. In this article, we review the current imaging techniques that are used to assess lymphocyte activation in different contexts, from whole animals to single molecules, and discuss the advantages and potential limitations of these methods. PMID:21179118

  3. Californium interrogation prompt neutron (CIPN) instrument for non-destructive assay of spent nuclear fuel-Design concept and experimental demonstration

    NASA Astrophysics Data System (ADS)

    Henzlova, D.; Menlove, H. O.; Rael, C. D.; Trellue, H. R.; Tobin, S. J.; Park, Se-Hwan; Oh, Jong-Myeong; Lee, Seung-Kyu; Ahn, Seong-Kyu; Kwon, In-Chan; Kim, Ho-Dong

    2016-01-01

    This paper presents results of the first experimental demonstration of the Californium Interrogation Prompt Neutron (CIPN) instrument developed within a multi-year effort launched by the Next Generation Safeguards Initiative Spent Fuel Project of the United States Department of Energy. The goals of this project focused on developing viable non-destructive assay techniques with capabilities to improve an independent verification of spent fuel assembly characteristics. For this purpose, the CIPN instrument combines active and passive neutron interrogation, along with passive gamma-ray measurements, to provide three independent observables. This paper describes the initial feasibility demonstration of the CIPN instrument, which involved measurements of four pressurized-water-reactor spent fuel assemblies with different levels of burnup and two initial enrichments. The measurements were performed at the Post-Irradiation Examination Facility at the Korea Atomic Energy Institute in the Republic of Korea. The key aim of the demonstration was to evaluate CIPN instrument performance under realistic deployment conditions, with the focus on a detailed assessment of systematic uncertainties that are best evaluated experimentally. The measurements revealed good positioning reproducibility, as well as a high degree of insensitivity of the CIPN instrument's response to irregularities in a radial burnup profile. Systematic uncertainty of individual CIPN instrument signals due to assembly rotation was found to be <4.5%, even for assemblies with fairly extreme gradients in the radial burnup profile. These features suggest that the CIPN instrument is capable of providing a good representation of assembly average characteristics, independent of assembly orientation in the instrument.

  4. An improvement of Barter's method for assaying plasma cholesterol ester transfer activity: experimental and clinical applications.

    PubMed

    Harvengt, C; Desager, J P; Mailleux, P; Heller, F R

    1989-01-01

    The use of a discontinuous density gradient and of a vertical rotor to separate plasma lipoproteins are modifications of Barter's described method for assaying cholesteryl ester transfer activity (CETA) in plasma. The original feature of our approach is the fast preparation of the labeled substrate by a physiologic-like process, which renders the assay easy and suitable for measurement of this activity in both man and animals. PMID:2730951

  5. Paraoxonase-1 Enzyme Activity Assay for Clinical Samples: Validation and Correlation Studies

    PubMed Central

    Garelnabi, Mahdi; Younis, Abdelmoneim

    2015-01-01

    Background Paraoxonase-1 (PON1) enzyme is reported in various types of tissues and linked to numerous pathophysiological disorders. It is a potential biomarker in many pathological conditions such as cardiovascular diseases. Material/Methods We conducted several small-scale studies to evaluate PON1 performance as affected by sample types, storage, and interferences. We also carried out short-term studies to compare the performance of the widely used PON1 assay to the similar commercially available PON1 kit assay method; sample size for the method comparison was N=40, and the number varied for other validation experiments. Results Our studies using various types of anticoagulants show that samples collected in tubes with NaF, citrate, EDTA, clot activator, and sodium heparin have increased PON1 levels that are 49%, 24.5%, 19.8%, 11.4%, and 8%, respectively, higher compared to serum samples collected in plain tubes. However, samples collected in lithium heparin tubes demonstrated 10.4% lower PON1 levels compared to serum collected in plain tubes. Biological interference such as hemolysis has little effect on PON1 levels; however, samples spiked with lipids have shown 13% lower PON 1 levels. Our studies comparing the PON1 method commonly available for PON1 assay and a similar non-ELISA commercially available PON1 kit method showed a weak Spearman correlation coefficient of R2=0.40 for the range of 104.9–245.6 U/L. Conclusions The current study provides new validation data on enzyme PON1 performance. While no appreciable change was seen with storage, samples type affects the enzyme performance. Our results should encourage additional clinical studies to investigate other aspects of factors known to affect PON1 enzyme function and performance. PMID:25814092

  6. A novel 96-well gel-based assay for determining antifungal activity against filamentous fungi.

    PubMed

    Troskie, Anscha Mari; Vlok, Nicolas Maré; Rautenbach, Marina

    2012-12-01

    In recent years the global rise in antibiotic resistance and environmental consciousness lead to a renewed fervour to find and develop novel antibiotics, including antifungals. However, the influence of the environment on antifungal activity is often disregarded and many in vitro assays may cause the activity of certain antifungals to be overestimated or underestimated. The general antifungal test assays that are economically accessible to the majority of scientists primarily rely on visual examination or on spectrophotometric analysis. The effect of certain morphogenic antifungals, which may lead to hyperbranching of filamentous fungi, unfortunately renders these methods unreliable. To minimise the difficulties experienced as a result of hyperbranching, we developed a straightforward, economical 96-well gel-based method, independent of spectrophotometric analysis, for highly repeatable determination of antifungal activity. For the calculation of inhibition parameters, this method relies on the visualisation of assay results by digitisation. The antifungal activity results from our novel micro-gel dilution assay are comparable to that of the micro-broth dilution assay used as standard reference test of The Clinical and Laboratory Standard Institute. Furthermore, our economical assay is multifunctional as it permits microscopic analysis of the preserved assay results, as well as rendering highly reliable data. PMID:23089670

  7. A new formula to calculate activity of superoxide dismutase in indirect assays.

    PubMed

    Zhang, Chen; Bruins, Marieke E; Yang, Zhi-Qiang; Liu, Shu-Tao; Rao, Ping-Fan

    2016-06-15

    To calculate superoxide dismutase (SOD) activity rapidly and accurately by indirect SOD assays, a formula based on the ratio of the catalytic speed of SOD to the reaction speed of the indicator with superoxide anion was deduced. The accuracy of this formula was compared with the conventional formula based on inhibition in five indirect SOD assays. The new formula was validated in nearly the entire SOD activity range, whereas the conventional formula was validated only during inhibition of 40-60%. This formula might also be used for the assays of other enzymes. PMID:27033009

  8. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    PubMed

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants. PMID:27424913

  9. A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2011-10-15

    Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors. PMID:21729692

  10. A Homogeneous Cell-Based Assay for Measurement of Endogenous PON1 Activity

    PubMed Central

    Ahmad, Syed; Carter, Jade J.; Scott, John E.

    2010-01-01

    PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. In order to identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in media and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells and tolerance to DMSO and serum. Aspirin, quercetin and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel, homogenous assay for detection of endogenous PON1 expression has been developed and is amenable to high throughput screening for the identification of small molecules that enhance PON1 expression. PMID:20096260

  11. Design of benign matrix drums for the non-destructive assay performance demonstration program for the National TRU Program

    SciTech Connect

    Becker, G.K.

    1996-09-01

    Regulatory compliance programs associated with the Department of Energy (DOE) Waste Isolation Pilot Plant (WIPP) Transuranic (TRU) Waste Characterization Program (the Program) require the collection of waste characterization data of known quality to support repository performance assessment, permitting, and associated activities. Blind audit samples, referred to as PDP (performance demonstration program) samples, are devices used in the NDA PDP program to acquire waste NDA system performance data per defined measurement routines. As defined under the current NDA PDP Program Plan, a PDP sample consists of a DOT 17C 55-gallon PDP matrix drum configured with insertable radioactive standards, working reference materials (WRMs). The particular manner in which the matrix drum and PDP standard(s) are combined is a function of the waste NDA system performance test objectives of a given cycle. The scope of this document is confined to the design of the PDP drum radioactive standard internal support structure, the matrix type and the as installed configuration. The term benign is used to designate a matrix possessing properties which are nominally non-interfering to waste NDA measurement techniques. Measurement interference sources are technique specific but include attributes such as: high matrix density, heterogeneous matrix distributions, matrix compositions containing high moderator/high Z element concentrations, etc. To the extent practicable the matrix drum design should not unduly bias one NDA modality over another due to the manner in which the matrix drum configuration manifests itself to the measurement system. To this end the PDP matrix drum configuration and composition detailed below is driven primarily by the intent to minimize the incorporation of matrix attributes known to interfere with fundamental waste NDA modalities, i.e. neutron and gamma based techniques.

  12. Adult Basic Learning in an Activity Center: A Demonstration Approach.

    ERIC Educational Resources Information Center

    Metropolitan Adult Education Program, San Jose, CA.

    Escuela Amistad, an activity center in San Jose, California, is now operating at capacity, five months after its origin. Average daily attendance has been 125 adult students, 18-65, most of whom are females of Mexican-American background. Activities and services provided by the center are: instruction in English as a second language, home…

  13. A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes.

    PubMed

    Lewis, Cody W; Taylor, Ryan G; Kubara, Philip M; Marshall, Kris; Meijer, Laurent; Golsteyn, Roy M

    2013-09-17

    We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 μM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity. PMID:23954627

  14. Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.

    PubMed

    Jung, Se-Hui; Kong, Deok-Hoon; Jeon, Hye-Yoon; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Min-Soo; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-08-15

    Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34μg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions. PMID:27040940

  15. Air Pollution and Weather: Activities and Demonstrations for Science Classes

    ERIC Educational Resources Information Center

    Cole, Henry S.

    1973-01-01

    Discusses a number of concepts (turbulence, dispersion, vertical temperature distribution, atmospheric stability and instability, and inversions) which are prerequisite to understanding how weather affects air quality. Describes classroom demonstrations effective in introducing these concepts to students at the elementary, secondary and college…

  16. Assay of Flippase Activity in Proteoliposomes Using Fluorescent Lipid Derivatives.

    PubMed

    Marek, Magdalena; Günther-Pomorski, Thomas

    2016-01-01

    Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level. PMID:26695033

  17. ACTIVE SOIL DEPRESSURIZATION (ASD) DEMONSTRATION IN A LARGE BUILDING

    EPA Science Inventory

    The report gives results of an evaluation of the feasibility of implementing radon resistant construction techniques -- especially active soil depressurization (ASD) -- in new large buildings in Florida. Indoor radon concentrations and radon entry were monitored in a finished bui...

  18. A simplified assay for the quantification of circulating activated protein C.

    PubMed

    Martos, Laura; Bonanad, Santiago; Ramón, Luis A; Cid, Ana-Rosa; Bonet, Elena; Corral, Javier; Miralles, Manuel; España, Francisco; Navarro, Silvia; Medina, Pilar

    2016-08-01

    Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories. PMID:27262823

  19. Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

    PubMed Central

    Chen, Lu; Ooi, Soon-Keat; Conaway, Joan W.; Conaway, Ronald C.

    2014-01-01

    Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes. PMID:25407555

  20. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  1. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  2. Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors.

    PubMed

    Frang, Heini; Mukkala, Veli-Matti; Syystö, Rita; Ollikka, Pia; Hurskainen, Pertti; Scheinin, Mika; Hemmilä, Ilkka

    2003-04-01

    GPCRs represent important targets for drug discovery because GPCRs participate in a wide range of cellular signaling pathways that play a role in a variety of pathological conditions. A large number of screening assays have been developed in HTS laboratories for the identification of hits or lead compounds acting on GPCRs. One type of assay that has found relatively widespread application, due to its at least in part generic nature, relies on the use of a radioactive GTP analogue, [(35)S]GTPgammaS. The G-protein alpha subunit is an essential part of the interaction between receptor and G proteins in transmembrane signaling, where the activated receptor catalyzes the release of GDP from Galpha, thereby enabling the subsequent binding of GTP or a GTP analogue. [(35)S]GTPgammaS allows the extent of this interaction to be followed quantitatively by determining the amount of radioactivity associated with cell membranes. However, with the increased desire to move assays to nonradioactive formats, there is a considerable need to develop a nonradioactive GTP binding assay to monitor ligand-induced changes in GPCR activity. The Eu-GTP binding assay described here is based on TRF that exploits the unique fluorescence properties of lanthanide chelates, and provides a powerful alternative to assays using radioisotopes. In this article, we have used the human alpha(2A)-AR as a model GPCR system to evaluate the usefulness of this Eu-GTP binding assay. PMID:15090192

  3. beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.

    PubMed

    Gong, Haibiao; Zhang, Bin; Little, Garrick; Kovar, Joy; Chen, Huaxian; Xie, Wen; Schutz-Geschwender, Amy; Olive, D Michael

    2009-03-01

    beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use. PMID:19103143

  4. A highly sensitive telomerase activity assay that eliminates false-negative results caused by PCR inhibitors.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Miyoshi, Daisuke; Sugimoto, Naoki

    2013-01-01

    An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity. PMID:24071983

  5. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

    PubMed Central

    Jońca, Joanna; Żuk, Monika; Wasąg, Bartosz; Janaszak-Jasiecka, Anna; Lewandowski, Krzysztof; Wielgomas, Bartosz; Waleron, Krzysztof; Jasiecki, Jacek

    2015-01-01

    Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. PMID:26444431

  6. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

    PubMed

    Jońca, Joanna; Żuk, Monika; Wasąg, Bartosz; Janaszak-Jasiecka, Anna; Lewandowski, Krzysztof; Wielgomas, Bartosz; Waleron, Krzysztof; Jasiecki, Jacek

    2015-01-01

    Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. PMID:26444431

  7. Active nondestructive assay of nuclear materials: principles and applications

    SciTech Connect

    Gozani, Tsahi

    1981-01-01

    The purpose of this book is to present, coherently and comprehensively, the wealth of available but scattered information on the principles and applications of active nondestructive analysis (ANDA). Chapters are devoted to the following: background and overview; interactions of neutrons with matter; interactions of ..gamma..-rays with matter; neutron production and sources; ..gamma..-ray production and sources; effects of neutron and ..gamma..-ray transport in bulk media; signatures of neutron- and photon-induced fissions; neutron and photon detection systems and electronics; representative ANDA systems; and instrument analysis, calibration, and measurement control for ANDA. Each chapter has an introductory section describing the relationship of the topic of that chapter to ANDA. Each chapter ends with a section that summarizes the main results and conclusions of the chapter, and a reference list.

  8. Suggested improvements to the standard filter paper assay used to measure cellulase activity.

    PubMed

    Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar; Holtzapple, Mark

    2003-06-20

    Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized. PMID:12673775

  9. Radiometric assay of ghrelin hydrolase activity and 3H-ghrelin distribution into mouse tissues.

    PubMed

    Chen, Vicky Ping; Gao, Yang; Geng, Liyi; Brimijoin, Stephen

    2015-12-15

    A high-throughput radiometric assay was developed to characterize enzymatic hydrolysis of ghrelin and to track the peptide's fate in vivo. The assay is based on solvent partitioning of [(3)H]-octanoic acid liberated from [(3)H]-octanoyl ghrelin during enzymatic hydrolysis. This simple and cost-effective method facilitates kinetic analysis of ghrelin hydrolase activity of native and mutated butyrylcholinesterases or carboxylesterases from multiple species. In addition, the assay's high sensitivity facilitates ready evaluation of ghrelin's pharmacokinetics and tissue distribution in mice after i.v. bolus administration of radiolabeled peptide. PMID:26514871

  10. An Experiential Learning Activity Demonstrating Normal and Phobic Anxiety

    ERIC Educational Resources Information Center

    Canu, Will H.

    2008-01-01

    This article describes an activity for an undergraduate abnormal psychology course that used student-generated data to illustrate normal versus clinically significant anxiety responses related to specific phobias. Students (N = 37) viewed 14 images of low- or high-anxiety valence and rated their subjective response to each. Discussion in a…

  11. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    SciTech Connect

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-09-15

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When /sup 125/I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. /sup 125/I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When (/sup 14/C)elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases.

  12. Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    PubMed Central

    Kazlauskaite, Agne; Kelly, Van; Johnson, Clare; Baillie, Carla; Hastie, C. James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R.; Pedrioli, Patrick G. A.; Muqit, Miratul M. K.

    2014-01-01

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser65 is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease. PMID:24647965

  13. How Do Detergents Work? A Qualitative Assay to Measure Amylase Activity

    ERIC Educational Resources Information Center

    Novo, M. Teresa; Casanoves, Marina; Garcia-Vallvé, Santi; Pujadas, Gerard; Mulero, Miquel; Valls, Cristina

    2016-01-01

    We present a practical activity focusing on two main goals: to give learners the opportunity to experience how the scientific method works and to increase their knowledge about enzymes in everyday situations. The exercise consists of determining the amylase activity of commercial detergents. The methodology is based on a qualitative assay using a…

  14. A novel live cell assay to measure diacylglycerol lipase α activity.

    PubMed

    Singh, Praveen K; Markwick, Rachel; Howell, Fiona V; Williams, Gareth; Doherty, Patrick

    2016-06-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays. PMID:27013337

  15. A novel live cell assay to measure diacylglycerol lipase α activity

    PubMed Central

    Singh, Praveen K.; Markwick, Rachel; Howell, Fiona V.; Williams, Gareth; Doherty, Patrick

    2016-01-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays. PMID:27013337

  16. Development and validation of a potentiometric biosensor assay for tylosin with demonstrated applicability for the detection of two other antimicrobial growth-promoter compounds in feedstuffs.

    PubMed

    Stead, S L; Wolodko-Cierniak, K B; Richmond, S F; Sharman, M; Driver, P; Teale, P; Leonardova, O; Purvis, D

    2011-01-01

    A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CCβ) for tylosin was determined as 0.2 mg kg(-1) in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg(-1) of ≤13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37°C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust. PMID:21574083

  17. Active Control of High-Frequency Combustor Instability Demonstrated

    NASA Technical Reports Server (NTRS)

    DeLaat, John C.; Chang, Clarence T.

    2003-01-01

    To reduce the environmental impact of aerospace propulsion systems, extensive research is being done in the development of lean-burning (low fuel-to-air ratio) combustors that can reduce emissions throughout the mission cycle. However, these lean-burning combustors have an increased susceptibility to thermoacoustic instabilities-high-pressure oscillations much like sound waves that can cause severe high-frequency vibrations in the combustor. These pressure waves can fatigue the combustor components and even the downstream turbine blades. This can significantly decrease the combustor and turbine safe operating life. Thus, suppression of the thermoacoustic combustor instabilities is an enabling technology for lean, low-emissions combustors. Under the Propulsion and Power Program, the NASA Glenn Research Center in partnership with Pratt & Whitney, United Technologies Research Center, and Georgia Institute of Technology is developing technologies for the active control of combustion instabilities.

  18. A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

    PubMed Central

    2014-01-01

    Background Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. Results We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. Conclusion The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional

  19. Measurement of filter paper activities of cellulase with microplate-based assay

    PubMed Central

    Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

    2015-01-01

    It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

  20. Measurement of filter paper activities of cellulase with microplate-based assay.

    PubMed

    Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

    2016-01-01

    It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

  1. Assaying ATE1 Activity in Yeast by β-Gal Degradation.

    PubMed

    Kashina, Anna S

    2015-01-01

    In 1980s it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta galactosidase (beta-Gal) because its activity can be easily measured using standardized colorimetric assays. Here we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species. PMID:26285881

  2. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate.

    PubMed

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F; Zhang, Kate

    2015-06-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  3. Activities of the OECD/NEA Expert Group on Assay Data for Spent Nuclear Fuel

    SciTech Connect

    Gauld, Ian C; Rugama, Yolanda

    2009-01-01

    Management of spent nuclear fuel is a key issue for many NEA member countries. In nuclear criticality safety, the decision of many countries to advance burnup credit as part of their licensing strategy has heightened recent interest in experimental data needed to validate computer codes used in burnup credit calculations. This paper discusses recent activities of an Expert Group on assay data, formed under the OECD/NEA/NSC/WPNCS (Working Party on Nuclear Criticality Safety) to help coordinate isotopic assay data activities and facilitate international collaboration between NEA member countries developing or implementing burnup credit methodologies. Recent activities of the Expert Group are described, focusing on the planned expansion of the Spent Fuel Isotopic Composition Database (SFCOMPO), and preparation of a state-of-the-art report on assay data that includes sections on recommended radiochemical analysis methods, techniques, and lessons learned from previous experiments.

  4. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

    PubMed Central

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S.; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F.; Zhang, Kate

    2015-01-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  5. In vitro and in vivo assays of protein kinase CK2 activity.

    PubMed

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  6. A modified ferrous oxidation-xylenol orange assay for lipoxygenase activity in rice grains.

    PubMed

    Timabud, Tarinee; Sanitchon, Jirawat; Pongdontri, Paweena

    2013-12-01

    Ferrous oxidation-xylenol orange assay reagent was reformulated by using spectral analysis of ferric-xylenol orange complex to detect low concentrations of lipoxygenase rice grain products. Reducing the levels of ferrous sulphate and xylenol orange in the FOX reagent enabled the detection of low concentrations of hydroperoxy fatty acid derived from lipoxygenase activity in the range of 0.1-1.5 μM. Protein, substrate and time courses of the modified FOX assay were studied to determine lipoxygenase activity in rice grain. The assay was also applicable as a high throughput technique for comparisons of lipoxygenase activity from various rice varieties. This has important implications for rapid screening for low-lipoxygenase containing rice cultivars in rice breeding program and grain quality during storage. PMID:23870974

  7. Activity-based assay of matrix metalloproteinase on nonbiofouling surfaces using time-of-flight secondary ion mass spectrometry.

    PubMed

    Kim, Young-Pil; Lee, Bong Soo; Kim, Eunkyung; Choi, Insung S; Moon, Dae Won; Lee, Tae Geol; Kim, Hak-Sung

    2008-07-01

    A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with nonbiofouling poly(oligo(ethylene glycol) methacrylate) (pOEGMA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atom-transfer radical polymerization of OEGMA on a Si/SiO2 substrate, the MMP activity was determined by analyzing the cleaved peptide fragments using TOF-SIMS on the peptide-conjugated AuNPs. The use of nonbiofouling pOEGMA films in conjunction with AuNPs synergistically enhanced the sensitivity of assays for MMP activity and its inhibition in human serum. The detection sensitivity of MMP-7 in serum was as low as 20 ng mL(-1) (1 pmol mL(-1)), and the half-maximal inhibitory concentration (IC50) of minocycline, which is a MMP-7 inhibitor, was estimated to be 450 nM. It is anticipated that the developed system will be broadly useful for conducting activity-based assays of serum proteases, as well as for screening of their inhibitors, with high sensitivity in a high-throughput manner. PMID:18505270

  8. A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

    PubMed

    Arnold, Laurence H; Kunzelmann, Simone; Webb, Martin R; Taylor, Ian A

    2015-01-01

    The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents. PMID:25331707

  9. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay.

    PubMed

    Sonavane, Manoj; Creusot, Nicolas; Maillot-Maréchal, Emmanuelle; Péry, Alexandre; Brion, François; Aїt-Aïssa, Selim

    2016-04-15

    Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish. PMID:26851879

  10. Characterizing and improving passive-active shufflers for assays of 208-Liter waste drums

    SciTech Connect

    Rinard, P.M.; Adams, E.L.; Menlove, H.O.; Sprinkle, J.K. Jr.

    1992-06-01

    A passive and active neutron shuffler for 208-L waste drums has been used to perform over 1500 active and 500 passive measurements on uranium and plutonium samples in 28 different matrices. The shuffler is now better characterized and improvements have been implemented or suggested. An improved correction for the effects of the matrix material was devised from flux-monitor responses. The most important cause of inaccuracies in assays is a localized instead of a uniform distribution of fissile material in a drum; a technique for deducing the distribution from the assay data and then applying a correction is suggested and will be developed further. A technique is given to detect excessive amounts of moderator that could make hundreds of grams of {sup 235}U assay as zero grams. Sensitivities (minimum detectable masses) for {sup 235}U with active assays and for {sup 240}Pu{sub eff} with passive assays are presented and the effects of moderators and absorbers on sensitivities noted.

  11. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  12. Modulating temporal control of NF-kappaB activation: implications for therapeutic and assay selection.

    PubMed

    Klinke, David J; Ustyugova, Irina V; Brundage, Kathleen M; Barnett, John B

    2008-06-01

    The activation of transcription factor NF-kappaB (nuclear factor-kappaB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-kappaB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-kappaB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-kappaB activation. Both assays detected damped oscillatory behavior of NF-kappaB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-kappaB after LPS stimulation. DCPA is known to inhibit the production of two NF-kappaB-inducible cytokines, IL-6 and tumor necrosis factor alpha, by reducing but not completely abrogating NF-kappaB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-kappaB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-kappaB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery. PMID:18281385

  13. Modulating Temporal Control of NF-κB Activation: Implications for Therapeutic and Assay Selection

    PubMed Central

    Klinke, David J.; Ustyugova, Irina V.; Brundage, Kathleen M.; Barnett, John B.

    2008-01-01

    The activation of transcription factor NF-κB (nuclear factor-κB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-κB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-κB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-κB activation. Both assays detected damped oscillatory behavior of NF-κB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-κB after LPS stimulation. DCPA is known to inhibit the production of two NF-κB-inducible cytokines, IL-6 and tumor necrosis factor α, by reducing but not completely abrogating NF-κB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-κB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-κB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery. PMID:18281385

  14. A protein chip membrane-capture assay for botulinum neurotoxin activity

    SciTech Connect

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-12-15

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

  15. Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

    SciTech Connect

    Kuo, Chun-Chen; Chu, Chi-Yuan; Lin, Jing-Jer; Lo, Lee-Chiang

    2010-01-01

    Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

  16. [Detection of viable metabolically active yeast cells using a colorimetric assay].

    PubMed

    Růzicka, F; Holá, V

    2008-02-01

    The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts. PMID:18318392

  17. Activity, assay and target data curation and quality in the ChEMBL database.

    PubMed

    Papadatos, George; Gaulton, Anna; Hersey, Anne; Overington, John P

    2015-09-01

    The emergence of a number of publicly available bioactivity databases, such as ChEMBL, PubChem BioAssay and BindingDB, has raised awareness about the topics of data curation, quality and integrity. Here we provide an overview and discussion of the current and future approaches to activity, assay and target data curation of the ChEMBL database. This curation process involves several manual and automated steps and aims to: (1) maximise data accessibility and comparability; (2) improve data integrity and flag outliers, ambiguities and potential errors; and (3) add further curated annotations and mappings thus increasing the usefulness and accuracy of the ChEMBL data for all users and modellers in particular. Issues related to activity, assay and target data curation and integrity along with their potential impact for users of the data are discussed, alongside robust selection and filter strategies in order to avoid or minimise these, depending on the desired application. PMID:26201396

  18. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease.

    PubMed

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F

    2016-09-01

    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. PMID:27261892

  19. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    PubMed

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment. PMID:27208079

  20. Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

    PubMed Central

    Figuera-Losada, Mariana; Rojas, Camilo; Slusher, Barbara S.

    2014-01-01

    Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor κB, and mitogen-activated protein kinases. PMID:23945875

  1. A sensitive assay for the biosynthesis and secretion of MANF using NanoLuc activity.

    PubMed

    Norisada, Junpei; Hirata, Yoko; Amaya, Fumimasa; Kiuchi, Kazutoshi; Oh-hashi, Kentaro

    2014-07-11

    Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent neuronal cell death caused by certain stimuli. Accordingly, the molecular features of MANF have been intensively investigated since the reporting of its cytoprotective actions. In addition to the characterization of the transcriptional regulation of MANF under pathophysiological conditions, it is important to understand its intracellular transport and secretion after translation. In this study, we developed a convenient and quantitative assay to evaluate the post-translational regulation of MANF using NanoLuc, a highly active and small luciferase. We inserted NanoLuc after the putative signal peptide sequence (SP) of MANF to construct NanoLuc-tagged MANF (SP-NL-MANF). Similar to wild-type (wt) MANF, SP-NL-MANF was secreted from transiently transfected HEK293 cells in a time-dependent manner. The overexpression of mutant Sar1 or wild-type GRP78, which has been reported to decrease wt MANF secretion, also attenuated the secretion of SP-NL-MANF. Using INS-1 cells stably expressing SP-NL-MANF, we found that the biosynthesis and secretion of SP-NL-MANF can be evaluated quantitatively using only a small number of cells. We further investigated the effects of several stimuli responsible for the expression of ER stress-induced genes on the secretion of SP-NL-MANF from INS-1 cells. Treatment with thapsigargin and high potassium significantly increased NanoLuc activity in the culture medium, but serum withdrawal dramatically down-regulated luciferase activity both inside and outside of the cells. Collectively, these results demonstrate that our method for measuring NanoLuc-tagged MANF as a secretory factor is highly sensitive and convenient not only for characterizing post-translational regulation but also for screening useful compounds that may be used to treat ER stress-related diseases such as neurodegenerative disease, ischemia and diabetes. PMID:24845376

  2. A minimally invasive assay for individual assessment of the ATM/CHEK2/p53 pathway activity

    PubMed Central

    Kabacik, Sylwia; Ortega-Molina, Ana; Efeyan, Alejo; Finnon, Paul; Bouffler, Simon; Serrano, Manuel

    2011-01-01

    Ionizing radiation induces DNA Double-Strand Breaks (DSBs), which activate the ATM/CHEK2/p53 pathway leading to cell cycle arrest and apoptosis through transcription of genes including CDKN1A (p21) and BBC3 (PUMA). This pathway prevents genomic instability and tumorigenesis as demonstrated in heritable syndromes [e.g., Ataxia Telangiectasia (AT); Li-Fraumeni syndrome (LFS)]. Here, a simple assay based on gene expression in peripheral blood to measure accurately ATM/CHEK2/p53 pathway activity is described. The expression of p21, Puma and Sesn2 was determined in blood from mice with different gene copy numbers of Atm, Trp53 (p53), Chek2 or Arf and in human blood and mitogen stimulated T-lymphocyte (MSTL) cultures from AT, AT carriers, LFS patients and controls, both before and after ex vivo ionizing irradiation. Mouse Atm/Chek2/p53 activity was highly dependent on the copy number of each gene except Arf. In human MSTL, an AT case, AT carriers and LFS patients showed responses distinct from healthy donors. The relationship between gene copy number and transcriptional induction upon radiation was linear for p21 and Puma and correlated well with cancer incidence in p53 variant mice. This reliable blood test provides an assay to determine ATM/CHEK2/p53 pathway activity and demonstrates the feasibility of assessing the activity of this essential cancer protection pathway in simple assays. These findings may have implications for the individualized prediction of cancer susceptibility. PMID:21389785

  3. Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

    PubMed Central

    Dillingham, Mark S.; Tibbles, Katherine L.; Hunter, Jackie L.; Bell, Jason C.; Kowalczykowski, Stephen C.; Webb, Martin R.

    2008-01-01

    The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases. PMID:18599625

  4. Neutron Activation Analysis for the Demonstration of Amphibolite Rock-Weathering Activity of a Yeast

    PubMed Central

    Rades-Rohkohl, E.; Hirsch, P.; Fränzle, O.

    1979-01-01

    Neutron activation analysis was employed in a survey of weathering abilities of rock surface microorganisms. A yeast isolated from an amphibolite of a megalithic grave was found actively to concentrate, in media and in or on cells, iron and other elements when grown in the presence of ground rock. This was demonstrated by comparing a spectrum of neutron-activated amphibolite powder (particle size, 50 to 100 μm) with the spectra of neutron-activated, lyophilized yeast cells which had grown with or without amphibolite powder added to different media. The most active yeast (IFAM 1171) did not only solubilize Fe from the rock powder, but significant amounts of Co, Eu, Yb, Ca, Ba, Sc, Lu, Cr, Th, and U were also mobilized. The latter two elements occurred as natural radioactive isotopes in this amphibolite. When the yeast cells were grown with neutron-activated amphibolite, the cells contained the same elements. Furthermore, the growth medium contained Fe, Co, and Eu which had been solubilized from the amphibolite. This indicates the presence, in this yeast strain, of active rockweathering abilities as well as of uptake mechanisms for solubilized rock components. PMID:16345472

  5. A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts.

    PubMed

    Bøjstrup, Marie; Marri, Lucia; Lok, Finn; Hindsgaul, Ole

    2015-12-23

    Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format. PMID:26615836

  6. Antioxidant activity evaluation by physiologically relevant assays based on haemoglobin peroxidase activity and cytochrome c-induced oxidation of liposomes.

    PubMed

    Mot, Augustin C; Bischin, Cristina; Muresan, Bianca; Parvu, Marcel; Damian, Grigore; Vlase, Laurian; Silaghi-Dumitrescu, Radu

    2016-06-01

    Two new protocols for exploring antioxidant-related chemical composition and reactivity are described: one based on a chronometric variation of a haemoglobin ascorbate peroxidase assay and one based on cytochrome c-induced oxidation of lecithin liposomes. Detailed accounts are given on their design, application, critical correlations with established methods and mechanisms. These assays are proposed to be physiologically relevant and bring new information regarding a real sample, both qualitative and quantitative. The well-known assays used for evaluation of antioxidant (re)activity are revisited and compared with these new methods. Extracts of the Hedera helix L. are examined as test case, with focus on seasonal variation and on leaf, fruit and flower with respect to chromatographic, spectroscopic and reactivity properties. According to the set of assays performed, winter are the most antioxidant, followed by summer leaves, and then by flowers and fruits. PMID:26208459

  7. Standardization of a micro-cytotoxicity assay for human natural killer cell lytic activity.

    PubMed

    Mariani, E; Monaco, M C; Sgobbi, S; de Zwart, J F; Mariani, A R; Facchini, A

    1994-06-24

    Cytotoxicity assays are widely used to evaluate the functional activity of NK and T cells against tumour target cells and the release of radioactive sodium chromate from labelled target cells is still the most commonly used marker of target lysis in culture supernatants. We describe here the standardization of a micro-cytotoxicity test in which the number of cytolytic effector and tumour target cells have been decreased by a factor of 10. The release obtained by 500 tumour target cells was compared with the release obtained by 5000 target cells in the standard cytotoxicity assay for target:effector cell ratios from 1:1 to 1:100. Both gamma and beta emissions of the 51Cr isotope were evaluated to determine the assay release. The results obtained by the micro-cytotoxicity assay (500 target cells) were comparable to those of the standard assay (5000 target cells) and 51Cr release evaluation using the gamma counter was the most sensitive method of determining lytic activity using 500 tumour target cells. beta counter evaluation using solid phase scintillation was found to be a reproducible alternative method, even if the lytic curves cannot be compared with those obtained using the traditional method. PMID:8034970

  8. Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13.

    PubMed

    Gogia, Shobhit; Lo, Chi Y; Neelamegham, Sriram

    2015-01-01

    Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly

  9. Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

    PubMed Central

    Gogia, Shobhit; Lo, Chi Y.; Neelamegham, Sriram

    2015-01-01

    Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly

  10. VAPORIZATION TECHNIQUE TO MEASURE MUTAGENIC ACTIVITY OF VOLATILE ORGANIC CHEMICALS IN THE AMES/'SALOMELLA' ASSAY

    EPA Science Inventory

    The purpose of the research was to develop and characterize a sensitive test method to detect mutagenic activity of volatile liquid organic chemicals (i.e., volatiles) in the Ames/Salmonella assay. A Tedlar bag vaporization technique was developed which increased contact time bet...

  11. Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of a biochar made from switchgrass on four soil enzymes (ß- glucosidase, ß-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Inconsistent results from enzyme assays of char-amended soils s...

  12. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Valenti, D; Gagliardi, S; Passarella, S

    2000-11-01

    Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. PMID:11086257

  13. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gold standard assay for measuring the activity and typing of Clostridium botulinum neurotoxins is the mouse bioassay. The mouse bioassay is sensitive, robust and does not require specialized equipment. However, the mouse bioassay is slow, not practical for many settings and results in the death ...

  14. Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay to Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

    PubMed Central

    Perez, Jonas W.; Pantazides, Brooke G.; Watson, Caroline M.; Thomas, Jerry D.; Blake, Thomas A.; Johnson, Rudolph C.

    2015-01-01

    Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intra-spot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intra-spot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10-times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intra-spot variability without the need to control for initial sample volume, and enhances analyte stability. PMID:25955132

  15. Antioxidant Activity/Capacity Measurement. 3. Reactive Oxygen and Nitrogen Species (ROS/RNS) Scavenging Assays, Oxidative Stress Biomarkers, and Chromatographic/Chemometric Assays.

    PubMed

    Apak, Reşat; Özyürek, Mustafa; Güçlü, Kubilay; Çapanoğlu, Esra

    2016-02-10

    There are many studies in which the antioxidant potential of different foods have been analyzed. However, there are still conflicting results and lack of information as a result of unstandardized assay techniques and differences between the principles of the methods applied. The measurement of antioxidant activity, especially in the case of mixtures, multifunctional or complex multiphase systems, cannot be evaluated satisfactorily using a simple antioxidant test due to the many variables influencing the results. In the literature, there are many antioxidant assays that are used to measure the total antioxidant activity/capacity of food materials. In this review, reactive oxygen and nitrogen species (ROS/RNS) scavenging assays are evaluated with respect to their mechanism, advantages, disadvantages, and potential use in food systems. On the other hand, in vivo antioxidant activity (AOA) assays including oxidative stress biomarkers and cellular-based assays are covered within the scope of this review. Finally, chromatographic and chemometric assays are reviewed, focusing on their benefits especially with respect to their time saving, cost-effective, and sensitive nature. PMID:26689748

  16. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  17. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    PubMed

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  18. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay. PMID:27366781

  19. [Demonstration of C3-binding circulating immune complexes using Raji, conglutinin and anti-C3 assays--a critical review].

    PubMed

    Arndt, R

    1984-02-01

    There remains no doubt at the present time, that the appearance of circulating immune complexes in illness accompanying vasculitis and for glomerulonephritis correlates with the severity of disease. Moreover, immune complexes are of diagnostic importance where infections with a chronic development or neoplastic diseases are concerned. The choice of IC test system should incorporate their essential biological functions and identify those IC that activate the complement cascade both by the classical and the alternative route. The detection of IC bound C3 cleavage products (C3b, C3bi, C3d) represents the key to identification of a wide range of IC. Of the presently available methods Raji cell test, conglutinin- and anti C3-IC assay, on critical appraisal, the anti C3-IC assay represents the most applicable way of defining complement binding IC. The advantage of this system is that appreciable disturbances and limitations that influence other systems do not affect the antigen-antibody reaction which is the core of the anti C3 assay. PMID:6241918

  20. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos.

    PubMed

    Raftery, Tara D; Isales, Gregory M; Yozzo, Krystle L; Volz, David C

    2014-01-01

    Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants. PMID:24328182

  1. Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.

    PubMed

    Ferrer, Marc; Zuck, Paul; Kolodin, Garrett; Mao, Shi Shan; Peltier, Richard R; Bailey, Carolyn; Gardell, Stephen J; Strulovici, Berta; Inglese, James

    2003-06-01

    An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening. PMID:12729605

  2. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

    PubMed Central

    Oberhaus, S M; Newbold, J E

    1993-01-01

    Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host. Images PMID:8411359

  3. In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

    PubMed

    Lieberman, M M; Patterson, G M; Moore, R E

    2001-11-01

    In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration. PMID:11578805

  4. Plant compounds enhance the assay sensitivity for detection of active Bacillus cereus toxin.

    PubMed

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-03-01

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety. PMID:25767986

  5. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

    PubMed Central

    James, Dominic I.; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A.; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D.; Ogilvie, Donald J.

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

  6. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

    PubMed

    James, Dominic I; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D; Ogilvie, Donald J

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years. PMID:27610220

  7. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  8. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity.

    PubMed

    Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  9. Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

    PubMed Central

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-01-01

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety. PMID:25767986

  10. Assay of insulator enhancer-blocking activity with the use of transient transfection.

    PubMed

    Smirnov, N A; Didych, D A; Akopov, S B; Nikolaev, L G; Sverdlov, E D

    2013-08-01

    We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators. PMID:24228877

  11. Colorimetric assay for heterogeneous-catalyzed lipase activity: enzyme-regulated gold nanoparticle aggregation.

    PubMed

    Zhang, Wei; Tang, Yan; Liu, Jia; Jiang, Ling; Huang, Wei; Huo, Feng-Wei; Tian, Danbi

    2015-01-14

    Lipase is a neglected enzyme in the field of gold nanoparticle-based enzyme assays. This paper reports a novel colorimetric probe to rapidly visualize lipase activities by using Tween 20 functioned GNPs (Tween 20-GNPs) as a reporter. The present strategy hence could overcome the limitations caused by the heterogeneous interface in lipase assay. Catalytic hydrolytic cleavage of the ester bond in Tween 20-GNPs by lipase will trigger the rapid aggregation of GNPs at a high salt solution. The color change from red to purple could be used to sense the activity of lipase. The detection limit (3σ) is as low as 2.8 × 10-2 mg/mL. A preliminary enzyme activity screening was carried out for seven commercially purchased lipase samples. It also has been successfully applied to detecting lipase in fermentation broth of Bacillus subtilis without any pretreatment. PMID:25516269

  12. Application of a hemolysis assay for analysis of complement activation by perfluorocarbon nanoparticles

    PubMed Central

    Pham, Christine T.N.; Thomas, Dennis G.; Beiser, Julia; Mitchell, Lynne M.; Huang, Jennifer L.; Senpan, Angana; Hu, Grace; Gordon, Mae; Baker, Nathan A.; Pan, Dipanjan; Lanza, Gregory M.; Hourcade, Dennis E.

    2013-01-01

    Nanoparticles offer new options for medical diagnosis and therapeutics with their capacity to specifically target cells and tissues with imaging agents and/or drug payloads. The unique physical aspects of nanoparticles present new challenges for this promising technology. Studies indicate that nanoparticles often elicit moderate to severe complement activation. Using human in vitro assays that corroborated the mouse in vivo results we previously presented mechanistic studies that define the pathway and key components involved in modulating complement interactions with several gadolinium-functionalized perfluorocarbon nanoparticles (PFOB). Here we employ a modified in vitro hemolysis-based assay developed in conjunction with the mouse in vivo model to broaden our analysis to include PFOBs of varying size, charge and surface chemistry and examine the variations in nanoparticle-mediated complement activity between individuals. This approach may provide the tools for an in-depth structure-activity relationship study that will guide the eventual development of biocompatible nanoparticles. PMID:24211337

  13. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    PubMed

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-01

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations. PMID:12109482

  14. An Alternative Procedure for the Glucose Oxidase Assay of Glucose as Applied to the Lactase Activity Assay

    NASA Astrophysics Data System (ADS)

    Corbin Mullis, T.; Winge, Jeffery T.; Deal, S. Todd

    1999-12-01

    The glucose oxidase assay of glucose has been modified to eliminate the use of micropipets. The modification involves the use of disposable Pasteur pipets and a specified number of drops of each reagent. This simplified technique gives accurate and reproducible results.

  15. A Continuous Spectrophotometric Assay for APS Reductase Activity with Sulfite-Selective Probes

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) adenosine 5′-phosphosulfate (APS) reductase (EC number 1.8.4.10), (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of TB infection. Despite the importance of APR to Mtb, and other bacterial pathogens, current assay methods depend on use of [35S]-labeled APS or shunt AMP to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off-on” fluorescent levulinate-based probes. The APR activity can thus be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michelis-Menten kinetic constants (Km, kcat, kcat/Km) and apparent inhibition constant (Ki) for adenosine 5′-diphosphate (ADP), which compared favorably to values obtained in the gold-standard radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer. PMID:23711725

  16. Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins

    PubMed Central

    Nicklisch, Sascha C.T.; Waite, J. Herbert

    2014-01-01

    The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility:•Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.•Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.•0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs. PMID:25530949

  17. Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

    PubMed Central

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q.; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

  18. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    PubMed

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

  19. Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    Roberts, Julia; Ling, Yan; Quezada, Landys Lopez; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Warren, J. David; Nathan, Carl

    2015-01-01

    The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic. PMID:26239979

  20. Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis.

    PubMed

    Gold, Ben; Roberts, Julia; Ling, Yan; Quezada, Landys Lopez; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Warren, J David; Nathan, Carl

    2015-10-01

    The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days-about 2 weeks sooner than required to count CFU-fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic. PMID:26239979

  1. Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth.

    PubMed

    Garcia, Eugenio José; Oldoni, Tatiane Luiza Cadorin; Alencar, Severino Matias de; Reis, Alessandra; Loguercio, Alessandro D; Grande, Rosa Helena Miranda

    2012-01-01

    The aim of this study was to assess, using the DPPH assay, the antioxidant activity of several substances that could be proposed to immediately revert the problems caused by bleaching procedures. The percentage of antioxidant activity (AA%) of 10% ascorbic acid solution (AAcidS), 10% ascorbic acid gel (AAcidG), 10% sodium ascorbate solution (SodAsS), 10% sodium ascorbate gel (SodAsG), 10% sodium bicarbonate (Bicarb), Neutralize(®) (NE), Desensibilize(®) (DES), catalase C-40 at 10 mg/mL (CAT), 10% alcohol solution of alpha-tocopherol (VitE), Listerine(®) (LIS), 0.12% chlorhexidine (CHX), Croton Lechleri (CL), 10 % aqueous solution of Uncaria Tomentosa (UT), artificial saliva (ArtS) and 0.05% sodium fluoride (NaF) was assessed in triplicate by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical assay. All substances exhibited antioxidant activity, except for CL. AAcidS, AAcidG and VitE exhibited the highest AA% (p<0.05). On the contrary, CHX, NE, LIS and NaF showed the lowest AA% (p<0.05). In conclusion, AAcidS, AAcidG, SodAsS, SodAsG and VitE presented the highest antioxidant activity among substances tested in this study. The DPPH assay provides an easy and rapid way to evaluate potential antioxidants. PMID:22460310

  2. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  3. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.

    PubMed

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-08-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  4. A description of an HPLC assay of coproporphyrinogen III oxidase activity in mononuclear cells.

    PubMed

    Gross, U; Gerlach, R; Kühnel, A; Seifert, V; Doss, M O

    2003-01-01

    Coproporphyrinogen III oxidase is deficient in hereditary coproporphyria. An activity assay for this enzyme in mononuclear cells, besides the preparation of the substrate, are presented. The separation conditions for the product of the test protoporphyrin IX by gradient, reversed-phase high-performance liquid chromatography are given. The normal value from mononuclear cells of healthy volunteers was 138 +/- 21 pkat/g total soluble protein (mean +/- SD). The enzyme activity of a family with hereditary coproporphyria was measured. The gene carriers exhibit a specific coproporphyrinogen III oxidase activity of 61-90 pkat/g total soluble protein. PMID:14605502

  5. Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay.

    PubMed

    Hannig, Christian; Spitzmüller, Bettina; Hannig, Matthias

    2009-03-01

    Lysozyme is among the most protective enzymes in the pellicle layer. The aim of the present study was to establish a precise fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle. For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 min) on buccal and palatal sites. The enzymatic assay was based on hydrolysis of cell walls from Micrococcus lysodeicticus linked to a fluorogenic substance. When the substrate is hydrolysed, a fluorescing product is released. Furthermore, the effects of chlorhexidine and black tea on lysozyme in the in situ pellicle were investigated. The fluorimetric method allowed direct determination of the enzyme activity with the slab inside the well of a microtiter plate. The mean immobilised activity over all samples amounted to 68.67 +/- 27.35 U/cm(2) (desorbed activity = 46.76 +/- 21.18 U/cm(2)). The enzyme activity exposed at the pellicles' surfaces increased in a time-dependant manner and showed a Michaelis-Menten kinetic. Chlorhexidine and black tea reduced lysozyme activity of the in situ pellicle significantly. After rinsing with tea or chlorhexidine, V(max) was reduced, whereas K(m) remained unaffected indicating a negative allosteric effect of the V type. The fluorimetric method is appropriate for determination of pellicle lysozyme activity. The influence of effectors on immobilised lysozyme activity can be monitored. PMID:18810509

  6. Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test

    PubMed Central

    2013-01-01

    Background Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as “rattle or rattlesnake” and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of the present study was to investigate the estrogenic potential of C. pallida leaves by means of the Recombinant Yeast Assay (RYA), seeking an alternative for estrogen replacement therapy during menopause; and to reflect on the safe use of natural products to assess the mutagenic activity of the crude extract from C. pallida leaves, the dichloromethane fraction and stigmasterol by means of the Ames test. Methods The recombinant yeast assay with the strain BY4741 of Saccharomyces cerevisiae, was performed with the ethanolic extract, dichloromethane fraction and stigmasterol isolated from the leaves of C. pallida. Mutagenic activity was evaluated by the Salmonella/microsome assay (Ames test), using the Salmonella typhimurium tester strains TA100, TA98, TA97 and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. Results All samples showed estrogenic activity, mainly stigmasterol. The ethanolic extract from C. pallida leaves showed mutagenic activity in the TA98 strain (-S9), whereas dichloromethane fraction and stigmasterol were found devoid of activity. Conclusion Considering the excellent estrogenic activity performed by stigmasterol in the RYA associated with the absence of mutagenic activity when evaluated by the Ames test, stigmasterol becomes a strong candidate to be used in hormone replacement therapy during menopause. PMID:24134316

  7. Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor-reporter gene assays.

    PubMed

    Balaguer, P; Joyeux, A; Denison, M S; Vincent, R; Gillesby, B E; Zacharewski, T

    1996-02-01

    In vitro recombinant receptor-reporter gene assays have been used to assess and rank the potency of chemicals and complex mixtures suspected of possessing estrogen and (or) aryl hydrocarbon receptor (AhR) mediated activity. The environmental estrogen (E2) bioassay consists of a Gal4-human estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) that have been stably integrated into HeLa cells. The assay exhibits 10-fold induction in luciferase reporter gene activity following treatment with 1 nM 17 beta-estradiol and has a detection limit of approximately 5 pg of 17 beta-estradiol/mL. The AhR bioassay uses Hepa 1c1c7 wild-type cells transiently transfected with a dioxin response element regulated luciferase reporter gene. These assays were used to assess the estrogen and dioxin-like activities of naringenin, atrazine, and simazine and complex mixtures such as pulp and paper mill black liquor and urban air particulates. The activities of these chemicals and complex mixtures are confirmed using the pure antiestrogen ICI 164,384 and in in vitro gel retardation assays. Results of this study demonstrate the utility of in vitro recombinant receptor-reporter gene assays in identifying and assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures. PMID:8723035

  8. Highly sensitive assay for acetylcholinesterase activity and inhibition based on a specifically reactive photonic nanostructure.

    PubMed

    Tian, Tian; Li, Xuesong; Cui, Jiecheng; Li, Jian; Lan, Yue; Wang, Chen; Zhang, Meng; Wang, Hui; Li, Guangtao

    2014-09-10

    Assays for acetylcholinesterase (AChE) with high sensitivity and high selectivity as well as facile manipulation have been urgently required in various fields. In this work, a reaction-based photonic strategy was developed for the efficient assay of AChE activity and inhibition based on the synergetic combination of the specific thiol-maleimide addition reaction with photonic porous structure. It was found that various applications including detection of AChE activity, measurement of the related enzymatic kinetics, and screening of inhibitors could be efficiently implemented using such strategy. Remarkably, the unique photonic nanostructure endows the constructed sensing platform with high sensitivity with a limit of detection (LOD) of 5 mU/mL for AChE activity, high selectivity, and self-reporting signaling. Moreover, the label-free solid film-based sensing approach described here has advantages of facile manipulation and bare-eye readout, compared with conventional liquid-phase methods, exhibiting promising potential in practical application for the AChE assay. PMID:25130420

  9. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma

    PubMed Central

    Goiffon, Reece J.; Martinez, Sara C.; Piwnica-Worms, David

    2015-01-01

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l−1 MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders. PMID:25666092

  10. Lack of biological activity of preproendothelin (110-130) in several endothelin assays

    SciTech Connect

    Cade, C.; Lumma, W.C. Jr.; Mohan, R.; Rubanyi, G.M.; Parker-Botelho, L.H. )

    1990-01-01

    A 21 amino acid peptide containing the prepropendothelin sequence from amino acids 110 to 130 and two intrachain disulfide bonds was synthesized and tested for biological activity in the following endothelin assays: (1) a competition binding assay using ({sup 125}I)ET-1 and dog heart membranes, (2) three RIA's using {sup 125}-ET-1, -2 and -3 and the respective anti-ET rabbit antisera; and (3) a contractile activity bioassay using hamster aortic rings. The synthetic peptide which has been referred to as the endothelin-like peptide occurs 36 amino acids C-terminal to endothelin in the prepro-protein sequence. It contains only 40% sequence homology to the three endothelin isoforms, but has the same sequence and cyclization pattern of cysteines at positions 1, 3, 11 and 15. Despite the overall similarity in secondary structure to the three isoforms of endothelin and sarafotoxin S6b, preproendothelin (110-130) had no activity in any of the assays when tested at concentrations of 10{sup {minus}10}M to 10{sup {minus}5}M.

  11. Reproducible quantification of osteoclastic activity: characterization of a biomimetic calcium phosphate assay.

    PubMed

    Maria, Salwa M; Prukner, Christiane; Sheikh, Zeeshan; Mueller, Frank; Barralet, Jake E; Komarova, Svetlana V

    2014-07-01

    Osteoclasts are responsible for bone and joint destruction in rheumatoid arthritis, periodontitis, and osteoporosis. Animal tusk slice assays are standard for evaluating the effect of therapeutics on these cells. However, in addition to batch-to-batch variability inherent to animal tusks, their use is clearly not sustainable. Our objective was to develop and characterize a biomimetic calcium phosphate assay based on the use of phase pure hydroxyapatite coated as a thin film on the surface of culture plates, to facilitate the reproducible quantification of osteoclast resorptive activity. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using a pro-resorptive cytokine RANKL (50 ng/mL). No change in substrate appearance was noted after culture with media without cells, or undifferentiated monocytes. Only in the presence of osteoclasts localized areas of calcium phosphate dissolution were observed. The total area resorbed positively correlated with the osteoclast numbers (R(2) = 0.99). The resorbed area was significantly increased by the addition of RANKL, and decreased after application of known inhibitors of osteoclast resorptive activity, calcitonin (10 μM), or alendronate (100 μM). Thus, calcium phosphate coated substrates allow reliable monitoring of osteoclast resorptive activity and offer an alternative to animal tusk slice assays. PMID:24259122

  12. Assessment of estrogenic activity in Tunisian water and wastewater by E-screen assay.

    PubMed

    Limam, Atef; Talorete, Terence P N; Ali, Mourad Ben Sik; Kawano, Mitsuko; Jenhani, Amel Ben Rejeb; Abe, Yukuo; Ghrabi, Ahmed; Isoda, Hiroko

    2007-01-01

    Wastewater and surface water samples from three wastewater treatment plants (WWTPs) and three rivers in Tunisia were assayed for estrogenic activity using the E-screen assay and enzyme-linked immunosorbent assay (ELISA). Results showed that all the Tunisian raw wastewater samples as well as the Roriche river water sample induced a strong proliferative response in human MCF-7 breast cancer cells. Tunisian raw wastewater had an average 17beta-estradiol content of 2,705.4 pg/ml, whereas that of the Roriche river was 36.7 pg/ml, which is sufficient for inducing endocrine-mediated responses in aquatic organisms. Results further showed that the Mornag WWTP, which uses the activated-sludge treatment system, has a higher estrogen removal efficiency than the stabilization ponds of the Gammart and pilot WWTPs. This study, which is the first of such studies in Tunisia, and probably the first in the North African region, underscores the need to detect and monitor the estrogenic activity of water and wastewater, given the scarcity of water in Tunisia and the detrimental impact of endocrine-disrupting compounds on the physiology of both animals and humans. PMID:18382414

  13. Estimation of the dermal carcinogenic activity of petroleum fractions using a modified Ames assay.

    PubMed

    Blackburn, G R; Deitch, R A; Schreiner, C A; Mehlman, M A; Mackerer, C R

    1984-10-01

    The Ames Salmonella/microsomal activation mutagenesis assay has been adapted to improve sensitivity to complex hydrocarbon mixtures produced by the refining of petroleum. Extraction of oil samples with dimethyl sulfoxide produces aqueous-compatible solutions that more easily interact with the tester bacteria. These extracts, therefore, produce higher revertant values than do equivalent volumes of oil delivered neat or dissolved in organic solvent. Parallel increases in the liver microsomal S-9 concentration further improve the sensitivity of the assay, allowing detection of mutagenicity in otherwise inactive samples. The effect of increased microsomal fraction from rodent liver is apparently attributable to the higher levels of activating enzymes rather than to the concomitant increase in the overall hydrophobicity of the test system. The modified assay has been used to rank thirteen petroleum-derived oils and a corn oil control for relative mutagenic activity. This ranking closely correlates (r = 0.97) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays. PMID:6401126

  14. Is there a role for estrogen activity assays? Recombinant cell bioassay for estrogen: Development and applications.

    PubMed

    Klein, Karen Oerter

    2015-07-01

    There are many questions which cannot be answered without a very sensitive estradiol assay. A recombinant cell bioassay (RCBA) for estradiol was developed in 1994. The sensitivity of the bioassay is 0.02-0.2 pg/ml (0.07-0.7 pmol/L), more than 20 times more sensitive than commercial RIAs and 10 times more sensitive than newer mass spectrometry assays. The RCBA for estradiol opened the door to study low levels of estradiol equivalents (EE) across the physiological spectrum of life from prepubertal children through menopause and across the spectrum from normal physiology, in boys as well as girls, to pathology, including: premature thelarche; estradiol suppression in children treated with GnRH analogues for precocious puberty; aromatase inhibition in boys with growth hormone deficiency; the differences between oral and transdermal routes of estrogen administration in girls with Turner's syndrome; women with breast cancer treated with aromatase inhibitors; and women with urogenital atrophy treated with low dose vaginal estrogen. A bioassay also allows study of endocrine disruptors, like phytoestrogens and other environmental compounds, which are relevant to public health and alternative medicine options. This paper reviews the assay and the last 20 years of applications. A bioassay for estrogen has a role because measuring biological effect is theoretically useful, increasing the understanding of physiology in addition to biochemical levels, giving different information than other assays, and opening the door to measure very low levels of estrogen activity in both humans and the environment. PMID:25159103

  15. Are the Opsonophagocytic Activities of Antibodies in Infant Sera Measured by Different Pneumococcal Phagocytosis Assays Comparable?

    PubMed Central

    Väkeväinen, Merja; Jansen, Wouter; Saeland, Eirikur; Jonsdottir, Ingileif; Snippe, Harm; Verheul, Andre; Käyhty, Helena

    2001-01-01

    Host protection against Streptococcus pneumoniae is mainly mediated by opsonin-dependent phagocytosis. Several techniques for measuring opsonophagocytic activity (OPA) of antibodies to S. pneumoniae have been standardized and used. These include the viable cell-assay, flow-cytometric assays, and an assay utilizing radiolabeled bacteria. Using these different methods, we measured the OPA of antibodies to S. pneumoniae types 6B and 19F from the sera of infants immunized with a pneumococcal conjugate vaccine, PncCRM. Generally, the results obtained by the various techniques correlated well, although serotype-specific differences were found (6B, r = 0.78 to 0.95, P < 0.001; 19F, r = 0.50 to 0.84, P < 0.001). The same serotype-specific differences were observed for the relationship between the concentrations of specific immunoglobulin G antibodies measured by enzyme immunoassay and the OPA. Since the sensitivities of the OPA assays differed, the most prominent discrepancies between the techniques were found at low antibody concentrations. PMID:11238223

  16. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

    PubMed

    Ouellette, Steven B; Noel, Brett M; Parker, Laurie L

    2016-01-01

    Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. PMID:27598410

  17. A nanostructure-initiator mass spectrometry-based enzyme activity assay

    SciTech Connect

    Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren; Yannone, Steven M.; Wong, Chi-Huey; Siuzdak, Gary

    2008-03-10

    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme

  18. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    PubMed

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets. PMID:26691755

  19. Sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin

    SciTech Connect

    Sawada, M.; Horikoshi, T.; Masada, M.; Akino, M.; Sugimoto, T.; Matsuura, S.; Nagatsu, T.

    1986-04-01

    A highly sensitive and simple assay for the activity of GTP cyclohydrolase I (EC 3.5.4.16) was established using a newly developed radioimmunoassay. D-erythro-7,8-Dihydroneopterin triphosphate formed from GTP by GTP cyclohydrolase I was oxidized by iodine and dephosphorylated by alkaline phosphatase to D-erythro-neopterin, and quantified by a radioimmunoassay for D-erythro-neopterin. This method was highly sensitive and required only 0.2 mg of rat liver tissues for the measurement of the activity. It was reproducible and can be applied for the simultaneous assay of many samples. The activity of GTP cyclohydrolase I was measured in several rat tissues. For example, the enzyme activity in rat striatum (n = 5) was 13.7 +/- 1.5 pmol/mg protein per hour (mean +/- SE), and agreed well with those obtained by high-performance liquid chromatography with fluorescence detection. The activity in the autopsy human brains (caudate nucleus) was measured by this new method for the first time. The activity in the caudate nucleus from parkinsonian patients (n = 6) was 0.82 +/- 0.56 pmol/mg protein per hour which was significantly lower than the control value, 4.22 +/- 0.43 pmol/mg protein per hour (n = 10).

  20. [Detection of endotoxin activity in water environment and analysis of influence factors for TAL assay].

    PubMed

    Zhang, Can; Liu, Wen-jun; Zhang, Ming-lu; Tian, Fang; Sun, Wen; Qian, Ling-jia; Zhan, Rui

    2013-09-01

    Endotoxins, derived from cell walls of most Gram-negative bacteria and some cyanobacteria, are common pyrogen and highly immunogenic molecules, and related to many diseases. In this paper, a detection method for endotoxin activity in water environment using kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL) was established, the influence of pH and salts on TAL assay was investigated. Results showed that it was favorable for TAL assay in the pH range of 6.0-8.4, at low pHs, inhibition results were observed and opposite results were obtained at high pHs. The pH should be adjusted by Tris-HCl (pH = 7.4) buffer before the endotoxin detection. No significant interference was shown in the detection of water containing NaCl, Na2SO4, CaCl2, MgCl2 and KCl with a concentration of less than 50 mg x L(-1), however, the inhibition occurred at the concentration up to 1000-10,000 mg x L(-1). Only 2. 5 mg x L(-1) of FeCl, Fe2(SO4)3, AlCl3 and Al2 (SO4)3 caused significant inhibition. Endotoxin activities of ultrapure water, tap water and recreational water were detected by TAL assay, and their endotoxin activities were < 0.06 EU x mL(-1), 0.46 EU x mL(-1) and 432. 68 EU x mL(-1), respectively. PMID:24288979

  1. "Singing in the Tube"--audiovisual assay of plant oil repellent activity against mosquitoes (Culex pipiens).

    PubMed

    Adams, Temitope F; Wongchai, Chatchawal; Chaidee, Anchalee; Pfeiffer, Wolfgang

    2016-01-01

    Plant essential oils have been suggested as a promising alternative to the established mosquito repellent DEET (N,N-diethyl-meta-toluamide). Searching for an assay with generally available equipment, we designed a new audiovisual assay of repellent activity against mosquitoes "Singing in the Tube," testing single mosquitoes in Drosophila cultivation tubes. Statistics with regression analysis should compensate for limitations of simple hardware. The assay was established with female Culex pipiens mosquitoes in 60 experiments, 120-h audio recording, and 2580 estimations of the distance between mosquito sitting position and the chemical. Correlations between parameters of sitting position, flight activity pattern, and flight tone spectrum were analyzed. Regression analysis of psycho-acoustic data of audio files (dB[A]) used a squared and modified sinus function determining wing beat frequency WBF ± SD (357 ± 47 Hz). Application of logistic regression defined the repelling velocity constant. The repelling velocity constant showed a decreasing order of efficiency of plant essential oils: rosemary (Rosmarinus officinalis), eucalyptus (Eucalyptus globulus), lavender (Lavandula angustifolia), citronella (Cymbopogon nardus), tea tree (Melaleuca alternifolia), clove (Syzygium aromaticum), lemon (Citrus limon), patchouli (Pogostemon cablin), DEET, cedar wood (Cedrus atlantica). In conclusion, we suggest (1) disease vector control (e.g., impregnation of bed nets) by eight plant essential oils with repelling velocity superior to DEET, (2) simple mosquito repellency testing in Drosophila cultivation tubes, (3) automated approaches and room surveillance by generally available audio equipment (dB[A]: ISO standard 226), and (4) quantification of repellent activity by parameters of the audiovisual assay defined by correlation and regression analyses. PMID:26412058

  2. Evaluation of estrogenic activities of aquatic herbicides and surfactants using an rainbow trout vitellogenin assay.

    PubMed

    Xie, Lingtian; Thrippleton, Kelly; Irwin, Mary Ann; Siemering, Geoffrey S; Mekebri, Abdou; Crane, David; Berry, Kevin; Schlenk, Daniel

    2005-10-01

    Estrogenic potencies of four herbicides (triclopyr, 2,4-dichlorophenoxyacetic acid (2,4-D), diquat dibromide, glyphosate), two alkylphenol ethoxylate-containing surfactants (R-11 and Target Prospreader Activator (TPA)), and the binary mixture of surfactants with the herbicides were evaluated using an in vivo rainbow trout vitellogenin assay. Juvenile rainbow trout exposed to 2,4-D (1.64 mg/l) for 7 days had a 93-fold increase in plasma vitellogenin (Vtg) levels compared with untreated fish, while rainbow trout exposed to other pesticides alone did not show elevated vitellogenin levels compared to the control fish. When combined with surfactants, trends indicated enhanced estrogenicity for all combinations, but only 2,4-D and triclopyr caused significant induction of Vtg. Concentration-response studies demonstrated that the lowest observed effect concentrations (LOECs) for 2,4-D and triclopyr were 0.164 mg/l and 1 mg/l, respectively. In terms of measured 4-nonylphenol (4-NP), the LOECs of R-11 and TPA were 20 micro/l and 9.5 microg/l, respectively. Binary mixtures of TPA and 2,4-D showed a greater than additive estrogenic response at the lowest concentrations tested, but a less than additive response at the highest combined concentrations. Binary mixtures of TPA with triclopyr also caused greater than additive Vtg responses in two middle concentrations when compared to TPA or triclopyr alone. When trout were exposed to water collected from a site where triclopyr was used in combination with TPA, a concentration-dependent increase in Vtg expression was observed. Measured values of 4-NP were 3.7 microg/l, and triclopyr concentrations were below detection (<5 ng/l). Estradiol equivalents (EEQs) of the lake water were calculated from an estradiol concentration-response curve and were similar (8.5 +/- 7.7 ng/l) to the mean values for the combined triclopyr + TPA treatments (9.9-12.2 ng/l) in the laboratory, suggesting the estrogenicity of the water may have been due to

  3. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  4. Highly Sensitive and Selective Immuno-capture/Electrochemical Assay of Acetylcholinesterase Activity in Red Blood Cells: A Biomarker of Exposure to Organophosphorus Pesticides and Nerve Agents

    SciTech Connect

    Chen, Aiqiong; Du, Dan; Lin, Yuehe

    2012-02-09

    Acetylcholinesterase (AChE) enzyme activity in red blood cells (RBCs) is a useful biomarker for biomonitoring of exposures to organophosphorus (OP) pesticides and chemical nerve agents. In this paper, we reported a new method for AChE activity assay based on selective immuno-capture of AChE from biological samples followed by enzyme activity assay of captured AChE using a disposable electrochemical sensor. The electrochemical sensor is based on multiwalled carbon nanotubes-gold nanocomposites (MWCNTs-Au) modified screen printed carbon electrode (SPCE). Upon the completion of immunoreaction, the target AChE (including active and inhibited) is captured onto the electrode surface and followed by an electrochemical detection of enzymatic activity in the presence of acetylthiocholine. A linear response is obtained over standard AChE concentration range from 0.1 to 10 nM. To demonstrate the capability of this new biomonitoring method, AChE solutions dosed with different concentration of paraoxon were used to validate the new AChE assay method. AChE inhibition in OP dosed solutions was proportional to its concentration from 0.2 to 50 nM. The new AChE activity assay method for biomonitoring of OP exposure was further validated with in-vitro paraoxon-dosed RBC samples. The established electrochemical sensing platform for AChE activity assay not only avoids the problem of overlapping substrate specificity with esterases by using selective antibody, but also eliminates potential interference from other electroactive species in biological samples. It offers a new approach for sensitive, selective, and rapid AChE activity assay for biomonitoring of exposures to OPs.

  5. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel . E-mail: octave@nchm.ucl.ac.be

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.

  6. Prompt activation of telomerase by chemical carcinogens in rats detected with a modified TRAP assay.

    PubMed

    Miura, M; Karasaki, Y; Abe, T; Higashi, K; Ikemura, K; Gotoh, S

    1998-05-01

    The maintenance of telomere length is crucial for survival of cells. Telomerase is an RNA-containing reverse transcriptase, which is responsible for elongation of shortened telomeres. Telomerase reactivation has been suggested to be involved in malignant progressions. To study on the involvement of telomerase activation in in vivo carcinogenesis, we first modified the original TRAP assay by changing the primer designs and the labeling method of PCR products to an end-labeling method. Second, we investigated the activation of telomerase in different organs after treatments of rats with various chemical carcinogens. Very early after the beginning of the treatment, telomerase activity in the liver, kidney, and lung was increased. In most cases, telomerase activation occurred in the primary or favorite target organs. The present results suggest that telomerase activation occurs promptly when animals are exposed to chemical carcinogens, which may contribute to in vivo chemical carcinogenesis. PMID:9600060

  7. A high content assay for biosensor validation and for examining stimuli that affect biosensor activity

    PubMed Central

    Slattery, Scott D.; Hahn, Klaus M.

    2015-01-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor’s maximally activated and inactivated state, and examine response to specific proteins. This involves considerable labor and expense, as expression conditions must be optimized to saturate the biosensor with the regulator, and multiple replicates and controls are required. We describe here a protocol for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays allows visual inspection (eg for cell health and biosensor or regulator localization). Optimization of single chain and dual chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for variations in upstream molecules. PMID:25447074

  8. A fluorescence-based assay for Core 1 β3galactosyltransferase (T-synthase) activity.

    PubMed

    Ju, Tongzhong; Cummings, Richard D

    2013-01-01

    Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6-10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays. PMID:23765650

  9. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    PubMed

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  10. Expression of human endostatin in larvae of silkworm (Bombyx mori) and in vitro activity assays.

    PubMed

    Yongfeng, Jin; Yingfei, Wang; Zhenhong, Zhu; Yaozhou, Zhang

    2002-08-01

    Human endostatin is a novel antiangiogenic molecule, which can inhibit the proliferation and development of new blood vessels, and experimentally can cause nearly complete regression of established tumors. In this paper, the cDNA encoding human endostatin was cloned into a baculovirus shuttle vector pBacPAK8 and co-infected with linearized Bm-BacPAK6 DNA into and BmN cells. The recombinant virus was screened and identified by PCR, DNA and RNA dot hybridization, and ELISA assay. The recombinant endostatin was expressed in culture cells, and the larvae and pupa of silkworm by inoculation of recombinant virus. The biological activity assay showed that the expression product in larvae was over 150 microg/ml, about 50-fold higher than that expressed in cultured cells. SDS-PAGE and Western blotting analysis showed a pattern of molecular weight of about 20 kDa. The bio-activity of the protein product was determined by human umbilical vein endothelial cells (ECV304) proliferation test in vitro and the chick chorioallantoic membrane (CAM) vascular inhibition test. Endostatin showed significant inhibitory effect on endothelial cells in a dose-dependent manner. Silkworm-produced endostatin induced apoptosis of endothelial cells and also inhibited angiogenesis in the CAM assay. Combination regimen using angiostatin and endostatin showed more than additive effect in angiogenic inhibition and increasing apoptosis when compared with treatment with the individual antiangiogenic protein. PMID:12186748

  11. A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators.

    PubMed

    Schumacher, Charlotte Helene; Körschen, Heinz G; Nicol, Christopher; Gasser, Carlos; Seifert, Reinhard; Schwärzel, Martin; Möglich, Andreas

    2016-01-01

    As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal definition, reversibility, and noninvasiveness. Sensory photoreceptors serve as genetically encoded, light-regulated actuators and hence embody the cornerstone of optogenetics. To expand the scope of optogenetics, ever more naturally occurring photoreceptors are being characterized, and synthetic photoreceptors with customized, light-regulated function are being engineered. Perturbational control over intracellular cyclic-nucleotide-monophosphate (cNMP) levels is achieved via sensory photoreceptors that catalyze the making and breaking of these second messengers in response to light. To facilitate discovery, engineering and quantitative characterization of such light-regulated cNMP actuators, we have developed an efficient fluorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantified with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-light-activated LAPD. Key benefits include potential for parallelization and automation, as well as suitability for both purified enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism. PMID:26965118

  12. Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay.

    PubMed

    Fic, Anja; Žegura, Bojana; Gramec, Darja; Mašič, Lucija Peterlin

    2014-10-01

    The present study investigated and compared the estrogenic and androgenic activities of the three different classes of environmental pollutants and their metabolites using the XenoScreen XL YES/YAS assay, which has advantages compared with the original YES/YAS protocol. Contrary to the parent brominated flame retardants TBB and TBPH, which demonstrated no or very weak (anti)estrogenic or (anti)androgenic activities, their metabolites, TBBA and TBMEPH, exhibited anti-estrogenic (IC50 for TBBA=31.75 μM and IC50 for TBMEPH=0.265 μM) and anti-androgenic (IC50 for TBBA=73.95 μM and IC50 for TBMEPH=2.92 μM) activities. These results reveal that metabolism can enhance the anti-estrogenic and anti-androgenic effects of these two novel brominated flame retardants. Based on the activities of BPAF, BPF, BPA and MBP, we can conclude that the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti)androgenic assays that are reported in the literature. For BPA, it was confirmed previously that the metabolite formed after an ipso-reaction (hydroxycumyl alcohol) exhibited higher estrogenic activity compared with the parent BPA, but this was not confirmed for BPAF and BPF ipso-metabolites, which were not active in the XenoScreen YES/YAS assay. Among the substituted BPA analogues, bis-GMA exhibited weak anti-estrogenic activity, BADGE demonstrated weak anti-estrogenic and anti-androgenic activities (IC50=13.73 μM), and the hydrolysed product BADGE·2H2O demonstrated no (anti)estrogenic or (anti)androgenic activities. PMID:25048928

  13. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  14. Arsenite as the probable active species in the human carcinogenicity of arsenic: Mouse micronucleus assays on Na and K arsenite, orpiment, and Fowler's solution

    SciTech Connect

    Tinwell, H.; Ashby, J. ); Stephens, S.C. )

    1991-11-01

    Sodium arsenite, potassium arsenite, and Fowler's solution (arsenic trioxide dissolved in potassium bicarbonate) are equally active in the mouse bone marrow micronucleus assay ({approximately} 10 mg/kg by IP injection). The natural ore orpiment (principally As{sub 2}S{sub 3}) was inactive despite blood levels of arsenic of 300 to 900 mg/mL in treated mice at 24 hr. Sodium arsenite was active in three strains of mice. It is suggested that the human lung cancer observed among arsenic ore smelters and the skin cancer among people exposed therapeutically to Fowler's solution, have, as their common origin, the genotoxic arsenite ion AsO{sub 2}{sup {minus}}. The difficulty experienced when attempting to demonstrate rodent carcinogenicity for derivatives of arsenic suggests that the bone marrow micronucleus assay may act as a useful assay for potentially carcinogenic arsenic derivatives.

  15. Determination of Rab5 activity in the cell by effector pull-down assay.

    PubMed

    Qi, Yaoyao; Liang, Zhimin; Wang, Zonghua; Lu, Guodong; Li, Guangpu

    2015-01-01

    Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins. PMID:25800849

  16. Research on the chemical inactivation of antibiotic activity in assays of sterility and contamination of pharmaceuticals.

    PubMed

    Negretti, F; Casetta, P

    1991-01-01

    Membrane filtration, frequently used for removing antibacterial activity in assays of sterility and contamination of the antibiotics, presents the drawback of adsorption of antibiotic to membrane. The washing with large volumes of peptone water removes partially interferences with microbial growth. We evaluated the inactivating action of some chemical substances (albumin, calcium pantothenate, heparin, hydroxylamine, tri-valent iron) on the antimicrobial activity of membranes employed for antibiotic filtration. The results are not positive for the use of chemical substances in the antibiotic activity neutralization. In fact the per cent reduction of inhibition zones ranges from -61.5% to +20.0% and the inhibiting activity on the growth of colony forming units (CFU) oscillates from 89.6% to 100%. Discovery of new neutralizing substances and severe measures of asepsis in pharmaceutical production are recommended. PMID:12041793

  17. Comparative analysis of cholinesterase activities in food animals using modified Ellman and Michel assays

    PubMed Central

    Askar, Kasim Abass; Kudi, A. Caleb; Moody, A. John

    2011-01-01

    This study investigated correlations between modified Ellman and Michel assay methods for measuring cholinesterase (ChE) activities. It also established a foundation for the applicability of measuring ChE activities in food animal species as biochemical biomarkers for evaluating exposure to and effects of organophosphorus and carbamate pesticides. Measuring ChE activities in blood and tissue is currently the most important method of confirming the diagnosis of such exposure. The study also characterized the level of ChE activity in the selected organs/tissues of these animals and determined the best organ/tissue in which to measure ChE activity. The ChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with ethylenediamine tetra-acetic acid (EDTA). Of the different tissues tested, the mean of ChE activities was found to be highest in tissue from liver, followed by lung, muscle, kidney, and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle, and heart. The highest activities of ChE were found in pigs, followed by cattle and sheep. There was no significant difference between the modified Ellman and Michel method, but the percentage coefficient of variance (%CV) values were higher when the Michel method was used. PMID:22468023

  18. Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

    PubMed Central

    Lascombe, I; Beffa, D; Rüegg, U; Tarradellas, J; Wahli, W

    2000-01-01

    Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:10903615

  19. Sensitive assay of glycogen phosphorylase activity by analysing the chain-lengthening action on a Fluorogenic [corrected] maltooligosaccharide derivative.

    PubMed

    Makino, Yasushi; Omichi, Kaoru

    2009-07-01

    The action of glycogen phosphorylase (GP) is essentially reversible, although GP is generally classified as a glycogen-degrading enzyme. In this study, we developed a highly sensitive and convenient assay for GP activity by analysing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc(4)) residue comprising the non-reducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method was used to measure the GP activities of crude and purified GP preparations, and was demonstrated to have about 1,000 times greater sensitivity than the spectrophotometric orthophosphate assay. PMID:19279194

  20. A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics

    PubMed Central

    Iqbal, Junaid; Kazmi, Shahana Urooj; Khan, Naveed Ahmed

    2013-01-01

    Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30%) in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics. PMID:23865073

  1. Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

    PubMed Central

    Strazza, Marianne; Azoulay-Alfaguter, Inbar; Pedoeem, Ariel; Mor, Adam

    2014-01-01

    T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin). PMID:24961998

  2. A fluorometric assay for measurement of mono-ADP-ribosyltransferase activity.

    PubMed

    Klebl, B M; Pette, D

    1996-08-01

    Using 1,N6-etheno NAD, a fluorescent analog of NAD, we extended an existing assay for NAD glycohydrolase to the measurement of mono-ADP-ribosyltransferase (mADP-RT) activity using agmatine as acceptor for ADP-ribose. The reaction products were analyzed by reversed-phase chromatography. In the presence of agmatine two newly formed fluorescent products were tentatively identified as ADP-ribosylagmatine anomers. Fluorescence intensity increased upon splitting the N-glycoside bondage of 1,N6-etheno NAD. Therefore, 1, N6-etheno AMP could be used for calibration. The nonradioactive assay yielded values nearly identical to those obtained with the [carbonyl-14C]NAD method. It proved to be highly reproducible, rapid, and suitable for an improved purification protocol yielding a 76,000-fold enriched mADP-RT preparation from rabbit skeletal muscle. The identity and high purity of the enzyme were confirmed immunochemically. The assay served to determine the pH optimum of the enzyme (pH 9.0) and its KM for 1,N6-etheno NAD (287 microM). PMID:8811894

  3. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  4. Quantifying microbial activity in deep subsurface sediments using a tritium based hydrognease enzyme assay

    NASA Astrophysics Data System (ADS)

    Adhikari, R.; Nickel, J.; Kallmeyer, J.

    2012-12-01

    Microbial life is widespread in Earth's subsurface and estimated to represent a significant fraction of Earth's total living biomass. However, very little is known about subsurface microbial activity and its fundamental role in biogeochemical cycles of carbon and other biologically important elements. Hydrogen is one of the most important elements in subsurface anaerobic microbial metabolism. Heterotrophic and chemoautotrophic microorganisms use hydrogen in their metabolic pathways. They either consume or produce protons for ATP synthesis. Hydrogenase (H2ase) is a ubiquitous intracellular enzyme that catalyzes the interconversion of molecular hydrogen and/or water into protons and electrons. The protons are used for the synthesis of ATP, thereby coupling energy generating metabolic processes to electron acceptors such as CO2 or sulfate. H2ase enzyme targets a key metabolic compound in cellular metabolism therefore the assay can be used as a measure for total microbial activity without the need to identify any specific metabolic process. Using the highly sensitive tritium assay we measured H2ase enzyme activity in the organic-rich sediments of Lake Van, a saline, alkaline lake in eastern Turkey, in marine sediments of the Barents Sea and in deep subseafloor sediments from the Nankai Trough. H2ase activity could be quantified at all depths of all sites but the activity distribution varied widely with depth and between sites. At the Lake Van sites H2ase activity ranged from ca. 20 mmol H2 cm-3d-1 close to the sediment-water interface to 0.5 mmol H2 cm-3d-1 at a depth of 0.8 m. In samples from the Barents Sea H2ase activity ranged between 0.1 to 2.5 mmol H2 cm-3d-1 down to a depth of 1.60 m. At all sites the sulfate reduction rate profile followed the upper part of the H2ase activity profile until sulfate reduction reached the minimum detection limit (ca. 10 pmol cm-3d-1). H2ase activity could still be quantified after the decline of sulfate reduction, indicating that

  5. An improved thyroid hormone reporter assay to determine the thyroid hormone-like activity of amiodarone, bithionol, closantel and rafoxanide.

    PubMed

    Matsubara, Kana; Sanoh, Seigo; Ohta, Shigeru; Kitamura, Shigeyuki; Sugihara, Kazumi; Fujimoto, Nariaki

    2012-01-01

    A number of environmental chemicals have been reported to exhibit thyroid hormone-like activity. Since thyroid hormones play a crucial role in development, it is important to identify chemicals in the environment that are capable of endocrine disruption of thyroid hormone homeostasis. In order to detect thyroid hormone-like activity, the growth of pituitary cell lines has been commonly used as a sensitive marker, albeit with limited specificity to thyroid hormones. Reporter gene assays using the thyroid hormone responsive element (TRE) connected to the luciferase reporter gene have also been developed. Thus far however, this type of assay appears to have limited sensitivity compared to cell growth assays. In the present study, we developed a highly sensitive TRE reporter gene assay by using a pituitary cell line, MtT/E-2, and by culturing cells in a serum-free medium. Our assay was developed in order to detect T3 activity at a concentration of 10(-11)M. This assay identified thyroid hormone-like activity from the antiarrhythmic drug, amiodarone, and from three anti-parasitic drugs, bithionol, closantel and rafoxanide, all commonly used in veterinary medicine. Thyroid hormone-like activity of these compounds was further confirmed by the induction of BCL3 gene expression in MtT/E-2, which is known to be regulated by thyroid hormones. Our improved assay was proved to be a sensitive tool for assessing thyroid hormone-like activity of environmental chemicals. PMID:22015988

  6. The relevance of chemical interactions with CYP17 enzyme activity: assessment using a novel in vitro assay.

    PubMed

    Roelofs, Maarke J E; Piersma, Aldert H; van den Berg, Martin; van Duursen, Majorie B M

    2013-05-01

    The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. PMID:23415678

  7. In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein

    PubMed Central

    Nair, Smita; Rein, Alan

    2016-01-01

    Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this “restriction” of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3. The present protocol (Nair et al., 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner. The protocol described here is mainly adapted from that described by Iwatani et al. (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.

  8. A Plaque Assay for Malignant Catarrhal Fever Virus and Virus Neutralizing Activity

    PubMed Central

    Hazlett, D. T. G.

    1980-01-01

    A cell-free strain of malignant catarrhal fever virus which produced a readily recognizable cytopathic effect was obtained by serial passage of the virus in a rabbit kidney cell line. Plaque assay of the virus was more rapid and gave higher titres 11 days postinoculation than tube titration, but the latter advantage decreased with a longer incubation period. Plaques were clear with sharp edges and measured 0.5 to 2 mm in diameter after 15 days. A plaque neutralization test was developed and successfully employed for the titration of malignant catarrhal fever virus neutralizing activity in the sera and nasal secretions of blue wildebeest. PMID:7427840

  9. A Soil-free System for Assaying Nematicidal Activity of Chemicals.

    PubMed

    Preiser, F A; Babu, J R; Haidri, A A

    1981-10-01

    A biological assay system for studying the nematicidal activity of chemicals has been devised using a model consisting of cucumber (Cucumis sativus L. cv. Long Marketer) seedlings growing in the diSPo(R) growth-pouch apparatus. Meloidogyne incognita was used as the test organism. The response was quantified in terms of the numbers of galls produced. Statistical procedures were applied to estimate the ED(50) values of currently available nematicides. This system permits accurate quantification of galling and requires much less space and effort than the currently used methods. PMID:19300800

  10. Serum acetaminophen assay using activated charcoal adsorption and gas chromatography without derivatization.

    PubMed

    Jeevanandam, M; Novic, B; Savich, R; Wagman, E

    1980-01-01

    A quantitative assay of acetaminophen in serum has been developed. The drug, together with an internal standard 2-acetamidophenol, is adsorbed on activated charcoal and then extracted into a mixture of ethyl acetate and isopropanol. This extract is then analyzed, without any derivatization, by gas chromatography. The isothermal analysis yielded a good, highly reproducible separation. The drug peak was symmetrical and without any tailing. The peak height response ratio was found to be linear with concentrations ranging from 25-500 ng/L. No interference was observed with the various drugs or metabolites which are commonly encountered in human serum. PMID:7421146

  11. A chromism-based assay (CHROBA) technique for in situ detection of protein kinase activity.

    PubMed

    Tomizaki, Kin-ya; Jie, Xu; Mihara, Hisakazu

    2005-03-15

    A unique chromism-based assay technique (CHROBA) using photochromic spiropyran-containing peptides has been firstly established for detection of protein kinase A-catalyzed phosphorylation. The alternative method has advantages that avoid isolation and/or immobilization of kinase substrates to remove excess reagents including nonreactive isotope-labeled ATP or fluorescently-labeled anti-phosphoamino acid antibodies from the reaction mixture. Such a novel protocol based on thermocoloration of the spiropyran moiety in the peptide can offer not only an efficient screening method of potent kinase substrates but also a versatile analytical tool for monitoring other post-translational modification activities. PMID:15745830

  12. A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima.

    PubMed

    Moustafa, Hanan M A; Zaghloul, Taha I; Zhang, Y-H Percival

    2016-05-15

    Phosphopentomutase (PPM) catalyzes the interconversion of α-D-(deoxy)-ribose 1-phosphate and α-D-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on D-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 μM α-D-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s(-1) at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date. PMID:26924489

  13. Comparative Antimicrobial Activities of Aerosolized Sodium Hypochlorite, Chlorine Dioxide, and Electrochemically Activated Solutions Evaluated Using a Novel Standardized Assay

    PubMed Central

    Thorn, R. M. S.; Robinson, G. M.

    2013-01-01

    The main aim of this study was to develop a standardized experimental assay to enable differential antimicrobial comparisons of test biocidal aerosols. This study represents the first chlorine-matched comparative assessment of the antimicrobial activities of aerosolized sodium hypochlorite, chlorine dioxide, and electrochemically activated solution (ECAS) to determine their relative abilities to decontaminate various surface-associated health care-relevant microbial challenges. Standard microbiological challenges were developed by surface-associating typed Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis spores, or a clinical methicillin-resistant S. aureus (MRSA) strain on stainless steel, polypropylene, or fabric. All test coupons were subjected to 20-min biocidal aerosols of chlorine-matched (100 ppm) sodium hypochlorite, chlorine dioxide, or ECAS within a standard aerosolization chamber using a commercial humidifier under defined conditions. Biocidal treatment type and material surface had a significant effect on the number of microorganisms recovered from various material surfaces following treatment exposure. Under the conditions of the assay, the order of antimicrobial efficacy of biocidal aerosol treatment was as follows: ECAS > chlorine dioxide > sodium hypochlorite. For all biocides, greater antimicrobial reductions were seen when treating stainless steel and fabric than when treating plastic-associated microorganisms. The experimental fogging system and assay protocol designed within this study were shown capable of differentiating the comparative efficacies of multiple chlorine-matched biocidal aerosols against a spectrum of target organisms on a range of test surface materials and would be appropriate for testing other biocidal aerosol treatments or material surfaces. PMID:23459480

  14. A new dye uptake assay to test the activity of antibiotics against intracellular Francisella tularensis

    PubMed Central

    Sutera, Vivien; Caspar, Yvan; Boisset, Sandrine; Maurin, Max

    2014-01-01

    Francisella tularensis, a facultative intracellular bacterium, is the aetiological agent of tularaemia. Antibiotic treatment of this zoonosis is based on the administration of a fluoroquinolone or a tetracycline for cases with mild to moderate severity, whereas an aminoglycoside (streptomycin or gentamicin) is advocated for severe cases. However, treatment failures and relapses remain frequent, especially in patients suffering from chronic lymph node suppuration. Therefore, new treatment alternatives are needed. We have developed a dye uptake assay for determination of minimal inhibitory extracellular concentrations (MIECs) of antibiotics against intracellular F. tularensis, and validated the method by comparing the results obtained using a CFU-enumerating method. We also compared MIECs with MICs of the same compounds determined using a CLSI broth microdilution method. We tested the activity of 11 antibiotics against two clinical strains of F. tularensis subsp. holarctica isolated in France. Both strains displayed low MICs (≤1 μg/mL) to fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin), gentamicin, doxycycline and rifampicin. Higher MICs (≥8 μg/mL) were found for carbapenems (imipenem and meropenem), daptomycin and linezolid. Erythromycin MICs were 4.0 and 16.0 μg/mL, respectively, for the two clinical strains. MIECs were almost the same with the two methods used. They were concordant with MICs, except for erythromycin and linezolid (respectively, four and eight times more active against intracellular F. tularensis) and gentamicin (four to eight times less active against intracellular F. tularensis). This study validated the dye uptake assay as a new tool for determination of the activity of a large panel of antibiotics against intracellular F. tularensis. This test confirmed the intracellular activity of first-line antibiotics used for tularaemia treatment, but also revealed significant activity of linezolid against intracellular F. tularensis

  15. SELENIUM TREATMENT/REMOVAL ALTERNATIVES DEMONSTRATION PROJECT - MINE WASTE TECHNOLOGY PROGRAM ACTIVITY III, PROJECT 20

    EPA Science Inventory

    This document is the final report for EPA's Mine WAste Technology Program (MWTP) Activity III, Project 20--Selenium Treatment/Removal Alternatives Demonstration project. Selenium contamination originates from many sources including mining operations, mineral processing, abandoned...

  16. Scaling-down the size and increasing the throughput of glycosyltransferase assays: Activity changes upon stem-cell differentiation

    PubMed Central

    Patil, Shilpa A.; Chandrasekaran, E. V.; Matta, Khushi L.; Parikh, Abhirath; Tzanakakis, Emmanuel S.; Neelamegham, Sriram

    2012-01-01

    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 since traditional methods require large reaction volumes and complex chromatography. Approaches tested utilized: i) Microarray pin-printing. This method was appropriate when glycoT activity was high; ii) Microwells and microcentrifuge tubes. This was suitable for studies with cell lysates when enzyme activity was moderate; iii) C18 pipette tips and solvent-extraction. This enriched reaction product when the extent of reaction was low. In all cases, reverse phase-thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrate 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 differentiation towards cardiomyocytes. Enzymatic and lectin binding data suggest a transition from LeX type structures in mESCs to sialylated Galβ1,3GalNAc type glycans upon differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated to cardiomyocytes. Overall, simple, rapid, quantitative and scalable glycoT activity analysis methods are presented. These utilize a range of natural and synthetic acceptors for the analysis of complex biological specimen that have limited availability. PMID:22449497

  17. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes: Enzyme assay for immunotoxicity of xenobiotics

    SciTech Connect

    Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C. . Dept. of Biological Sciences); Venables, B.J. . Dept. of Biological Sciences TRAC Laboratories Inc., Denton, TX )

    1994-04-01

    Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes appears sufficiently sensitive for use as a nonmammalian biomarker to detect toxic effects of sublethal body burdens of Cu[sup 2+]. Lysozyme, a phylogenetically conserved enzyme, is capable of bactericidal activity via action on peptidoglycan of gram-positive bacterial cell walls and functions as a component of an organism's innate antimicrobial defense mechanism. Coelomic fluid and coelomocyte lysozyme activities, which exhibit temperature-response patterns similar to those of human saliva, plasma, serum and leukocyte extracts, were sensitive to Cu[sup 2+] exposure. Lysozyme activity of coelomic fluid and coelomocyte extracts from earthworms exposed for 5 d to CuSO[sub 4], using filter paper contact exposure, decreased with increasing sublethal Cu[sup 2+] concentrations of 0.05 and 0.1 [mu]g/cm[sup 2]. Compared to controls, coelomic fluid lysozyme activity was suppressed significantly at both exposure concentrations, whereas coelomocyte extract lysozyme activity was suppressed significantly at the 0.1-[mu]g/cm[sup 2] exposure concentration. Low inherent natural variability and sensitivity to sublethal Cu[sup 2+] body burdens indicate that lysozyme activity has potential as a biomarker for assaying immunotoxicity of metals.

  18. H2Oh!: Classroom demonstrations and activities for improving student learning of water concepts

    NASA Astrophysics Data System (ADS)

    Chan-Hilton, A.; Neupauer, R. M.; Burian, S. J.; Lauer, J. W.; Mathisen, P. P.; Mays, D. C.; Olson, M. S.; Pomeroy, C. A.; Ruddell, B. L.; Sciortino, A.

    2012-12-01

    Research has shown that the use of demonstrations and hands-on activities in the classroom enhances student learning. Students learn more and enjoy classes more when visual and active learning are incorporated into the lecture. Most college-aged students prefer visual modes of learning, while most instruction is conducted in a lecture, or auditory, format. The use of classroom demonstrations provides opportunities for incorporating visual and active learning into the classroom environment. However, while most instructors acknowledge the benefits of these teaching methods, they typically do not have the time and resources to develop and test such activities and to develop plans to incorporate them into their lectures. Members of the Excellence in Water Resources Education Task Committee of the Environmental and Water Resources Institute (EWRI) of the American Society of Civil Engineers (ASCE) have produced a publication that contains a collection of activities aimed to foster excellence in water resources and hydrology education and improve student learning of principles. The book contains forty-five demonstrations and activities that can be used in water-related classes with topics in fluid mechanics, hydraulics, surface water hydrology, groundwater hydrology, and water quality. We present examples of these activities, including topics such as conservation of momentum, buoyancy, Bernoulli's principle, drag force, pipe flow, watershed delineation, reservoir networks, head distribution in aquifers, and molecular diffusion in a porous medium. Unlike full laboratory exercises, these brief demonstrations and activities (most of which take less than fifteen minutes) can be easily incorporated into classroom lectures. For each demonstration, guidance for preparing and conducting the activity, along with a brief overview of the principles that are demonstrated, is provided. The target audience of the activities is undergraduate students, although the activities also may be

  19. Investigation of magnetic nanoparticles for the rapid extraction and assay of alpha-emitting radionuclides from urine: Demonstration of a novel radiobioassay method

    SciTech Connect

    O'Hara, Matthew J.; Carter, Jennifer C.; Maclellan, Jay A.; Warner, Cynthia L.; Warner, Marvin G.; Addleman, Raymond S.

    2011-08-01

    In the event of an accidental or intentional release of radionuclides into a populated area, three things must occur in a timely manner: food and drinking water supplies must be determined to be safe to eat / drink, civilians and/or military personnel must be surveyed to ensure that they do not have external contamination, and they must be screened to ensure that significant ingestion or inhalation of radionuclides has not occurred (this paper is concerned with the latter). In the event of such a disaster, the volume of radiobioassays to be performed would be tremendous. If the event released significant levels of β- or α-emitting radionuclides, in vivo assays would be ineffective. Therefore, highly efficient and rapid analytical methods for radionuclide detection from submitted spot urine samples (≤ 50 mL) would be required. At present, the quantitative determination of α-emitting radionuclides from urine samples is highly labor intensive, and requires significant sample preparation and analysis time. Sorbent materials that provide effective collection and enable rapid assay could significantly streamline the radioanalytical process. We have demonstrated the use of paramagnetic nanoparticles as a novel class of extracting media for four α-emitting radionuclides of concern (Po, Ra, Am, and U) from chemically unmodified and pH 2 human urine. Herein the initial experimental sorption results are presented along with a novel method that utilizes paramagnetic nanoparticles for the extraction of radionuclides from unmodified human urine followed by the magnetic field-induced collection of the particles for subsequent α-counting-source preparation. Additionally, we construct a versatile human dose model that determines the detector count times required to estimate internal human dose at specific protective action thresholds. The model provides a means to assess a method’s detection capabilities and use fundamental health physics parameters and actual experimental

  20. ASSESSMENT OF STANDARD REFERENCE COMPOUNDS FOR COMPARATIVE STUDIES USING THE SALMONELLA TYPHIMURIUM MUTAGENICITY ASSAY: II. WITH EXOGENOUS ACTIVATION

    EPA Science Inventory

    The purpose of this project was to evaluate the variability in the mutagenic response of potential standard reference chemicals that require exogenous metabolic activation in the standard plate; incorporation Salmonella mutagenicity assay, and to develop ranking criteria for muta...

  1. Multi-tiered Approach to Development of Increased Throughput Assay Models to Assess Endocrine-Disrupting Activity of Chemicals

    EPA Science Inventory

    Screening for endocrine-disrupting chemicals (EDCs) requires sensitive, scalable assays. Current high-throughput screening (HTPS) approaches for estrogenic and androgenic activity yield rapid results, but many are not sensitive to physiological hormone concentrations, suggesting ...

  2. A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

    EPA Science Inventory

    AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

  3. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

    PubMed Central

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O.; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  4. EC50 estimation of antioxidant activity in DPPH· assay using several statistical programs.

    PubMed

    Chen, Zheng; Bertin, Riccardo; Froldi, Guglielmina

    2013-05-01

    DPPH(·) assay is a reliable method to determine the antioxidant capacity of biological substrates. The DPPH(·) radical scavenging activity is generally quantified in terms of inhibition percentage of the pre-formed free radical by antioxidants, and the EC(50) (concentration required to obtain a 50% antioxidant effect) is a typically employed parameter to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC(50) estimation was performed using a comparative approach based on various regression models implemented in six specialized computer programs: GraphPad Prism® version 5.01, BLeSq, OriginPro® 8.5.1, SigmaPlot® 12, BioDataFit® 1.02, and IBM SPSS Statistics® Desktop 19.0. For this project, quercetin, catechin, ascorbic acid, caffeic acid, chlorogenic acid and acetylcysteine were screened as antioxidant standards with DPPH(·) assay to define the EC(50) parameters. All the statistical programs gave similar EC(50) values, but GraphPad Prism® five-parameter analysis pointed out a best performance, also showing a minor variance in relation to the actual EC(50). PMID:23265506

  5. Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity.

    PubMed

    Chemburu, Sireesha; Ji, Eunkyung; Casana, Yosune; Wu, Yang; Buranda, Tione; Schanze, Kirk S; Lopez, Gabriel P; Whitten, David G

    2008-11-20

    A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range. PMID:18808092

  6. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

    PubMed

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  7. Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity

    PubMed Central

    Kashima, Hajime; Momose, Fumiyasu; Umehara, Hiroshi; Miyoshi, Nao; Ogo, Naohisa; Muraoka, Daisuke; Shiku, Hiroshi; Harada, Naozumi; Asai, Akira

    2016-01-01

    Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors. PMID:27284967

  8. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  9. Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

    PubMed Central

    Çağlayan, Melike; Horton, Julie K.; Wilson, Samuel H.

    2016-01-01

    We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5′-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits. PMID:27390764

  10. mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay.

    PubMed Central

    Kroath, H; Shatkin, A J

    1982-01-01

    Reovirus mRNA 5'-terminal caps were 3'-radiolabeled with pCp and as affinity probes for proteins with cap binding activity. A rapid, simple, and sensitive blot assay was devised that could detect cellular cap binding protein in a complex polypeptide mixture. By using this method, cap binding activity was found in detergent-treated influenza virus but not in reovirus or vaccinia virus. Preincubation of capped reovirus mRNA with purified cellular cap binding protein reduced its primer effect on influenza transcriptase, whereas priming by ApG was not affected. The results indicate that influenza transcriptase complexes include cap-recognizing proteins that are involved in the formation of chimeric mRNAs. Images PMID:7097854

  11. The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

    SciTech Connect

    Roelofs, Maarke J.E.; Piersma, Aldert H.; Berg, Martin van den; Duursen, Majorie B.M. van

    2013-05-01

    The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

  12. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay

    PubMed Central

    Zhu, Shu; Diamond, Scott L.

    2014-01-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm2)/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s−1), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s−1) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s−1 revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s−1 or 1000 s−1, and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s−1 (compared to fibrin formed at 100 s−1) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions. PMID:25303860

  13. Quantification of microbial activity in subsurface environments using a hydrogenase enzyme assay

    NASA Astrophysics Data System (ADS)

    Adhikari, R. R.; Nickel, J.; Kallmeyer, J.

    2012-04-01

    The subsurface biosphere is the largest microbial ecosystem on Earth. Despite its large size and extensive industrial exploitation, very little is known about the role of microbial activity in the subsurface. Subsurface microbial activity plays a fundamental role in geochemical cycles of carbon and other biologically important elements. How the indigenous microbial communities are supplied with energy is one of the most fundamental questions in subsurface research. It is still an enigma how these communities can survive with such recalcitrant carbon over geological time scales. Despite its usually very low concentration, hydrogen is an important element in subsurface environments. Heterotrophic and chemoautotrophic microorganisms use hydrogen in their metabolic pathways; they either obtain protons from the radiolysis of water and/or cleavage of hydrogen generated by the alteration of basaltic crust, or they dispose of protons by formation of water. Hydrogenase (H2ase) is a ubiquitous intracellular enzyme that catalyzes the interconversion of molecular hydrogen and/or water into protons and electrons. The protons are used for the synthesis of ATP, thereby coupling energy-generating metabolic processes to electron acceptors such as carbon dioxide or sulfate. H2ase activity can therefore be used as a measure for total microbial activity as it targets a key metabolic compound rather than a specific turnover process. Using a highly sensitive tritium assay we measured H2ase enzyme activity in the organic-rich sediments of Lake Van, a saline, alkaline lake in eastern Turkey and in marine subsurface sediments of the Barents Sea. Additionally, sulfate reduction rates (SRRs) were measured to compare the results of the H2ase enzyme assay with the quantitatively most important electron acceptor process. H2ase activity was found at all sites, measured values and distribution of activity varied widely with depth and between sites. At the Lake Van sites H2ase activity ranged from

  14. Classroom Activity Connections: Demonstrating Various Flame Tests Using Common Household Materials

    ERIC Educational Resources Information Center

    Baldwin, Bruce W.; Hasbrouck, Scott; Smith, Jordan; Kuntzleman, Thomas S.

    2010-01-01

    In "JCE" Activity #67, "Flame Tests: Which Ion Causes the Color?", Michael Sanger describes how to conduct flame tests with household items. We have used this activity in outreach settings, and have extended it in a variety of ways. For example, we have demonstrated large-scale strontium (red), copper (green), and carbon (blue) flames using only…

  15. Teaching with Movement: Using the Health Privilege Activity to Physically Demonstrate Disparities in Society

    ERIC Educational Resources Information Center

    Irby-Shasanmi, Amy; Oberlin, Kathleen C.; Saunders, Tiffani N.

    2012-01-01

    This article describes and evaluates an activity designed to demonstrate how biological factors (e.g., genetics), individual-level behaviors (e.g., smoking), and social factors (e.g., socioeconomic status) shape health status and access to health care. Active learning techniques were utilized to introduce the sociological imagination as it…

  16. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    PubMed

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer. PMID:26119290

  17. A Novel Cell-Associated Protection Assay Demonstrates the Ability of Certain Antibiotics To Protect Ocular Surface Cell Lines from Subsequent Clinical Staphylococcus aureus Challenge▿†

    PubMed Central

    Wingard, J. B.; Romanowski, E. G.; Kowalski, R. P.; Mah, F. S.; Ling, Y.; Bilonick, R. A.; Shanks, R. M. Q.

    2011-01-01

    In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens. PMID:21628536

  18. Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp.

    PubMed

    Ngando Ebongue, G F; Dhouib, R; Carrière, F; Amvam Zollo, P-H; Arondel, V

    2006-10-01

    The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase. PMID:17064925

  19. Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.

    PubMed

    Nauen, Ralf; Stumpf, Natascha

    2002-04-15

    Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane-glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae). PMID:11950219

  20. Effect of Lead stress on phosphatase activity and reducing power assay of Triticum aestivum.

    PubMed

    Gubrelay, U; Agnihotri, R K; Shrotriya, S; Sharma, R

    2015-01-01

    Lead (Pb) is a highly toxic heavy metal for both plants and animals; the environment is increasingly polluted with heavy metals and reduces crop productivity. Plants possess homeostatic mechanisms that allow them to keep correct concentrations of essential metal ions in cellular compartments and to minimize the damaging effects of an excess of nonessential ones. One of their adverse effects on plants are the generation of harmful active oxygen species, leading to oxidative stress and the antioxidative activity seems to be of fundamental importance for adaptive response of plant against environmental stress. The present study explores the effects of lead (soil treated twice/ week) with (10, 30 and 60 mM) on the specific activities of phosphatases which might lead to reducing power assay in (Triticum aestivum PBW344) seedling. A significant decrease in the redox potential of shoot compared to root was observed at the similar concentration of lead. A similar trend on leaves was also noted. Acid and alkaline phosphatase activities were significantly higher in roots than in shoot at all the three concentration of lead i.e. 10, 30 and 60 mM, compared to controls. The above mentioned changes were more pronounced at 60 mM concentration of lead than two other concentrations. These results lead us to suggest that increased lead concentration in soil might lead to adverse effects on plant growth and phosphatase activities. PMID:26107501

  1. Crocin bleaching antioxidant assay revisited: application to microplate to analyse antioxidant and pro-oxidant activities.

    PubMed

    Prieto, M A; Vázquez, J A; Murado, M A

    2015-01-15

    The crocin bleaching assay (CBA) is a common method for evaluating the antioxidant activity of hydrosoluble samples. It is criticised due to its low reproducibility, problematic quantification of results, differences in reagent preparation, doubtful need for a preheating phase and sensitivity to factors such as temperature, pH, solvents and metals. Here, the critical points of the method were extensively revised, and a highly reproducible procedure for microplate readers redeveloped. The problems of using quantification procedures, disregarding kinetic considerations, are discussed in detail and a model is proposed for quantifying simultaneously anti- and pro-oxidant activities as function of concentration and time. Thus, the combined use of a reproducible procedure and robust mathematical modeling produced consistent and meaningful criteria for comparative characterization of any oxidation modifier, taking into account the dose-time-dependent behaviour. The method was verified by characterising several commercial antioxidants and some metal compounds using the parametric values of the proposed models. The activity of the tested antioxidants decreased in the order ETX>TR>PG>AA>TBHQ>BHA. Others, such as the lipophilic antioxidants of BHT and α-Tocopherol did not show any activity. Interference from metals were for Fe(2+), Fe(3+), Cd(2+), Ni(2+), Mg(2+), Zn(2+) and Sr(2+), slightly antioxidant for Cu(1+) and Cu(2+), and strongly antioxidant for Mn(2+). None of the tested metals showed a pro-oxidant activity. PMID:25148992

  2. Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.

    PubMed

    Prefontaine, M; Shih, C; Pan, C C; Bhavnani, B R

    1990-12-01

    The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used

  3. A SPECTROPHOTOMETRIC ASSAY TO MEASURE RUBISCO ACTIVASE ACTIVATION ACTIVITY UNDER VARYING ATP:ADP RATIOS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratio of ATP to ADP in the stroma is an important regulatory mechanism for controlling the activation state of Rubisco via Rubisco activase (activase). Understanding the response of activase to a varying ATP:ADP ratio should reveal insights into the regulation of photosynthesis. However, the cur...

  4. A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.)

    PubMed Central

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud’homme, Marie-Pascale; Roitsch, Thomas

    2015-01-01

    Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding. PMID:26734049

  5. Modified procedure for labelling target cells in a europium release assay of natural killer cell activity.

    PubMed

    Pacifici, R; Di Carlo, S; Bacosi, A; Altieri, I; Pichini, S; Zuccaro, P

    1993-05-01

    Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for the detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuCl3. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium. PMID:8486925

  6. PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS

    EPA Science Inventory

    A bactericidal assay developed to assess the ability of oyster (Crassostrea virginica) hemocytes to kill the human pathogen Vibrio parahaemolyticus was optimized to estimate killing of the oyster parasite Perkinsus marinus. Assay variables, temperature, hemocyte:parasite ratio, i...

  7. Tested Demonstrations.

    ERIC Educational Resources Information Center

    Gilbert, George L., Ed.

    1988-01-01

    Details three demonstrations for use in chemistry classrooms. Includes: "A Demonstration of Corrosion by Differential Aeration"; "A Simple Demonstration of the Activation Energy Concept"; and "A Boiling Demonstration at Room Temperature." Each description includes equipment, materials, and methods. (CW)

  8. Are fish and standardized FETAX assays protective enough for amphibians? A case study on Xenopus laevis larvae assay with biologically active substances present in livestock wastes.

    PubMed

    Martini, Federica; Tarazona, José V; Pablos, M Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC(50)) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  9. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    PubMed Central

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  10. Eupolyphaga sinensis Walker demonstrates angiogenic activity and inhibits A549 cell growth by targeting the KDR signaling pathway.

    PubMed

    Dai, Bingling; Qi, Junpeng; Liu, Rui; Zhang, Yanmin

    2014-09-01

    Eupolyphaga sinensis Walker has been reported to have anticoagulation, antithrombotic, liver protective and antitumor effects. In the present study, the inhibitory effects on proliferation of A549 human non‑small cell lung cancer cells and the underlying mechanisms were examined. Firstly, three solvents, 70% ethanol, distilled water and 95% ethanol, were used to extract Eupolyphaga sinensis Walker. The MTT assay results demonstrated that the 70% ethanol extract more potently reduced the growth of A549 cells and it was therefore adopted in the subsequent experiments. Eupolyphaga sinensis Walker 70% ethanol extract significantly inhibited A549 cell migration in a time‑ and dose‑dependent manner and inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. Furthermore, Eupolyphaga sinensis Walker 70% ethanol extract effectively inhibited blood vessel formation in the established tissue model for angiogenesis. In addition, Eupolyphaga sinensis Walker 70% ethanol extract was demonstrated to inhibit the autophosphorylation of KDR, and downregulate the subsequent activation of AKT and extracellular signal regulated kinase (ERK)1/2 in A549 cells. In conclusion, these findings demonstrated that the antitumor mechanism of Eupolyphaga sinensis Walker 70% ethanol extract was through inhibiting angiogenesis. It functioned by interrupting the autophosphorylation of KDR and subsequently, AKT and ERK1/2. PMID:25059654

  11. Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc).

    EPA Science Inventory

    The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity i...

  12. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  13. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    PubMed Central

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-01-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells. PMID:24603274

  14. Integrated gasification combined-cycle research development and demonstration activities in the US

    SciTech Connect

    Ness, H.M.; Brdar, R.D.

    1996-09-01

    The United States Department of Energy (DOE)`s Office of Fossil Energy, Morgantown Energy Technology Center, is managing a research development and demonstration (RD&D) program that supports the commercialization of integrated gasification combined-cycle (IGCC) advanced power systems. This overview briefly describes the supporting RD&D activities and the IGCC projects selected for demonstration in the Clean Coal Technology (CCT) Program.

  15. Integrated gasification combined-cycle research development and demonstration activities in the U.S.

    SciTech Connect

    Ness, H.M.

    1994-12-31

    The United States Department of Energy (DOE) has selected seven integrated gasification combined-cycle (IGCC) advanced power systems for demonstration in the Clean Coal Technology (CCT) Program. DOE`s Office of Fossil Energy, Morgantown Energy Technology Center, is managing a research development and demonstration (RD&D)program that supports the CCT program, and addresses long-term improvements in support of IGCC technology. This overview briefly describes the CCT projects and the supporting RD&D activities.

  16. A simple and efficient diffusion technique for assay of endo β-1,4-xylanase activity

    PubMed Central

    Samanta, A.K.; Kolte, Atul P.; Senani, S.; Sridhar, Manpal.; Jayapal, Natasha.

    2011-01-01

    Endo-β-1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo-β-1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±10C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured. Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo-β-1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein. The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo-β-1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride. PMID:24031763

  17. Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts.

    PubMed

    Chen, Feng; Zhang, Dexin; Zhang, Qing; Zuo, Xiaolei; Fan, Chunhai; Zhao, Yongxi

    2016-06-16

    Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts. PMID:26690725

  18. Gamma interferon release assay for monitoring of treatment response for active tuberculosis: an explosion in the spaghetti factory.

    PubMed

    Denkinger, Claudia M; Pai, Madhukar; Patel, Meena; Menzies, Dick

    2013-02-01

    Few studies have correlated the results of interferon (gamma interferon) release assays (IGRAs) with known markers of tuberculosis (TB) treatment response. We report the results of serial QuantiFERON-TB gold in-tube assay (QFT) testing on 149 patients with active tuberculosis and correlate the results with smear and culture conversion. We show that QFT results do not offer much value for treatment monitoring of TB disease. PMID:23175268

  19. Generation of recombinant rabies viruses encoding NanoLuc luciferase for antiviral activity assays.

    PubMed

    Anindita, Paulina Duhita; Sasaki, Michihito; Nobori, Haruaki; Sato, Akihiko; Carr, Michael; Ito, Naoto; Sugiyama, Makoto; Orba, Yasuko; Sawa, Hirofumi

    2016-04-01

    Rabies is an invariably fatal disease caused by Rabies virus (RABV), a member of the family Rhabdoviridae, genus Lyssavirus. Once central nervous infection occurs and symptoms develop, the case fatality rate approaches 100% despite availability of post-exposure prophylaxis. Therefore, new antiviral therapies for rabies are urgently required. Antivirals which can inhibit virus replication can be identified through screening of small compounds, however, as RABV infection does not generate easily discernible cytopathic effects in vitro, cell viability assays may not be feasible to observe antiviral activity of small compounds against RABV. In this study, recombinant RABVs (rRABVs) encoding NanoLuc luciferase (NanoLuc) were generated to facilitate the screening of small compound libraries. NanoLuc expression was confirmed in single-step growth cures of virus infection and showed that the rRABVs were capable of viral replication without decrease of luciferase activity through ten serial passages. Furthermore, the rRABVs were able to quantify the antiviral activity of the nucleoside analogue ribavirin against RABV in vitro. These findings confirm the potential of the rRABV encoding NanoLuc system to facilitate screening of small compounds to inhibit RABV infection. PMID:26869397

  20. Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays

    PubMed Central

    2011-01-01

    Background Abelmoschus moschatus Medik. leaves and seeds are considered as valuable traditional medicine. The aromatic seeds of this plant are aphrodisiac, ophthalmic, cardio tonic, antispasmodic and used in the treatment of intestinal complaints and check queasiness. To give a scientific basis for traditional usage of this medicinal plant, the seed and leaf extracts were evaluated for their antioxidant, free radical scavenging, antimicrobial and antiproliferative activities. Methods In this study, antioxidant, antimicrobial and antiproliferative activities of A. moschatus extracts were evaluated in a series of in vitro assay involving free radicals, reactive oxygen species and their IC50 values were also determined. The antioxidant activities of the seed and leaf extracts of A. moschatus were determined by total antioxidant, DPPH, and ferrous reducing antioxidant property (FRAP) methods. In addition, the antiproliferative activity was also evaluated using colorectal adenocarcinoma and retinoblastoma human cancer cell lines. Moreover, six bacterial reference strains, two gram-positive (Bacillus subtilis and Staphylococcus aureus), four gram-negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella enterica paratyphi) and one fungal strain (Candida albicans) were used to evaluate its antimicrobial activity. Results The results from this study showed that the antioxidant activities of A. moschatus as determined by the total phenol, flavonoids, total antioxidant and FRAP methods were higher in leaf than that of the seed extracts. On the other hand, the aqueous overnight seed extract (AMS-I) has shown significant radical scavenging activity as in 1, 1- Diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, hydroxyl radical, superoxide and lipid peroxidation as compared to other seed and leaf extracts. The AMS-I and AML-IV have shown activity against six and seven microorganisms respectively. Simulteneously, AMS-IV and AML-IV have demonstrated

  1. Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

    PubMed

    Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

    2002-11-01

    In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different

  2. Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

    PubMed Central

    Al Khamici, Heba; Brown, Louise J.; Hossain, Khondker R.; Hudson, Amanda L.; Sinclair-Burton, Alxcia A.; Ng, Jane Phui Mun; Daniel, Elizabeth L.; Hare, Joanna E.; Cornell, Bruce A.; Curmi, Paul M. G.; Davey, Mary W.; Valenzuela, Stella M.

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function. PMID:25581026

  3. Chromogenic assay for the prothrombin activator ecarin from the venom of the saw-scaled viper (Echis carinatus).

    PubMed

    Stocker, K; Fischer, H; Brogli, M

    1986-01-01

    Ecarin, by limited proteolysis and subsequent autocatalytic reactions, causes the conversion of prothrombin into three products with amidolytic activity: meizothrombin, meizothrombin 1 and lpha-thrombin. Ecarin action may be abolished by ethylenediaminetetraacetic acid and the activity of alpha-thrombin can, with a high degree of selectivity, be inhibited by heparin. Thus, ecarin potency may be assayed by measuring the meizothrombin activity generated by ecarin action on human plasma in the presence of heparin. The chromogenic substrate Tosyl-glycyl-L-prolyl-L-arginine-p-nitroanilide (Chromozym TH) is used in this assay. PMID:3082039

  4. Measurement of HCV-Specific CD8(+) Cytotoxic T-Cell Activities in the Peripheral Blood by Europium Release Assay.

    PubMed

    Imawari, M

    1999-01-01

    Peripheral blood mononuclear cells (PBMC) contain NK cells, cytotoxic T-lymphocytes (CTL), helper T-cells, and B-cells that respond to viral infection and act to eliminate the virus from infected individuals. CTLs are not only thought to be a major host defense against viral infection, but are also implicated in the immunopathogenesis. Classical CTLs are CD8(+) and recognize endogenously synthesized and processed antigen in association with a human leukocyte antigen (HLA) class I molecule. The antigens are usually 8-10 amino acids long. HCV-specific CTLs have been demonstrated in the peripheral blood of some of patients with HCV infection by stimulating PBMC with the HCV synthetic peptides (1). The peptides were synthesized as overlapping peptides to encompass a certain region of the HCV antigen (1), on the basis of antigenicity prediction from the amino acid composition of HCV (2), or on the basis of the HLA binding motifs in the HCV antigen (3). Several minimal and optimal epitopes in the HCV antigen and their HLA restriction of recognition by CTLs have been defined. Recently, it has been reported that HCV-specific CTLs may suppress the outgrowth of HCV (4). In this chapter, methods will be discussed that demonstrate HCV-specific CTLs in the peripheral blood of patients with HCV infection. We use nonradioisotope europium (Eu) for assay of CTL activities. PMID:21374383

  5. Comparison of In Vitro Activities of 17 Antifungal Drugs against a Panel of 20 Dermatophytes by Using a Microdilution Assay

    PubMed Central

    Favre, Bertrand; Hofbauer, Bettina; Hildering, Kwang-Soo; Ryder, Neil S.

    2003-01-01

    The in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes comprising 6 different species were determined using a microdilution assay according to the NCCLS M38-P method with some modifications. Terbinafine was the most potent systemic drug while tolnaftate and amorolfine were the most active topical agents. PMID:14532230

  6. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    PubMed

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity. PMID:16834534

  7. A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.

    PubMed

    Biswas, Tapan; Resto-Roldán, Esteban; Sawyer, Sean K; Artsimovitch, Irina; Tsodikov, Oleg V

    2013-02-01

    Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase-pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z' = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery. PMID:23267008

  8. A novel non-radioactive primase–pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

    PubMed Central

    Biswas, Tapan; Resto-Roldán, Esteban; Sawyer, Sean K.; Artsimovitch, Irina; Tsodikov, Oleg V.

    2013-01-01

    Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase–pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z′ = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery. PMID:23267008

  9. FULL-SCALE DEMONSTRATION OF OPEN TANK OXYGEN ACTIVATED SLUDGE TREATMENT

    EPA Science Inventory

    This report presents an operating summary of a full-scale demonstration of the FMC open tank pure oxygen (FMC O2) activated sludge system, conducted at the facilities of the Metropolitan Denver Sewage Disposal District No. 1 (Metro) in Denver, Colorado. The system was operated ov...

  10. 34 CFR 350.15 - What must a grantee do in carrying out a demonstration activity?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true What must a grantee do in carrying out a demonstration activity? 350.15 Section 350.15 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION DISABILITY AND REHABILITATION RESEARCH PROJECTS...

  11. A Classroom Activity to Demonstrate Self-Other Agreement in Personality Judgments

    ERIC Educational Resources Information Center

    Kaplan, Seth A.; Stachowski, Alicia A.; Bradley-Geist, Jill C.

    2012-01-01

    This article describes a classroom activity to demonstrate (dis)agreement in personality judgments, using an exercise derived from Watson's research on the accuracy of rating strangers' personalities. On the first day of class, undergraduate students in psychology courses rated their own personality and the personality of a classmate, using items…

  12. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  13. Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation.

    PubMed

    Cieniewicz, Anne M; Cooper, Philip R; McGehee, Jennifer; Lingham, Russell B; Kihm, Anthony J

    2016-08-01

    Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize

  14. Active learning of introductory optics: real-time physics labs, interactive lecture demonstrations and magic

    NASA Astrophysics Data System (ADS)

    Sokoloff, David R.

    2005-10-01

    Widespread physics education research has shown that most introductory physics students have difficulty learning essential optics concepts - even in the best of traditional courses, and that well-designed active learning approaches can remedy this problem. This mini-workshop and the associated poster session will provide direct experience with methods for promoting students' active involvement in the learning process in lecture and laboratory. Participants will have hands-on experience with activities from RealTime Physics labs and Interactive Lecture Demonstrations - a learning strategy for large (and small) lectures, including specially designed Optics Magic Tricks. The poster will provide more details on these highly effective curricula.

  15. Complete validation of a unique digestion assay to detect Trichinella larvae in horsemeat demonstrates its reliability for meeting food safety and trade requirements.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A tissue digestion assay using a double separatory funnel (DSF) procedure for the detection of Trichinella larvae in horsemeat was validated for application in food safety programs and trade. It consisted of a pepsin-HCl digestion step to release larvae from muscle tissue followed by two sequential ...

  16. Honokiol bis-dichloroacetate (Honokiol DCA) demonstrates activity in vemurafenib-resistant melanoma in vivo

    PubMed Central

    Bonner, Michael Y.; Karlsson, Isabella; Rodolfo, Monica; Arnold, Rebecca S.; Vergani, Elisabetta; Arbiser, Jack L.

    2016-01-01

    The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo. PMID:26871475

  17. Enzyme activity demonstrates multiple pathways of innate immunity in Indo-Pacific anthozoans.

    PubMed

    Palmer, C V; Bythell, J C; Willis, B L

    2012-09-22

    Coral reefs are threatened by increasing levels of coral disease and the functional loss of obligate algal symbionts (bleaching). Levels of immunity relate directly to susceptibility to these threats; however, our understanding of fundamental aspects of coral immunology is lacking. We show that three melanin-synthesis pathway components (mono-phenoloxidase, ortho-diphenoloxidase (tyrosinase-type pathway) and para-diphenoloxidase (laccase-type pathway)) are present in both their active (phenoloxidase, PO) and inactive (prophenoloxidase, PPO) forms across a diverse range of 22 species of healthy Indo-Pacific anthozoans. We also demonstrate transglutaminase activity of the coagulation cascade for, to our knowledge, the first time in a coral. Melanin-synthesis enzyme activities varied among taxa, although they were generally lowest in the coral family Acroporidae and highest in the Poritidae and Oculinidae. Inactive tyrosinase-type activity (PPO) and active laccase-type activity (PO) correlate with taxonomic patterns in disease resistance, whereas the converse pattern in activity levels correlates with bleaching resistance. Overall, we demonstrate the presence of several melanin-synthesis pathways in Indo-Pacific corals, co-regulation among some pathway components, and highlight their potential roles in coral health. PMID:22810430

  18. Enzyme activity demonstrates multiple pathways of innate immunity in Indo-Pacific anthozoans

    PubMed Central

    Palmer, C. V.; Bythell, J. C.; Willis, B. L.

    2012-01-01

    Coral reefs are threatened by increasing levels of coral disease and the functional loss of obligate algal symbionts (bleaching). Levels of immunity relate directly to susceptibility to these threats; however, our understanding of fundamental aspects of coral immunology is lacking. We show that three melanin-synthesis pathway components (mono-phenoloxidase, ortho-diphenoloxidase (tyrosinase-type pathway) and para-diphenoloxidase (laccase-type pathway)) are present in both their active (phenoloxidase, PO) and inactive (prophenoloxidase, PPO) forms across a diverse range of 22 species of healthy Indo-Pacific anthozoans. We also demonstrate transglutaminase activity of the coagulation cascade for, to our knowledge, the first time in a coral. Melanin-synthesis enzyme activities varied among taxa, although they were generally lowest in the coral family Acroporidae and highest in the Poritidae and Oculinidae. Inactive tyrosinase-type activity (PPO) and active laccase-type activity (PO) correlate with taxonomic patterns in disease resistance, whereas the converse pattern in activity levels correlates with bleaching resistance. Overall, we demonstrate the presence of several melanin-synthesis pathways in Indo-Pacific corals, co-regulation among some pathway components, and highlight their potential roles in coral health. PMID:22810430

  19. Towards a Better Understanding of the Psychopharmacology of Nutmeg: Activities in the Mouse Tetrad Assay

    PubMed Central

    El-Alfy, Abir; Wilson, Lisa; ElSohly, Mahmoud A.; Abourashed, Ehab A.

    2009-01-01

    Ethnopharmacological relevance Nutmeg, the seeds of Myritica fragrans (family Myristicaceae), is a well known kitchen spice with a long-standing reputation as a psychoactive herb. Nutmeg at high doses is considered a cheap substitute to several drugs of abuse. Earlier reports have attributed amphetamine-like activities to nutmeg. Aim of the study To characterize the neuropharmacological effects of different nutmeg extracts, administered orally and intraperitoneally, in comparison to Δ9-terahydrocannabinol, amphetamine, and morphine. Materials and methods Methanolic (ME), dichloromethane (DE), and hexane (HE) extracts were obtained from a chromatographically fingerprinted batch of nutmeg. Biological evaluation was conducted in sets of 6–8 mice in the tetrad assay at doses ranging from 100–500 and 500–1000 mg/kg for i.p. and oral administration, respectively. Results While oral administration of all the nutmeg extracts at 500 mg/kg caused a significant increase in locomotor activity, the i.p. administration of DE showed significant reduction in rectal temperature along with a significant increase in tail flick latency at 300 mg/kg. A significant decrease in core body temperature was observed with HE at 100 mg/kg, while higher doses caused significant increases in hot plate latency. Conclusion Different behavioral effects were observed that varied by the type of extract as well as by the route of administration. PMID:19703539

  20. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed Central

    Prestwich, G D; Wawrzeńczyk, C

    1985-01-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites. PMID:3860862

  1. Electrochemical assay of α-glucosidase activity and the inhibitor screening in cell medium.

    PubMed

    Zhang, Juan; Liu, Ying; Wang, Xiaonan; Chen, Yangyang; Li, Genxi

    2015-12-15

    An electrochemical method is established in this work for the assay of α-glucosidase activity and the inhibitor screening through one-step displacement reaction, which can be directly used in cell medium. The displacement reaction can be achieved via strong binding of 4-aminophenyl-α-D-glucopyranoside (pAPG)/magnetic nanoparticles (MNPs) to pyrene boric acid (PBA) immobilized on the surface of graphite electrode (GE), compared to that of dopamine (DA)/sliver nanoparticles (AgNPs). Since α-glucosidase can specifically catalyze MNPs/pAPG into MNPs/pAP which has no binding capacity with PBA, the activity of both isolated and membrane bound enzyme can be well evaluated by using this proposed method. Meanwhile, signal amplification can be accomplished via the immobilization of DA at the outer layer of AgNPs, and the accuracy can be strengthened through magnetic separation. Moreover, this method can also be utilized for inhibitor screening not only in the medium containing the enzyme but also in cell medium. With good precision and accuracy, it may be extended to other proteases and their inhibitors as well. PMID:26201984

  2. Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay

    PubMed Central

    Chen, Fangfang; Qi, Wen; Sun, Jiahong; Simpkins, James W.; Yuan, Dan

    2015-01-01

    Isorhynchophylline is one of the major alkaloids from the Uncaria hook possessing the effects of lowered blood pressure, vasodilatation and protection against ischemia-induced neuronal damage. However, the metabolic pathway of isorhynchophylline has not been fully reported yet. In this paper, the metabolism of isorhynchophylline was investigated in rats. Five metabolites were isolated by using solvent extraction and repeated chromatographic methods, and identified by spectroscopic methods including UV, MS, NMR and CD experiments. Three new compounds were identified as 5-oxoisorhynchophyllic acid-22-O-β-D-glucuronide (M1), 17-O-demethyl-16,17-dihydro isorhynchophylline (M2) and 5-oxoisorhynchophyllic acid (M4) together with two known compounds isorhynchophylline (M0) and rhynchophylline (M3). Possible metabolic pathways of isorhynchophylline are proposed. Furthermore, the activity assay for all the metabolites showed that isorhynchophylline (M0) exhibited potent neuroprotective effects against glutamate-induced HT22 cell death. However, little or weak neuroprotective activities were observed for M1–M4. Our present study is important to further understand its metabolic fate and disposition in humans. PMID:24910000

  3. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay

    SciTech Connect

    Prestwich, G.D.; Wawrzenczyk, C.

    1985-08-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.

  4. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  5. Liquid crystal based sensors monitoring lipase activity: a new rapid and sensitive method for cytotoxicity assays.

    PubMed

    Hussain, Zakir; Zafiu, Christian; Küpcü, Seta; Pivetta, Lucineia; Hollfelder, Nadine; Masutani, Akira; Kilickiran, Pinar; Sinner, Eva-Kathrin

    2014-06-15

    In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity. PMID:24508543

  6. A simple purification and activity assay of the coagulant protein from Moringa oleifera seed.

    PubMed

    Ghebremichael, Kebreab A; Gunaratna, K R; Henriksson, Hongbin; Brumer, Harry; Dalhammar, Gunnel

    2005-06-01

    Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1--4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications. PMID:15921719

  7. Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay.

    PubMed

    Chen, Fangfang; Qi, Wen; Sun, Jiahong; Simpkins, James W; Yuan, Dan

    2014-09-01

    Isorhynchophylline is one of the major alkaloids from the Uncaria hook possessing the effects of lowered blood pressure, vasodilatation and protection against ischemia-induced neuronal damage. However, the metabolic pathway of isorhynchophylline has not been fully reported yet. In this paper, the metabolism of isorhynchophylline was investigated in rats. Five metabolites were isolated by using solvent extraction and repeated chromatographic methods, and identified by spectroscopic methods including UV, MS, NMR and CD experiments. Three new compounds were identified as 5-oxoisorhynchophyllic acid-22-O-β-D-glucuronide (M1), 17-O-demethyl-16,17-dihydro isorhynchophylline (M2) and 5-oxoisorhynchophyllic acid (M4) together with two known compounds isorhynchophylline (M0) and rhynchophylline (M3). Possible metabolic pathways of isorhynchophylline are proposed. Furthermore, the activity assay for all the metabolites showed that isorhynchophylline (M0) exhibited potent neuroprotective effects against glutamate-induced HT22 cell death. However, little or weak neuroprotective activities were observed for M1-M4. Our present study is important to further understand its metabolic fate and disposition in humans. PMID:24910000

  8. In vitro demonstration of anti-lipogenic activity in serum from obese rats

    SciTech Connect

    Harris, R.B.S.; Martin, R.J.

    1986-03-01

    Studies with parabiosed rats provide evidence for a humoral factor, originating in obese animals, that specifically inhibits adipose lipogenesis. A bioassay was developed that allows serum from obese rats to be tested for this factor in vitro. Adipocytes are isolated from epididymal fat of 250g Sprague-Dawley rats. The cells are preincubated at 37/sup 0/C for 1 or 12 hrs, in TC199 media containing 1.1 mg/ml glucose, 0.1 M Hepes and 2% serum. Following preincubation, the cells are washed 3 times and resuspended in serum-free media. Aliquots of cells are tested for metabolic activity in a subsequent 2 hour radiolabelled incubation in serum-free media with the addition of 0.5 ..mu..Ci/ml U-/sup 14/C-glucose. Basal, insulin (100 ..mu..U/ml) and norepinephrine (0.1 ..mu..g/ml) stimulated rates of glucose oxidation and conversion to triglyceride fatty acids are measured. Using serum from ad libitum fed rats as control, preincubation with serum from obese rats (20 days at 2 x normal intake) depressed basal and insulin stimulated glucose oxidation, and basal fatty acid synthesis. Serum from obese parabiotic rats and parabiotic partners of obese rats depressed basal fatty acid synthesis. This assay allows us to test serum for anti-lipogenic activity and may be used to identify the factor responsible for this activity in obese animals.

  9. Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson's disease and in hepatic encephalopathy.

    PubMed

    Siotto, Mariacristina; Pasqualetti, Patrizio; Marano, Massimo; Squitti, Rosanna

    2014-10-01

    Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks. PMID:24663495

  10. Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads

    PubMed Central

    Wu, Ding; Nair-Gill, Evan; Sher, Dorie A.; Parker, Laurie L.; Campbell, Jennifer M.; Siddiqui, Mariah; Stock, Wendy; Kron, Stephen J.

    2015-01-01

    There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats. PMID:16236241

  11. Activation characteristics of a solid breeder blanket for a fusion power demonstration reactor

    NASA Astrophysics Data System (ADS)

    Fischer, Ulrich; Tsige-Tamirat, Haileyesus

    2002-12-01

    Activation characteristics have been assessed for a helium cooled solid breeder blanket on the basis of three-dimensional activation calculations for a 2200 MW fusion power demonstration reactor. FISPACT inventory calculations were performed for the beryllium neutron multiplier, the Li 4SiO 4 breeder ceramics and the Eurofer low activation steel. Neutron flux spectra distributions were provided by a previous MCNP calculation. Detailed spatial distributions have been obtained for the nuclide inventories and related quantities such as activity, decay heat and contact dose rate. These data are available form the authors upon request. On the basis of the calculated contact gamma dose rates, the waste quality was assessed with regard to a possible re-use of the activated materials following the remote or the hands-on handling recycling options.

  12. A novel protease activity assay method based on an engineered autoinhibited protein using an enzyme-linked immunoassay.

    PubMed

    Yoon, Hyun Kyung; Yoo, Tae Hyeon

    2013-12-01

    Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification. PMID:24106734

  13. Establishment of a New Cell-Based Assay To Measure the Activity of Sweeteners in Fluorescent Food Extracts

    PubMed Central

    2011-01-01

    Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca2+ concentration using Ca2+-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca2+-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca2+ indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. PMID:21981007

  14. Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

    PubMed

    Toda, Yasuka; Okada, Shinji; Misaka, Takumi

    2011-11-23

    Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca(2+) concentration using Ca(2+)-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca(2+)-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca(2+) indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. PMID:21981007

  15. Active capture and transport of virus particles using a biomolecular motor-driven, nanoscale antibody sandwich assay.

    SciTech Connect

    Hess, Henry; Bachand, Marlene; Bachand, George David; Carroll-Portillo, Amanda; Rivera, Susan B.

    2005-07-01

    Virus particles are captured and transported using kinesin-driven, antibody-functionalized microtubules. The functionalization was achieved through covalent crosslinking, which consequently enhanced the microtubule stability. The capture and transport of the virus particles was subsequently demonstrated in gliding motility assays in which antibody-coated microtubules functioned as capture elements, and antibody-coated microspheres served as fluorescent reporters.

  16. Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay.

    PubMed

    Hellum, Marit; Øvstebø, Reidun; Trøseid, Anne-Marie S; Berg, Jens P; Brandtzaeg, Petter; Henriksson, Carola E

    2012-09-01

    There is increasing clinical interest for measuring microparticle (MP)-associated tissue factor (TF) activity owing to its possible role as a prothrombotic biomarker in a variety of diseases. However, the methods used are to various extents hampered by lack of (pre)analytical standardization as well as limited published documentation. The objective of this study was to evaluate the performance of the Zymuphen MP-TF kit and the calibrated automated thrombogram (CAT) assay in measuring MP-associated TF activity in plasma using a Neisseria meningitidis (Nm)-stimulated whole blood model. In addition, (pre)analytical variables like centrifugation procedures, freezing/thawing and the effect of addition of exogenous phosphatidylserine in plasma were evaluated in the CAT assay. Citrate-anticoagulated blood was stimulated with Nm bacteria for 4 h before platelet-poor plasma (PPP) or platelet-free plasma (PFP) were prepared and assayed with either of the two methods. Nm dose-dependently (10-10 bacteria/ml) induced TF-specific activity, measured as decreased lagtimes, in the CAT assay. The Zymuphen MP-TF kit also detected TF activity, although much higher Nm doses (10 bacteria/ml) were required to achieve measurable levels. Neither freezing/thawing nor the use of PPP vs. PFP influenced the TF activity, measured over a broad range of lagtimes, in the CAT assay. In conclusion, changes in lagtime in the CAT assay reflected levels of MP-associated TF activity in a more sensitive manner than the Zymuphen MP-TF kit did, in our Nm-stimulated whole blood system. PMID:22732249

  17. A novel, sensitive assay for high-throughput molecular detection of plasmodia for active screening of malaria for elimination.

    PubMed

    Cheng, Zhibin; Sun, Xiaodong; Yang, Ye; Wang, Heng; Zheng, Zhi

    2013-01-01

    Although malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 μl of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three false-negative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/μl. The assay's simple work flow, high throughput, and sensitivity make it suitable for active malaria screening. PMID:23100347

  18. KiC assay: a quantitative mass spectrometry-based approach for kinase client screening and activity analysis [corrected].

    PubMed

    Huang, Yadong; Thelen, Jay J

    2012-01-01

    Protein phosphorylation is one of the most important posttranslational modifications (PTMs) involved in the transduction of cellular signals. The number of kinases in eukaryotic genomes ranges from several hundred to over one thousand. And with rapidly evolving mass spectrometry (MS)-based approaches, thousands to tens of thousands of phosphorylation sites (phosphosites) have been reported from various eukaryotic organisms, from man to plants. In this relative context, few bona fide kinase-client relationships have been identified to date. To merge the gap between these phosphosites and the cognate kinases that beget these events, comparable large-scale methodologies are required. We describe in detail a MS-based method for identifying kinase-client interactions and quantifying kinase activity. We term this novel Kinase-Client assay, the KiC assay. The KiC assay relies upon the fact that substrate specificities of many kinases are largely determined by primary amino acid sequence or phosphorylation motifs, which consist of key amino acids surrounding the phosphorylation sites. The workflow for detecting kinase-substrate interactions includes four major steps: (1) preparation of purified kinases and synthetic peptide library, (2) in vitro kinase peptide library assay, (3) liquid chromatography (LC)-tandem MS (MS/MS) analysis, and (4) data processing and interpretation. Kinase activity is quantified with the KiC assay by monitoring spectral counts of phosphorylated and unphosphorylated peptides as the readout from LC-tandem mass spectrometry. The KiC assay can be applied as a discovery assay to screen kinases against a synthetic peptide library to find kinase-client relationships or as a targeted assay to characterize kinase kinetics. PMID:22665311

  19. Evaluation of bioluminescence-based assays of anti-malarial drug activity

    PubMed Central

    2013-01-01

    Background Transgenic Plasmodium falciparum expressing luciferase offers an attractive bioluminescence-based assay platform for the investigation of the pharmacological properties of anti-malarial drugs. Here a side-by-side comparison of bioluminescence and fluorescence-based assays, utilizing a luciferase reporter cassette that confers a strong temporal pattern of luciferase expression during the S-phase of intraerythrocytic development, is reported. Methods Key assay parameters for a range of commercially available luminogenic substrates are determined and compared to those measured using a Malaria Sybr Green I fluorescence assay. In addition, the short-term temporal effects of anti-malarial compounds are evaluated using both bioluminescent and fluorescent assay platforms. Results The Z’, % coefficient of variation and 50% inhibition concentrations are essentially the same for bioluminescent and fluorescent assays in transgenic parasites generated in both chloroquine-sensitive and -resistant genetic backgrounds. Bioluminescent assays, irrespective of the luminogenic agent employed, do, however, offer significantly enhanced signal-to-noise ratios. Moreover, the bioluminescent assay is more dynamic in terms of determining temporal effects immediately following drug perturbation. Conclusion This study suggests that opportunities for bioluminescence-based assays lie not in the measurement of 50% inhibition concentrations, where the cheaper fluorescence assay performs excellently and is not restricted by the need to genetically modify the parasite clone under investigation. Instead, assays that use the dynamic response of the luciferase reporter for semi-automated screening of additional pharmacological properties, such as relative rate-of-kill and lethal dose estimation, are a more attractive development opportunity. PMID:23394077

  20. Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

    PubMed

    Huber, Warren J; Marohnic, Christopher C; Peters, Michelle; Alam, Jawed; Reed, James R; Masters, Bettie Sue Siler; Backes, Wayne L

    2009-04-01

    Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment. PMID:19131520

  1. Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

    PubMed

    Kumrungsee, Thanutchaporn; Akiyama, Sayaka; Guo, Jian; Tanaka, Mitsuru; Matsui, Toshiro

    2016-12-15

    Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1μM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300μM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6μM, respectively. PMID:27451188

  2. Tested Demonstrations.

    ERIC Educational Resources Information Center

    Gilbert, George L., Ed.

    1987-01-01

    Presented are three demonstrations for chemical education. The activities include: (1) demonstration of vapor pressure; (2) a multicolored luminol-based chemiluminescence demonstration; and (3) a Charles's Law/Vapor pressure apparatus. (RH)

  3. Reporter Phage and Breath Tests: Emerging Phenotypic Assays for Diagnosing Active Tuberculosis, Antibiotic Resistance, and Treatment Efficacy

    PubMed Central

    Jain, Paras; Thaler, David S.; Maiga, Mamoudou; Timmins, Graham S.; Bishai, William R.; Hatfull, Graham F.; Larsen, Michelle H.; Jacobs, William R.

    2011-01-01

    The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics. PMID:21996696

  4. An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions

    PubMed Central

    Pulkkinen, Elsi; Haapa-Paananen, Saija; Savilahti, Harri

    2014-01-01

    Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications. PMID:26442171

  5. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  6. 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. PMID:23654197

  7. A transcriptomic reporter assay employing neutrophils to measure immunogenic activity of septic patients’ plasma

    PubMed Central

    2014-01-01

    the other independent set. This subset included several genes previously established in sepsis pathogenesis as well as novel genes. Conclusions These results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for studying plasma, in this case from septic patients. The distinctive transcriptional signature we found could potentially help predict severity of disease and guide treatment. Our findings also shed new light on mechanisms of immune dysregulation in sepsis. PMID:24612859

  8. Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease.

    PubMed

    Liu, Xue-Guo; Xing, Xiao-Jing; Li, Bo; Guo, Yong-Ming; Zhang, Ye-Zhen; Yang, Yan; Zhang, Lian-Feng

    2016-07-15

    A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3'-end (F-DNA) and its complementary sequence modified with a 5'-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5'-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field. PMID:27015149

  9. Development of a new catalase activity assay for biological samples using optical CUPRAC sensor

    NASA Astrophysics Data System (ADS)

    Bekdeşer, Burcu; Özyürek, Mustafa; Güçlü, Kubilay; Alkan, Fulya Üstün; Apak, Reşat

    2014-11-01

    A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor-based ‘cupric reducing antioxidant capacity' (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-colored Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 μM). This optical sensor-based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC.

  10. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    PubMed

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved. PMID:27041274

  11. A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

    PubMed Central

    Rand, Matthew D.

    2014-01-01

    The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules. PMID:25046169

  12. Metabolic activation of organic extracts from diesel, coke oven, roofing tar, and cigarette smoke emissions in the Ames Assay

    SciTech Connect

    Williams, K.; Lewtas, J.

    1985-01-01

    Four environmental emissions samples were ranked by their genotoxic potency in several bioassays. Although the relative potency of a series of automotive emissions (diesel and gasoline) in the Ames assay correlated well with the relative potency in mammalian cell and mouse skin, this was not the case for the coke oven, roofing tar, and cigarette smoke condensate (CSC) emissions. This study examines the role of metabolic activation in determining the difference between a microbial and a mammalian bioassay in ranking the genotoxic potency of these environmental emissions. Uninduced and Aroclor 1254-induced S9 from both rat and hamster liver were compared as the metabolic activator in the Ames assay with Salmonella typhimurium TA98. The diesel emissions sample was direct-acting while the other samples required activation. The standard S9 concentration also produced the maximum mutagenic activity. The relative potency of these four samples was not significantly different between the microbial (Ames), mammalian cell (mouse lymphoma), and tumor initiation (mouse skin) assays. These results suggest that the differences observed between the relative mutagenic activity of these emissions in the mammalian cell and microbial assays was not due to a lack of optimization of the S9 system but may be inherent in the different response of the indicator cells to different chemical classes.

  13. An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

    PubMed

    Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing; Marko, John F; Matouschek, Andreas

    2016-09-15

    The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. PMID:27296635

  14. Active Closed-Loop Stator Vane Flow Control Demonstrated in a Low-Speed Multistage Compressor

    NASA Technical Reports Server (NTRS)

    Bright, Michelle M.; Culley, Dennis E.; Strazisar, Anthony J.

    2004-01-01

    Closed-loop flow control was successfully demonstrated on the surface of stator vanes in NASA Glenn Research Center's Low-Speed Axial Compressor (LSAC) facility. This facility provides a flow field that accurately duplicates the aerodynamics of modern highly loaded compressors. Closed-loop active flow control uses sensors and actuators embedded within engine components to dynamically alter the internal flow path during off-nominal operation in order to optimize engine performance and maintain stable operation.

  15. Field Demonstrations of Active Laser Ranging with Sub-mm Precision

    NASA Technical Reports Server (NTRS)

    Chen, Yijiang; Birnbaum, Kevin M.; Hemmati, Hamid

    2011-01-01

    Precision ranging between planets will provide valuable information for scientific studies of the solar system and fundamental physics. Current passive ranging techniques using retro-reflectors are limited to the Earth-Moon distance due to the 1/R? losses. We report on a laboratory realization and field implementation of active laser ranging in real-time with two terminals, emulating interplanetary distance. Sub-millimeter accuracy is demonstrated.

  16. An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

    PubMed

    King, Brian C; Donnelly, Marie K; Bergstrom, Gary C; Walker, Larry P; Gibson, Donna M

    2009-03-01

    Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production. PMID:18973283

  17. [Ecological demonstration activity and eco-civilization construction mode: review and prospects].

    PubMed

    Mao, Hui-ping; He, Xuan; He, Jia; Niu, Dong-jie; Bao, Cun-kuan

    2013-04-01

    Ecological civilization is to normalize human development behaviors to harmonize the relationships between social and ecological development and eco-environment protection. In this paper, a comparative analysis was made on the ecological demonstration activities of ecological demonstration areas led by the Ministry of Environmental Protection, exemplar cities of national environmental protection, and ecological provinces, cities, and counties. It was considered that all the ecological demonstration activities had the problems of lacking pertinence of construction goals, disordered construction subjects, inefficient construction processes, and lacking continuous incentive mechanisms of assessment. In the meantime, through the analysis of the connotations of eco-civilization, the relationships between eco-civilization and eco-demonstration constructions were approached, and the eco-civilization construction mode was put forward in terms of construction goal, construction subject, and construction processes and assessment. The construction mode included the construction goal based on regional characteristics; the synergistic cooperation of construction subjects, the expanding ways of public participation, and the establishment of evaluation system for comprehensively measuring the 'actions and results'. PMID:23898681

  18. Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry.

    PubMed

    Murase, Takayo; Nampei, Mai; Oka, Mitsuru; Ashizawa, Naoki; Matsumoto, Koji; Miyachi, Atsushi; Nakamura, Takashi

    2016-01-01

    Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter

  19. Macroautophagic cargo sequestration assays.

    PubMed

    Seglen, Per O; Luhr, Morten; Mills, Ian G; Sætre, Frank; Szalai, Paula; Engedal, Nikolai

    2015-03-01

    Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy. PMID:25576638

  20. In Vitro Activity of Ceftazidime-Avibactam Combination in In Vitro Checkerboard Assays

    PubMed Central

    Melchers, Maria J.; van Mil, Anita C.; Nichols, Wright W.

    2014-01-01

    To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobacter cloacae) and 26 strains of Pseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n = 69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC50 and MIC90 being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases. PMID:25487794

  1. Colorimetrical rate assay for urinary dipeptidyl peptidase IV (DPPIV) activity using a new substrate.

    PubMed

    Shibuya-Saruta, H; Sugiyama, M; Kasahara, Y

    1995-01-01

    We synthesized a new substrate glycyl-L-proline 3,5-dibromo-4-hydroxyanilide (Gly-Pro-DBAP), for dipeptidyl peptidase IV (DPPIV). Its hydrolysis by DPPIV resulted in the formation of a chromophore, 2,6-dibromophenol-indo-p-xylenol, and its maximal absorption wavelength (600 nm) was longer than that of p-nitroaniline (415 nm) released from conventional substrate, glycyl-L-proline p-nitroanilide (Gly-Pro-pNA). We also established the rate assay for urinary DPPIV activity using Gly-Pro-DBAP. The optimum pH was between 8.5 and 9.0. The apparent Km was 1.1 mmol/1. The detectable range was 2.5-350 U/l. No changes in blank values occurred throughout the enzyme reaction in the optimum pH. Its value was also much lower than Gly-Pro-pNA. CVs for within-run and between-run were 1.1% (n = 10) and 3.0% (n = 10), respectively. Among tested peptidases, only DPPIV could hydrolyze Gly-Pro-DBAP. Among the protease inhibitors, only two, diprotin-A and phenylmethylsulfonyl fluoride (PMSA), could inhibit DPPIV activity. The present method did not interfere with urinary ingredients such as hemoglobin. The correlation between the present (y) and conventional (x) methods is presented by the equation y = 1.121x + 0.096 (r = 0.993). Thus the present method provides practical advantages over the conventional method for routine laboratory use. PMID:7714663

  2. T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium, mercury, and zinc.

    PubMed

    Li, Jian; Liu, Yun; Kong, Dongdong; Ren, Shujuan; Li, Na

    2016-05-01

    In the present study, a two-hybrid yeast bioassay and a T-screen were used to screen for the thyroid receptor (TR)-disrupting activity of select metallic compounds (CdCl2, ZnCl2, HgCl2, CuSO4, MnSO4, and MgSO4). The results reveal that none of the tested metallic compounds showed TR-agonistic activity, whereas ZnCl2, HgCl2, and CdCl2 demonstrated TR antagonism. For the yeast assay, the dose-response relationship of these metallic compounds was established, and the concentrations producing 20 % of the maximum effect of ZnCl2, HgCl2, and CdCl2 were 9.1 × 10(-5), 3.2 × 10(-6), and 1.2 × 10(-6) mol/L, respectively. The T-screen also supported the finding that ZnCl2, HgCl2, and CdCl2 decreased the cell proliferation at concentrations ranging from 10(-6) to 10(-4) mol/L. Furthermore, the thyroid-disrupting activity of metallic compounds in environmental water samples collected from the Guanting Reservoir, Beijing, China was evaluated. Solid-phase extraction was used to separate the organic extracts, and a modified two-hybrid yeast bioassay revealed that the metallic compounds in the water samples could affect thyroid hormone-induced signaling by decreasing the binding of the thyroid hormone. The addition of ethylenediaminetetraacetic acid (30 mg/L) could eliminate the effects. Thus, the cause(s) of the thyroid toxicity in the water samples appeared to be partly related to the metallic compounds. PMID:26856863

  3. A versatile biosensing system for DNA-related enzyme activity assay via the synthesis of silver nanoclusters using enzymatically-generated DNA as template.

    PubMed

    Yuan, Yijia; Li, Wenhua; Liu, Zhuoliang; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2014-11-15

    In the present day, oligonucleotide-encapsulated silver clusters (DNA-AgNCs) have been widely applied into bio-analysis as a signal producer. Herein, we developed a novel method to synthesize DNA-AgNCs encapsulated by long-chain cytosine (C)-rich DNA. Such DNA was polymerized in a template-free way by terminal deoxynucleotidyl transferase (TdT). We demonstrated that TdT-polymerized long chain C-rich DNA can serve as an excellent template for AgNCs synthesis. Based on this novel synthesis strategy, we developed a label-free and turn-on fluorescence assay to detect TdT activity with ultralow limit of detection (LOD) of 0.0318 U and ultrahigh signal to background (S/B) of 46.7. Furthermore, our proposed method was extended to a versatile biosensing strategy for turn-on nucleases activity assay based on the enzyme-activated TdT polymerization. Two nucleases, EcoRI and ExoIII as model of endonuclease and exonuclease, respectively, have been detected with high selectivity and competitive low LOD of 0.0629 U and 0.00867 U, respectively. Our work demonstrates the feasibility of TdT polymerization-based DNA-AgNCs synthesis strategy as a versatile and potent biosensing platform to detect the activity of DNA-related enzymes. PMID:24907540

  4. Analysis of Flavonoids in Lotus (Nelumbo nucifera) Leaves and Their Antioxidant Activity Using Macroporous Resin Chromatography Coupled with LC-MS/MS and Antioxidant Biochemical Assays.

    PubMed

    Zhu, Ming-Zhi; Wu, Wei; Jiao, Li-Li; Yang, Ping-Fang; Guo, Ming-Quan

    2015-01-01

    Lotus (Nelumbo nucifera) leaves, a traditional Chinese medicinal herb, are rich in flavonoids. In an effort to thoroughly analyze their flavonoid components, macroporous resin chromatography coupled with HPLC-MS/MS was employed to simultaneously enrich and identify flavonoids from lotus leaves. Flavonoids extracted from lotus leaves were selectively enriched in the macroporous resin column, eluted subsequently as fraction II, and successively subjected to analysis with the HPLC-MS/MS and bioactivity assays. Altogether, fourteen flavonoids were identified, four of which were identified from lotus leaves for the first time, including quercetin 3-O-rhamnopyranosyl-(1→2)-glucopyranoside, quercetin 3-O-arabinoside, diosmetin 7-O-hexose, and isorhamnetin 3-O-arabino- pyranosyl-(1→2)-glucopyranoside. Further bioactivity assays revealed that these flavonoids from lotus leaves possess strong antioxidant activity, and demonstrate very good potential to be explored as food supplements or even pharmaceutical products to improve human health. PMID:26060918

  5. Total esterase activity in human saliva: Validation of an automated assay, characterization and behaviour after physical stress.

    PubMed

    Tecles, Fernando; Tvarijonaviciute, Asta; De Torre, Carlos; Carrillo, José M; Rubio, Mónica; García, Montserrat; Cugat, Ramón; Cerón, José J

    2016-07-01

    Although saliva has esterase activity, this activity has not been characterized or studied in individuals subjected to physical stress. The aim of this report was to develop and validate an automated spectrophotometric assay for total esterase activity measurement in human saliva, as well as to study the contribution of different enzymes on this activity and its behaviour under physical stress in healthy subjects. The assay used 4-nitrophenyl acetate as substrate and was precise, accurate and provided low limits of detection and quantification. Inhibition with diisopropylfluorophosphate showed that cholinesterase, carboxylesterase and cholesterol esterase contributions not represented more than 20% of total esterase. Addition of standards of lipase and albumin to saliva samples showed that both proteins significantly contributed to esterase activity only when equal or higher than 11.6 IU/L and 250 μg/mL, respectively. Western blot analyses showed absence of paraoxonase-1 and high amount of carbonic anhydrase-VI. The high affinity of purified carbonic anhydrase-VI for the substrate supported a major contribution of this enzyme. Total esterase activity and alpha-amylase was measured in saliva samples from 12 healthy male students before and after participation in an indoor football match. The activity significantly increased after match and positively correlated with salivary alpha-amylase. This method could be used as a biomarker of physical stress in humans, with carbonic anhydrase-VI being the esterase that contributed more to the activity of the assay. PMID:27045801

  6. Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity.

    PubMed

    Fu, Suneng; Yalcin, Abdullah; Lee, Grace Y; Li, Ping; Fan, Jason; Arruda, Ana Paula; Pers, Benedicte M; Yilmaz, Mustafa; Eguchi, Kosei; Hotamisligil, Gökhan S

    2015-06-17

    The endoplasmic reticulum (ER) plays a critical role in protein, lipid, and glucose metabolism as well as cellular calcium signaling and homeostasis. Perturbation of ER function and chronic ER stress are associated with many pathologies ranging from diabetes and neurodegenerative diseases to cancer and inflammation. Although ER targeting shows therapeutic promise in preclinical models of obesity and other pathologies, the available chemical entities generally lack the specificity and other pharmacological properties required for effective clinical translation. To overcome these challenges and identify new potential therapeutic candidates, we first designed and chemically and genetically validated two high-throughput functional screening systems that independently measure the free chaperone content and protein-folding capacity of the ER. With these quantitative platforms, we characterized a small-molecule compound, azoramide, that improves ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress in multiple systems. This compound also exhibited potent antidiabetic efficacy in two independent mouse models of obesity by improving insulin sensitivity and pancreatic β cell function. Together, these results demonstrate the utility of this functional, phenotypic assay platform for ER-targeted drug discovery and provide proof of principle for the notion that specific ER modulators can be potential drug candidates for type 2 diabetes. PMID:26084805

  7. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.

    PubMed

    Jia, Nan; Nakazawa, Yuka; Guo, Chaowan; Shimada, Mayuko; Sethi, Mieran; Takahashi, Yoshito; Ueda, Hiroshi; Nagayama, Yuji; Ogi, Tomoo

    2015-01-01

    DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete. PMID:25474029

  8. A sensitive, accurate assay for extrinsic pathway inhibitor (EPI) activity in rabbit plasma: Paradoxical effect of excess exogenous factor X

    SciTech Connect

    Warr, T.A.; Rao, L.V.; Rapaport, S.I. )

    1990-08-15

    A sensitive assay is described for the measurement of rabbit plasma EPI activity in experimental studies of induced hypercoagulable states in this species. It is based upon the ability of a dilution of rabbit plasma to inhibit human factor VIIa/rabbit brain tissue factor (TF) catalyzed activation of human factor IX (tritiated activation peptide release assay). Addition of {sup 3}H-factor IX to the reaction mixture is delayed for 45 minutes to allow full inhibition by EPI/factor Xa complex before the residual catalytic activity of factor VIIa/TF is measured. Although the diluted rabbit plasma test sample contains both EPI and factor X, supplemental factor X is added to the reaction mixture to assure that only EPI content of the test sample affects the assay result. However, the final concentration of factor X in the reaction mixture is critical. Too high a concentration of factor X diminishes the sensitivity of the assay. The reason for this phenomenon, which was observed with both human and rabbit factor X preparations, is unknown.

  9. In Vitro Assays for Assessment of Androgenic and Estrogenic Activity of Defined Mixtures and Complex Environmental Samples

    EPA Science Inventory

    Point sources of endocrine active compounds to aquatic environments such as waste water treatment plants, pulp and paper mills, and animal feeding operations invariably contain complex mixtures of chemicals. The current study investigates the use of targeted in vitro assays des...

  10. METABOLIC ACTIVATION OF ORGANIC EXTRACTS FROM DIESEL, COKE OVEN, ROOFING TAR, AND CIGARETTE SMOKE EMISSIONS IN THE AMES ASSAY

    EPA Science Inventory

    The role of metabolic activation in the difference between a microbial and mammalian bioassays in the ranking of genotoxic potency of several environmental emissions was investigated. Although the relative potency in the Ames assay correlated well with the relative potency in mam...

  11. Ultrasensitive detection of protease activity of anthrax and botulinum toxins by a new PCR-based assay.

    PubMed

    Kolesnikov, Alexander V; Kozyr, Arina V; Ryabko, Alyona K; Shemyakin, Igor G

    2016-02-01

    Anthrax and botulism are dangerous infectious diseases that can be fatal unless detected and treated quickly. Fatalities from these diseases are primarily due to endopeptidase toxins secreted by the pathogens. Rapid and sensitive detection of the presence of active toxins is the key element for protection from natural outbreaks of anthrax and botulism, as well as from the threat of bioterrorism. We describe an ultrasensitive polymerase chain reaction (PCR)-based assay for detecting proteolytic activity of anthrax and botulinum toxins using composite probes consisting of covalent peptide-DNA conjugate for the detection of anthrax, and noncovalent protein-aptamer assembly to assay botulinum toxin activity. Probes immobilized on the solid-phase support are cleaved by toxins to release DNA, which is detected by real-time PCR. Both assays can detect subpicogram quantities of active toxins isolated from composite matrices. Special procedures were developed to isolate intact toxins from the matrices under mild conditions. The assay is rapid, uses proven technologies, and can be modified to detect other proteolytic and biopolymer-degrading enzymes. PMID:26620058

  12. Novel "hybrid" iron chelators derived from aroylhydrazones and thiosemicarbazones demonstrate selective antiproliferative activity against tumor cells.

    PubMed

    Lovejoy, David B; Richardson, Des R

    2002-07-15

    We previously demonstrated that 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) and other aroylhydrazone chelators possess potent antineoplastic activity because of their ability to bind iron (Fe). From these studies, we identified structural components of the hydrazones that provide antineoplastic activity, namely the salicylaldehyde and 2-hydroxy-1-naphthylaldehyde moieties. A related group of chelators known as the thiosemicarbazones also show pronounced antitumor activity because of their ability to inhibit ribonucleotide reductase. Considering this, we designed a new series of "hybrid ligands" by condensation of the aldehydes described above with a range of thiosemicarbazides. The parent compound of these ligands is 2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT). Of 8 NT analogues, 3 chelators, namely NT, N4mT (2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone), and N44mT (2-hydroxy-1-naphthylaldehyde-4,4-dimethyl-3-thiosemicarbazone), showed high antiproliferative activity against SK-N-MC neuroepithelioma cells (50% inhibitory concentration [IC(50)] = 0.5-1.5 microM). Indeed, their activity was significantly (P <.0001) greater than that of desferrioxamine (DFO) (IC(50) = 22 microM). We demonstrate that 311, a 311 analogue (311m), and several NT-series chelators have significantly (P <.001) greater antiproliferative activity against tumor cells than against a range of normal cell types. For example, the IC(50) values of NT and N4mT in SK-N-MC neuroepithelioma cells were 0.5 microM, whereas for fibroblasts the IC(50) values were greater than 25 microM. Further, the effect of one of the most potent chelators (311m) on preventing the growth of bone marrow stem cell cultures was far less than that of doxorubicin and similar to that of cisplatin. These studies support the further development of these chelators as antiproliferative agents. PMID:12091363

  13. Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances

    EPA Science Inventory

    In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized appro...

  14. Smart phone: a popular device supports amylase activity assay in fisheries research.

    PubMed

    Thongprajukaew, Karun; Choodum, Aree; Sa-E, Barunee; Hayee, Ummah

    2014-11-15

    Colourimetric determinations of amylase activity were developed based on a standard dinitrosalicylic acid (DNS) staining method, using maltose as the analyte. Intensities and absorbances of red, green and blue (RGB) were obtained with iPhone imaging and Adobe Photoshop image analysis. Correlation of green and analyte concentrations was highly significant, and the accuracy of the developed method was excellent in analytical performance. The common iPhone has sufficient imaging ability for accurate quantification of maltose concentrations. Detection limits, sensitivity and linearity were comparable to a spectrophotometric method, but provided better inter-day precision. In quantifying amylase specific activity from a commercial source (P>0.02) and fish samples (P>0.05), differences compared with spectrophotometric measurements were not significant. We have demonstrated that iPhone imaging with image analysis in Adobe Photoshop has potential for field and laboratory studies of amylase. PMID:24912700

  15. Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens.

    PubMed

    Spinler, Jennifer K; Taweechotipatr, Malai; Rognerud, Cheryl L; Ou, Ching N; Tumwasorn, Somying; Versalovic, James

    2008-06-01

    Lactobacillus reuteri is a commensal-derived anaerobic probiotic that resides in the human gastrointestinal tract. L. reuteri converts glycerol into a potent broad-spectrum antimicrobial compound, reuterin, which inhibits the growth of gram-positive and gram-negative bacteria. In this study, we compared four human-derived L. reuteri isolates (ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and ATCC PTA 5289) in their ability to produce reuterin and to inhibit the growth of different enteric pathogens in vitro. Reuterin was produced by each of the four L. reuteri strains and assessed for biological activity. The minimum inhibitory concentration (MIC) of reuterin derived from each strain was determined for the following enteric pathogens: enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, Salmonella enterica, Shigella sonnei and Vibrio cholerae. We also analyzed the relative abilities of L. reuteri to inhibit enteric pathogens in a pathogen overlay assay. The magnitude of reuterin production did not directly correlate with the relative ability of L. reuteri to suppress the proliferation of enteric pathogens. Additional antimicrobial factors may be produced by L. reuteri, and multiple factors may act synergistically with reuterin to inhibit enteric pathogens. PMID:18396068

  16. Quantitative Signaling and Structure-Activity Analyses Demonstrate Functional Selectivity at the Nociceptin/Orphanin FQ Opioid Receptor.

    PubMed

    Chang, Steven D; Mascarella, S Wayne; Spangler, Skylar M; Gurevich, Vsevolod V; Navarro, Hernan A; Carroll, F Ivy; Bruchas, Michael R

    2015-09-01

    Comprehensive studies that consolidate selective ligands, quantitative comparisons of G protein versus arrestin-2/3 coupling, together with structure-activity relationship models for G protein-coupled receptor (GPCR) systems are less commonly employed. Here we examine biased signaling at the nociceptin/orphanin FQ opioid receptor (NOPR), the most recently identified member of the opioid receptor family. Using real-time, live-cell assays, we identified the signaling profiles of several NOPR-selective ligands in upstream GPCR signaling (G protein and arrestin pathways) to determine their relative transduction coefficients and signaling bias. Complementing this analysis, we designed novel ligands on the basis of NOPR antagonist J-113,397 [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] to explore structure-activity relationships. Our study shows that NOPR is capable of biased signaling, and further, the NOPR selective ligands MCOPPB [1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-(3R)-3-piperidinyl-1H-benzimidazole trihydrochloride] and NNC 63-0532 [8-(1-naphthalenylmethyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-3-acetic acid, methyl ester] are G protein-biased agonists. Additionally, minor structural modification of J-113,397 can dramatically shift signaling from antagonist to partial agonist activity. We explore these findings with in silico modeling of binding poses. This work is the first to demonstrate functional selectivity and identification of biased ligands at the nociceptin opioid receptor. PMID:26134494

  17. Renal adenylate cyclase assay for biologically active parathyroid hormone: clinical utility and physiological significance.

    PubMed

    Auf'mkolk, B; Hesch, R D

    1986-01-01

    The stimulation of cyclic AMP production by human renal cortical membranes in the presence of the GTP analogue 5'-guanylimidodiphosphate and a calcium chelator represents a homologous assay system for the evaluation of biologically active parathyroid hormone (bioPTH) in human serum. Bioactive PTH was raised above normal (normal range: undetectable to 4.6 pmol human PTH(1-34) per 1) in 13/17 (76%) patients with primary hyperparathyroidism, in 5/6 (83%) patients with surgically proven hyperparathyroidism secondary to chronic renal failure, in 4/5 (80%) patients with hyperparathyroidism secondary to hypocalcaemia, in all three patients with pseudohypoparathyroidism, in 5/17 (29%) patients with osteoporosis and in 1/9 (11%) patients with renal stones and/or hypercalciuria. Bioactive PTH correlated positively with immunoreactive PTH (iPTH) measured with a radioimmunoassay predominantly recognizing the middle- and carboxyl-terminal region of the PTH molecule (r = 0.503, P less than 0.001). A positive correlation (r = 0.572, P less than 0.05) was found between values of serum calcium and bioPTH in the group with primary hyperparathyroidism. Immunoreactive PTH did not correlate significantly with calcium in this group. In the other patients except those who had chronic renal failure, a negative correlation between serum calcium and both bioPTH and iPTH was observed (P less than 0.01). When alkaline phosphatase was compared with bioPTH in all patients, the correlation was positive (r = 0.390, P less than 0.01); no significant correlation existed between iPTH and alkaline phosphatase in the patients studied.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3944539

  18. Demonstration of prosthetic activation of central auditory pathways using ( sup 14 C)-2-deoxyglucose

    SciTech Connect

    Evans, D.A.; Niparko, J.K.; Altschuler, R.A.; Frey, K.A.; Miller, J.M. )

    1990-02-01

    The cochlear prosthesis is not applicable to patients who lack an implantable cochlea or an intact vestibulocochlear nerve. Direct electrical stimulation of the cochlear nucleus (CN) of the brain stem might provide a method for auditory rehabilitation of these patients. A penetrating CN electrode has been developed and tissue tolerance to this device demonstrated. This study was undertaken to evaluate metabolic activation of central nervous system (CNS) auditory tracts produced by such implants. Regional cerebral glucose use resulting from CN stimulation was estimated in a series of chronically implanted guinea pigs with the use of ({sup 14}C)-2-deoxyglucose (2-DG). Enhanced 2-DG uptake was observed in structures of the auditory tract. The activation of central auditory structures achieved with CN stimulation was similar to that produced by acoustic stimulation and by electrical stimulation of the modiolar portion of the auditory nerve in control groups. An interesting banding pattern was observed in the inferior colliculus following CN stimulation, as previously described with acoustic stimulation. This study demonstrates that functional metabolic activation of central auditory pathways can be achieved with a penetrating CNS auditory prosthesis.

  19. Radioactive Demonstrations Of Fluidized Bed Steam Reforming (FBSR) With Hanford Low Activity Wastes

    SciTech Connect

    Jantzen, C. M.; Crawford, C. L.; Burket, P. R.; Bannochie, C. J.; Daniel, W. G.; Nash, C. A.; Cozzi, A. D.; Herman, C. C.

    2012-10-22

    Several supplemental technologies for treating and immobilizing Hanford low activity waste (LAW) are being evaluated. One immobilization technology being considered is Fluidized Bed Steam Reforming (FBSR) which offers a low temperature (700-750?C) continuous method by which wastes high in organics, nitrates, sulfates/sulfides, or other aqueous components may be processed into a crystalline ceramic (mineral) waste form. The granular waste form produced by co-processing the waste with kaolin clay has been shown to be as durable as LAW glass. The FBSR granular product will be monolithed into a final waste form. The granular component is composed of insoluble sodium aluminosilicate (NAS) feldspathoid minerals such as sodalite. Production of the FBSR mineral product has been demonstrated both at the industrial, engineering, pilot, and laboratory scales on simulants. Radioactive testing at SRNL commenced in late 2010 to demonstrate the technology on radioactive LAW streams which is the focus of this study.

  20. HPLC-Analysis of Polyphenolic Compounds in Gardenia jasminoides and Determination of Antioxidant Activity by Using Free Radical Scavenging Assays

    PubMed Central

    Uddin, Riaz; Saha, Moni Rani; Subhan, Nusrat; Hossain, Hemayet; Jahan, Ismet Ara; Akter, Raushanara; Alam, Ashraful

    2014-01-01

    Purpose: Gardenia jasminoides is a traditional medicinal plant rich in anti-inflammatory flavonoids and phenolic compounds and used for the treatment of inflammatory diseases and pain. In this present study, antioxidant potential of Gardenia jasminoides leaves extract was evaluated by using various antioxidant assays. Methods: Various antioxidant assays such as 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, reducing power and total antioxidant capacity expressed as equivalent to ascorbic acid were employed. Moreover, phenolic compounds were detected by high-performance liquid chromatography (HPLC) coupled with diode-array detection. Results: The methanol extract showed significant free radical scavenging activities in DPPH radical scavenging antioxidant assays compared to the reference antioxidant ascorbic acid. Total antioxidant activity was increased in a dose dependent manner. The extract also showed strong reducing power. The total phenolic content was determined as 190.97 mg/g of gallic acid equivalent. HPLC coupled with diode-array detection was used to identify and quantify the phenolic compounds in the extracts. Gallic acid, (+)-catechin, rutin hydrate and quercetin have been identified in the plant extracts. Among the phenolic compounds, catechin and rutin hydrate are present predominantly in the extract. The accuracy and precision of the presented method were corroborated by low intra- and inter-day variations in quantitative results in leaves extract. Conclusion: These results suggest that phenolic compounds and flavonoids might contribute to high antioxidant activities of Gardenia jasminoides leaves. PMID:24754012

  1. Pit disassembly and conversion demonstration environmental assessment and research and development activities

    SciTech Connect

    1998-08-01

    A significant portion of the surplus plutonium is in the form of pits, a nuclear weapons component. Pits are composed of plutonium which is sealed in a metallic shell. These pits would need to be safely disassembled and permanently converted to an unclassified form that would be suitable for long-term disposition and international inspection. To determine the feasibility of an integrated pit disassembly and conversion system, a Pit Disassembly and Conversion Demonstration is proposed to take place at the Los Alamos National Laboratory (LANL). This demonstration would be done in existing buildings and facilities, and would involve the disassembly of up to 250 pits and conversion of the recovered plutonium to plutonium metal ingots and plutonium dioxide. This demonstration also includes the conversion of up to 80 kilograms of clean plutonium metal to plutonium dioxide because, as part of the disposition process, some surplus plutonium metal may be converted to plutonium dioxide in the same facility as the surplus pits. The equipment to be used for the proposed demonstration addressed in this EA would use some parts of the Advanced Recovery and Integrated Extraction System (ARIES) capability, other existing equipment/capacities, plus new equipment that was developed at other sites. In addition, small-scale R and D activities are currently underway as part of the overall surplus plutonium disposition program. These R and D activities are related to pit disassembly and conversion, MOX fuel fabrication, and immobilization (in glass and ceramic forms). They are described in Section 7.0. On May 16, 1997, the Office of Fissile Materials Disposition (MD) notified potentially affected states and tribes that this EA would be prepared in accordance with NEPA. This EA has been prepared to provide sufficient information for DOE to determine whether a Finding of No Significant Impact (FONSI) is warranted or whether an EIS must be prepared.

  2. Coinfection of Human Herpesviruses 6A (HHV-6A) and HHV-6B as Demonstrated by Novel Digital Droplet PCR Assay

    PubMed Central

    Leibovitch, Emily C.; Brunetto, Giovanna S.; Caruso, Breanna; Fenton, Kaylan; Ohayon, Joan; Reich, Daniel S.; Jacobson, Steven

    2014-01-01

    The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences between HHV-6A and HHV-6B has led to their recent reclassification as separate species. As it is now especially relevant to investigate each virus, our objectives were to first design a multiplex HHV-6A and HHV-6B ddPCR assay and then to investigate the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and patients with MS, a disease in which HHV-6 is thought to play a role. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus, which has important implications in clinical settings. Interestingly, upon comparing the saliva from MS patients and healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva; increased detection of HHV-6A in MS patients is consistent with other studies suggesting that this viral species (thought to be more neurotropic than HHV-6B) is more prevalent among MS patients compared to healthy donors. As the biology and disease associations between these two viral species differ, identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of each in health and disease. PMID:24663487

  3. Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells

    PubMed Central

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae. PMID:25658121

  4. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    PubMed

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae. PMID:25658121

  5. Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression

    PubMed Central

    Crucian, Brian; Sams, Clarence

    2015-01-01

    Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture

  6. Experimental demonstration of an active phase randomization and monitor module for quantum key distribution

    NASA Astrophysics Data System (ADS)

    Sun, Shi-Hai; Liang, Lin-Mei

    2012-08-01

    Phase randomization is a very important assumption in the BB84 quantum key distribution (QKD) system with weak coherent source; otherwise, eavesdropper may spy the final key. In this Letter, a stable and monitored active phase randomization scheme for the one-way and two-way QKD system is proposed and demonstrated in experiments. Furthermore, our scheme gives an easy way for Alice to monitor the degree of randomization in experiments. Therefore, we expect our scheme to become a standard part in future QKD systems due to its secure significance and feasibility.

  7. Substrate conditions that influence the assays used for determining the beta-glucosidase activity of cellulolytic microorganisms.

    PubMed

    Breuil, C; Mayers, P; Saddler, J N

    1986-11-01

    Culture filtrates from Trichoderma harzianum E58, T. reesei CL 847 and Penicillium sp. C 462 were assayed for beta-glucosidase activity using a range of substrates and sugar analysis methods. Although sugar analyses by the dinitrosalicylic acid (DNS) and Nelson-Somogyi methods gave a similar profile, when increasing concentrations of salicin were assayed, considerably higher values were obtained with the DNS assay. The salicin concentration used for the assay greatly influenced the final beta-glucosidase values with higher values obtained for T. harzianum E58 and T. reesei CL 847 at substrate concentrations of 1 mg/mL while optimum values for Penicillium sp. C 462 were obtained at substrate concentrations greater than 3 mg/mL. Low concentrations of salicin and p-nitro-phenyl-beta-D-glucopyranoside (PNPG) gave the same response as cellobiose. Cellobiose should be used at concentrations greater than 3.74 mg/mL to avoid substrate limitation of the beta-glucosidase assay. PMID:18555279

  8. Potential Role of M. tuberculosis Specific IFN-γ and IL-2 ELISPOT Assays in Discriminating Children with Active or Latent Tuberculosis

    PubMed Central

    Chiappini, Elena; Della Bella, Chiara; Bonsignori, Francesca; Sollai, Sara; Amedei, Amedeo; Galli, Luisa; Niccolai, Elena; Singh, Mahavir; D'Elios, Mario M.; de Martino, Maurizio

    2012-01-01

    Background Although currently available IGRA have been reported to be promising markers for TB infection, they cannot distinguish active tuberculosis (TB) from latent infection (LTBI). Objective Children with LTBI, active TB disease or uninfected were prospectively evaluated by an in-house ELISPOT assay in order to investigate possible immunological markers for a differential diagnosis between LTBI and active TB. Methods Children at risk for TB infection prospectively enrolled in our infectious disease unit were evaluated by in-house IFN-γ and IL-2 based ELISPOT assays using a panel of Mycobacterium tuberculosis antigens. Results Twenty-nine children were classified as uninfected, 21 as LTBI and 25 as active TB cases (including 5 definite and 20 probable cases). Significantly higher IFN-γ ELISPOT responses were observed in infected vs. uninfected children for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p = 0.003), and AlaDH (p = 0.001), while differences were not significant considering Ag85B (p = 0.063), PstS1 (p = 0.512), and HspX (16 kDa) (p = 0.139). IL-2 ELISPOT assay responses were different for ESAT-6 (p<0.0001), CFP-10 (p<0.0001), TB 10.3 (p<0.0001), HspX (16 kDa) (p<0.0001), PstS1 (p<0.0001) and AlaDH (p = 0.001); but not for Ag85B (p = 0.063). Comparing results between children with LTBI and those with TB disease differences were significant for IFN-γ ELISPOT only for AlaDH antigen (p = 0.021) and for IL-2 ELISPOT assay for AlaDH (p<0.0001) and TB 10.3 antigen (p = 0.043). ROC analyses demonstrated sensitivity of 100% and specificity of 81% of AlaDH-IL-2 ELISPOT assay in discriminating between latent and active TB using a cut off of 12.5 SCF per million PBMCs. Conclusion Our data suggest that IL-2 based ELISPOT with AlaDH antigen may be of help in discriminating children with active from those with latent TB. PMID:23029377

  9. Adolescents at Risk for Alcohol Abuse Demonstrate Altered Frontal Lobe Activation during Stroop Performance

    PubMed Central

    Silveri, Marisa M.; Rogowska, Jadwiga; McCaffrey, Alexandra; Yurgelun-Todd, Deborah A.

    2010-01-01

    Background Children and adolescents, family history positive (FH+) for alcoholism, exhibit differences in brain structure and functional activation when compared to family history negative (FH-) counterparts. Given that frontal brain regions, and associated reciprocal connections with limbic structures, undergo the most dramatic maturational changes during adolescence, the objective of this study was to compare functional brain activation during a frontally-mediated test of response inhibition in 32 adolescents separated into low-risk (FH-) and high-risk (FH+) groups. Methods Functional magnetic resonance (fMRI) blood oxygen level dependent (BOLD) data were acquired at 1.5 Tesla during performance of Stroop Color Naming, Word Reading and Interference. Preprocessing and statistical analyses, covaried for age, were conducted in SPM99 using a search territory that included superior, middle, and inferior frontal gyri (trigone region), anterior cingulate gyrus, and left and right amygdala. Results Significantly greater activation in the fronto-limbic search territory was observed in FH+ relative to FH- subjects during Stroop Interference. In addition, a significant regression between brain activation and family history density was observed, with a greater density being associated with increased activation in regions including middle frontal gyrus (BA9) and cingulate gyrus (BA24). Conclusions These data demonstrate a significant influence of FH status on brain activation during the performance of a response inhibition task, perhaps reflecting a neurobiological vulnerability associated with FH status that may include reduced neuronal efficiency and/or recruitment of additional neuronal resources. These findings are important given that the adolescent developmental period is already associated with reduced inhibitory capacity, even prior to the onset of alcohol use. PMID:21073483

  10. The impact of initiation: Early onset marijuana smokers demonstrate altered Stroop performance and brain activation.

    PubMed

    Sagar, K A; Dahlgren, M K; Gönenç, A; Racine, M T; Dreman, M W; Gruber, S A

    2015-12-01

    Marijuana (MJ) use is on the rise, particularly among teens and emerging adults. This poses serious public health concern, given the potential deleterious effects of MJ on the developing brain. We examined 50 chronic MJ smokers divided into early onset (regular MJ use prior to age 16; n=24) and late onset (age 16 or later; n=26), and 34 healthy control participants (HCs). All completed a modified Stroop Color Word Test during fMRI. Results demonstrated that MJ smokers exhibited significantly poorer performance on the Interference subtest of the Stroop, as well as altered patterns of activation in the cingulate cortex relative to HCs. Further, early onset MJ smokers exhibited significantly poorer performance relative to both HCs and late onset smokers. Additionally, earlier age of MJ onset as well as increased frequency and magnitude (grams/week) of MJ use were predictive of poorer Stroop performance. fMRI results revealed that while late onset smokers demonstrated a more similar pattern of activation to the control group, a different pattern was evident in the early onset group. These findings underscore the importance of assessing age of onset and patterns of MJ use and support the need for widespread education and intervention efforts among youth. PMID:25936584

  11. Low Speed, 2-D Rotor/Stator Active Noise Control at the Source Demonstration

    NASA Technical Reports Server (NTRS)

    Simonich, John C.; Kousen, Ken A.; Zander, Anthony C.; Bak, Michael; Topol, David A.

    1997-01-01

    Wake/blade-row interaction noise produced by the Annular Cascade Facility at Purdue University has been modeled using the LINFLO analysis. Actuator displacements needed for complete cancellation of the propagating acoustic response modes have been determined, along with the associated actuator power requirements. As an alternative, weighted least squares minimization of the total far-field sound power using individual actuators has also been examined. Attempts were made to translate the two-dimensional aerodynamic results into three-dimensional actuator requirements. The results lie near the limit of present actuator technology. In order to investigate the concept of noise control at the source for active rotor/stator noise control at the source, various techniques for embedding miniature actuators into vanes were examined. Numerous miniature speaker arrangements were tested and analyzed to determine their suitability as actuators for a demonstration test in the Annular Cascade Facility at Purdue. The best candidates demonstrated marginal performance. An alternative concept to using vane mounted speakers as control actuators was developed and tested. The concept uses compression drivers which are mounted externally to the stator vanes. Each compression driver is connected via a tube to an air cavity in the stator vane, from which the driver signal radiates into the working section of the experimental rig. The actual locations and dimensions of the actuators were used as input parameters for a LINFLO computational analysis of the actuator displacements required for complete cancellation of tones in the Purdue experimental rig. The actuators were designed and an arrangement determined which is compatible with the Purdue experimental rig and instrumentation. Experimental tests indicate that the actuators are capable of producing equivalent displacements greater than the requirements predicted by the LINFLO analysis. The acoustic output of the actuators was also found

  12. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

    PubMed

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

    2014-07-01

    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. PMID:24687155

  13. 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

  14. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  15. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    PubMed Central

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

  16. Chemical quantification and antioxidant assay of four active components in Ficus hirta root using UPLC-PAD-MS fingerprinting combined with cluster analysis

    PubMed Central

    2013-01-01

    Background Root of Ficus hirta (RFH) is widely consumed in China as a plant-derived popular food. However, contents of the active constituents of RFH are unknown, and the chemical as well as bioactive properties of RFH may be affected by growing area. In order to ensure the standard efficacy of health products made with RFH, its active constituents should firstly be determined and, secondly, a means of assessing samples for their contents of these constituents is needed. Results Four active components, including two coumarins, namely psoralen and bergapten, and two flavonoids, namely luteolin and apigenin, in twenty RFH samples were quantified using a new ultra performance liquid chromatography coupled with photodiode array detector and mass spectrometry (UPLC-PAD-MS) method, and the content level in descending order was psoralen > bergapten > luteolin > apigenin. Chromatographic fingerprint similarity evaluation and cluster analysis were used to assess geographical origin of RFH, and the results revealed a high level of similarity for the tested RFH samples obtained from Hainan, Guangdong, Guangxi provinces and Hong Kong. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was conducted to evaluate the antioxidant potencies of the four components, and the results clearly demonstrated that luteolin was most effective; apigenin exhibited a moderate potency, whereas psoralen and bergapten possessed little effect against free radical reactions. Structure-activity relationship of the components was elucidated, and the 3′-hydroxyl group of luteolin was found to be directly responsible for its antioxidant activity. Conclusion The present UPLC-PAD-MS method and DPPH radical scavenging assay performed well for the purpose of constituent quantification and antioxidant assay. Global profiles were highly similar for RFH samples from different origins. Both the coumarins and flavonoids were involved in the health benefit of RFH. PMID:23835498

  17. Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation.

    PubMed

    Tárnok, A; Mahnke, A; Müller, M; Zotz, R J

    1999-02-15

    Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents. PMID:10088974

  18. Establishment of a novel experimental protocol for drug-induced seizure liability screening based on a locomotor activity assay in zebrafish.

    PubMed

    Koseki, Naoteru; Deguchi, Jiro; Yamashita, Akihito; Miyawaki, Izuru; Funabashi, Hitoshi

    2014-08-01

    As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery. PMID:25056783

  19. Direct conscious telemetry recordings demonstrate increased renal sympathetic nerve activity in rats with chronic kidney disease

    PubMed Central

    Salman, Ibrahim M.; Sarma Kandukuri, Divya; Harrison, Joanne L.; Hildreth, Cara M.; Phillips, Jacqueline K.

    2015-01-01

    Chronic kidney disease (CKD) is associated with sympathetic hyperactivity and impaired blood pressure control reflex responses, yet direct evidence demonstrating these features of autonomic dysfunction in conscious animals is still lacking. Here we measured renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) using telemetry-based recordings in a rat model of CKD, the Lewis Polycystic Kidney (LPK) rat, and assessed responses to chemoreflex activation and acute stress. Male LPK and Lewis control animals (total n = 16) were instrumented for telemetric recording of RSNA and MAP. At 12–13 weeks-of-age, resting RSNA and MAP, sympathetic and haemodynamic responses to both peripheral (hypoxia: 10% O2) and central chemoreflex (hypercapnia: 7% CO2) activation and acute stress (open-field exposure), were measured. As indicators of renal function, urinary protein (UPro) and creatinine (UCr) levels were assessed. LPK rats had higher resting RSNA (1.2 ± 0.1 vs. 0.6 ± 0.1 μV, p < 0.05) and MAP (151 ± 8 vs. 97 ± 2 mmHg, p < 0.05) compared to Lewis. MAP was negatively correlated with UCr (r = −0.80, p = 0.002) and positively correlated with RSNA (r = 0.66, p = 0.014), with multiple linear regression modeling indicating the strongest correlation was with Ucr. RSNA and MAP responses to activation of the central chemoreflex and open-field stress were reduced in the LPK relative to the Lewis (all p < 0.05). This is the first description of dual conscious telemetry recording of RSNA and MAP in a genetic rodent model of CKD. Elevated RSNA is likely a key contributor to the marked hypertension in this model, while attenuated RSNA and MAP responses to central chemoreflex activation and acute stress in the LPK indicate possible deficits in the neural processing of autonomic outflows evoked by these sympathoexcitatory pathways. PMID:26300784

  20. Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

    PubMed

    Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Lejbkowicz, Flavio; Rennert, Hedy S; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

    2015-09-01

    The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT. PMID:26045303

  1. Comparison of Tuberculin Activity in the Interferon-gamma Assay for the Diagnosis of Bovine Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle infected with bovine tuberculosis still represent a serious regulatory and health concern in a variety of countries. Early diagnosis using the in vitro interferon gamma (IFN-gamma) assay has been applied for more than a decade. Briefly, IFN-gamma responses in whole blood cultures stimulated w...

  2. Comparison of Tuberculin Activity in the Interferon-gamma Assay for the Diagnosis of Bovine Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle infected with bovine tuberculosis still represent a serious regulatory and health concern in a variety of countries. Early diagnosis using the in vitro interferon gamma (IFN-g) assay has been applied for more than a decade. Briefly, IFN-g responses in whole blood cultures stimulated with puri...

  3. Laboratory Assay of Soil Microbial Activities is Congruent with In Situ Conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We used microtiter plates loaded with an oxygen-sensitive fluorophore to assay respiration of organic substrates by soil microbial communities. The respiration of soil slurries was measured at low substrate concentrations (0.5 mg substrate per g soil) with no additional nutrients over a short seven...

  4. Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.

    PubMed

    Randak, Christoph O; Ver Heul, Amanda R; Welsh, Michael J

    2012-10-19

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

  5. Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity*

    PubMed Central

    Randak, Christoph O.; Ver Heul, Amanda R.; Welsh, Michael J.

    2012-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl− channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the 32P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

  6. Avian extraintestinal Escherichia coli exhibits enterotoxigenic-like activity in the in vivo rabbit ligated ileal loop assay.

    PubMed

    Maluta, Renato Pariz; Gatti, Maria Silvia Viccari; Joazeiro, Paulo Pinto; de Paiva, Jacqueline Boldrin; Rojas, Thaís Cabrera Galvão; Silveira, Flávio; Houle, Sébastien; Kobayashi, Renata Katsuko Takayama; Dozois, Charles M; Dias da Silveira, Wanderley

    2014-06-01

    Avian pathogenic Escherichia coli (APEC) strains harbor a number of virulence genes and cause extraintestinal diseases, such as septicemia, swollen-head syndrome, salpingitis, and omphalitis in poultry. APEC strains are not known to cause intestinal diseases. Herein, for the first time, it is reported that APEC strains were able to induce an enterotoxigenic-like effect in rabbit ligated ileal loops. Strain SEPT362 caused cell detachment of the intestinal villi, which also showed a flattened and wilted appearance, but the integrity of the tight junctions was maintained. Additionally, this strain did not adhere to enterocytes in vivo, although adhesin encoding genes ( fimH, csgA, lpfA2-3, and ECP) were present while other lpfA types, sfa, afa, papC, and ral genes were not. This enterotoxigenic-like activity was conserved after thermal treatment of the supernatant at 65°C but not at 100°C. Moreover, experiments based on filtering with different molecular weight cut-off (MWCO) pore sizes demonstrated that the component associated with the observed biological effect has a molecular weight >100 kDa. Blast search and polymerase chain reaction assays for known E. coli virulence factors showed that strain SEPT362 harbors the gene encoding for the toxin EAST-1 and the serine protease autotransporter (SPATE) Tsh, but is negative for genes encoding for the toxins LT-I, STh, STp, Stx1, Stx2, CNF-1, CNF-2, CDT and the SPATEs Sat, Pic, Vat, SigA, SepA, EatA, EspP, or EspC. A cloned copy of the tsh gene in E. coli K-12 was also tested and was shown to have an enterotoxic effect. These results suggest that APEC might induce fluid accumulation in the rabbit gut. The Tsh autotransporter seems to be one of the factors associated with this phenotype. PMID:24673684

  7. SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents.

    PubMed

    Lenin, Raji; Mohan, Viswanathan; Balasubramanyam, Muthuswamy

    2015-06-01

    Endoplasmic reticulum (ER) stress is emerging as a unifying paradigm and one of the underlying mechanisms in the genesis of diabetes and its complications. While this has prompted the development of ER stress inhibitors, there is a limitation in monitoring of ER stress in vitro and in vivo by reliable methodologies. We validated the secreted alkaline phosphatase (SEAP) activity as a surrogate marker of ER stress in mouse β-TC6 cells exposed to glucolipotoxicity or tunicamycin and studied insulin secretion along with alterations in ER stress markers. SEAP activity assay was measured using the Great EscAPe SEAP kit, insulin levels were determined by Mercodia reagents and mRNA expression of ER stress markers was quantified by real-time PCR. SEAP activity in β-cells was significantly decreased (indicating increased ER stress) on exposure either to glucolipotoxicity or tunicamycin. This was accompanied by an increased mRNA expression of ER stress markers (GRP-78, PERK, IRE1α, ATF6, XBP-1, and CHOP) and decreased insulin secretion. Treating the cells with phenylbutyric acid normalized SEAP activity, decreased mRNA expression of ER stress markers and improved insulin secretion. Interestingly, cells exposed to different classes of anti-diabetes agents or compounds such as resveratrol resisted ER stress. Methylglyoxal also induces ER stress and this was counteracted by aminoguanidine. Out study demonstrates SEAP activity as a novel ER stress monitoring assay to investigate the therapeutic value of agents with ER stress inhibitory potential. Future studies should focus on the exercise of adopting this reporter assay for high-throughput screening mode of drug discovery. PMID:25776571

  8. Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

    PubMed

    Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

    2016-03-01

    A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. PMID:26706798

  9. Effect of metals and other inorganic ions on soil microbial activity: soil dehydrogenase assay as a simple toxicity test

    SciTech Connect

    Rogers, J.E.; Li, S.W.

    1985-06-01

    The purpose of this report is to illustrate the utility of the soil dehydrogenase assay as an effective primary test for assessing the potential toxicity of chemicals to soil microbial activity. In this manuscript the authors describe their use of the soil dehydrogenase assay in determining the effects of a number of potential toxic inorganic ions on soil microbial activity. The ions include Cu/sup 2 +/, Mg/sup 2 +/, Ni/sup 2 +/, Zn/sup 2 +/, NH/sub 4//sup +/, Cd/sup 2 +/, Cr/sup 32/, F/sup -/, AsO/sub 4//sup 3 -/, BO/sub 3//sup 3 -/, and SO/sub 4//sup 2 -/.

  10. A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

    PubMed

    Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

    2016-01-01

    The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations. PMID:27581492

  11. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay

    PubMed Central

    2013-01-01

    Background Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts. Methods The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF). Results MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis. Conclusions In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets

  12. Kinetic Demonstration.

    ERIC Educational Resources Information Center

    Burgardt, Erik D.; Ryan, Hank

    1996-01-01

    Presents a unit on chemical reaction kinetics that consists of a predemonstration activity, the demonstration, and a set of postdemonstration activities that help students transfer the concepts to actual chemical reactions. Simulates various aspects of chemical reaction kinetics. (JRH)

  13. Active Control of Rotating Stall Demonstrated for a Multistage Compressor With Inlet Distortion

    NASA Technical Reports Server (NTRS)

    VanSchalkwyk, Christian; Bright, Michelle M.; Suder, Kenneth L.; Straziar, Anthony J.; Thorp, Scott A.

    2001-01-01

    Aircraft compressors can suffer debilitating consequences as a result of rotating stall and surge events caused by inlet distortions. This is particularly true of aircraft during takeoff, when the compressor is operating at peak performance close to the surge line. Significant research has been conducted by the NASA Glenn Research Center in the area of compressor stability enhancement through active and passive control methods. Most recently, an experiment was conducted at the Wright Patterson Air Force Base Research Laboratory on a two-stage fan with inlet guide vanes and inlet distortion. In this joint Small Business Innovation Research effort between Scientific Systems and Glenn, control of rotating stall was demonstrated in a multistage transonic fan. This twostage fan with inlet guide vanes was tested under clean and distorted inlet conditions. The compressor was also configured with a circumferential distortion screen capable of 180 of distortion and with 14 high-velocity injectors upstream of the first rotor. Twelve of these injectors could oscillate up to frequencies of 450 Hz. The additional two injectors were located next to each other and were used in concert with each other as a single, on/off, high-authority actuator. In a first test of injection in this multistage environment, 12 of the valves were opened 50 percent of their full stroke to assess steady injection through the compressor. This baseline injection is shown in the compressor characteristic of the following figure, and stall margin improvements are tracked from this baseline condition. The compressor was then tested with clean inlet conditions using 12 injectors and active control. Pressure disturbances were tracked before rotating stall, and a constant gain control scheme reduced the stalling mass flow by 10.8 percent over the baseline. With the distortion screen present in the inlet, a pole-zero cancellation control scheme was used to achieve a 6.4-percent decrease in stalling mass flow

  14. Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes.

    PubMed

    Iwai, Atsushi; Yoshimura, Teruki; Wada, Keiji; Watabe, Satoshi; Sakamoto, Yuki; Ito, Etsuro; Miura, Toshiaki

    2013-01-01

    A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR. PMID:23574674

  15. Toxicity of 8-Hydroxyquinoline in Cryprinus carpio Using the Acute Toxicity Test, Hepatase Activity Analysis and the Comet Assay.

    PubMed

    Yan, Shuaiguo; Chen, Lili; Dou, Xiaofei; Qi, Meng; Du, Qiyan; He, Qiaoqiao; Nan, Mingge; Chang, Zhongjie; Nan, Ping

    2015-08-01

    To evaluate the environmental toxicity of 8-hydroxyquinoline (8-HOQ), an important industrial raw material found in China's major ornamental fish, Cryprinus carpio, using the acute toxicity test, hepatase activity analysis and the comet assay. The results indicated that 8-HOQ had significant acute toxicity in adult C. carpio with a 96 h-LC50 of 1.15 and 0.22 mg L(-1) hepatic quinoline residues as assessed by HPLC. 8-HOQ also induced genotoxicity in the form of strand breaks in the DNA of hepatic cells as shown by the comet assay. With regard to physiological toxicity, 8-HOQ induced a decrease in the activities of hepatic GOT and GPT with increased exposure concentration and time. These data suggest that 8-HOQ may be toxic to the health of aquatic organisms when accidentally released into aquatic ecosystems. The data also suggest that the comet assay may be used in biomonitoring to determine 8-HOQ genotoxicity and hepatic GPT and GOT activities may be potential biomarkers of physiological toxicity. PMID:26067700

  16. Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates.

    PubMed

    Haghbeen, Kamahldin; Wue Tan, Eng

    2003-01-01

    Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented. PMID:12479831

  17. Correlation of In Vivo and In Vitro Assay Results for Assessment of Free Radical Scavenging Activity of Green Tea Nutraceuticals.

    PubMed

    Abd-ElSalam, Heba-Alla H; Al-Ghobashy, Medhat A; Al-Shorbagy, Muhammad; Nassar, Noha; Zaazaa, Hala E; Ibrahim, Mohamed A

    2016-07-01

    Green tea (GT)-derived catechins; epigallocatechin gallate (EGCG) in particular are commonly used nutraceuticals for their free-radical scavenging activity (FRSA). The influence of photodegradation on the protective power of GT nutracenticals against oxidative stress was thoroughly explored. Photodegradation of GT extracts was carried out and monitored using orthogonal stability-indicating testing protocol; in vitro and in vivo assays. Total polyphenol content (TPC) and FRSA were determined spectrophotometrically while EGCG was selectively monitored using SPE-HPLC. In vivo assessment of photodegraded samples was investigated via measuring a number of biomarkers for hepatic oxidative stress and apoptosis (caspase-3, inducible nitric oxide synthase, nitric oxide, mitogen-activated protein kinase, glutathione, thiobarbituric acid reactive substances, nuclear factor kappa beta, and nuclear factor erythroid 2-related factor) as well as liver damage (alanine transaminase and aspartate transaminase) in serum of rats previously subjected to oxidative stress. Results showed complete degradation of EGCG in photodegraded green tea samples with no correlation with either TPC or FRSA. On the other hand, in vivo assay results revealed not only loss of activity but formation of harmful pro-oxidants. Photostability was found crucial for the protective effect of GT extract against lead acetate insult. Results confirmed that careful design of quality control protocols requires correlation of chemical assays to bioassays to verify efficacy, stability, and most importantly safety of nutraceuticals. PMID:27275932

  18. A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

    PubMed Central

    Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

    2010-01-01

    Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

  19. Application of non-radioactive europium (Eu3+) release assay to a measurement of human natural killer activity of healthy and patient populations.

    PubMed

    Nagao, F; Yabe, T; Xu, M; Yokoyama, K; Saito, K; Okumura, K

    1996-01-01

    Europium (Eu3+) release assay is a non-radioactive method for a measurement of cytotoxicity of lymphocytes and has several advantages compared with a conventional 51Cr release assay. However, the Eu3+ release assay has not been applied to a natural killer (NK) activity measurement of a large number of the human population mainly due to a lack of comparability with the 51Cr release assay. With some modifications of the procedures and careful manipulation of cells, constant and reproducible results were obtained by the Eu3+ release assay. NK activity of several individuals was measured by the Eu3+ release assay and was compared with data obtained by 51Cr release assay performed simultaneously. The obtained values by the two methods were almost identical. We applied the Eu3+ method to measure NK activity of a large number of individuals, including 68 apparently healthy donors and 36 autoimmune and 21 cancer patients. Some of these diseases are known to show abnormal NK activity. The obtained cytotoxicities were mostly consistent with the previously reported data obtained by the 51Cr release assay. These results indicated that the Eu3+ release assay could be used as an alternative method for a measurement of human NK activity of mass population including patients. PMID:8915687

  20. Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

    SciTech Connect

    Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

    1983-10-01

    The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

  1. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay

    EPA Science Inventory

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms o...

  2. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay##

    EPA Science Inventory

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms of...

  3. The Fibrin slide assay for detecting urokinase activity in human fetal kidney cells

    NASA Technical Reports Server (NTRS)

    Sedor, K.

    1985-01-01

    The Fibrin Slide Technique of Hau C. Kwaan and Tage Astrup is discussed. This relatively simple assay involves two steps: the formation of an artificial clot and then the addition of an enzyme (UKOKINASE) to dissolve the clot. The actual dissolving away of the clot is detected by the appearance of holes (lysis zones) in the stained clot. The procedure of Kwaan and Astrup is repeated, along with modifications and suggestions for improvements based on experience with the technique.

  4. Rectification of SEMG as a tool to demonstrate synchronous motor unit activity during vibration.

    PubMed

    Sebik, Oguz; Karacan, Ilhan; Cidem, Muharrem; Türker, Kemal S

    2013-04-01

    The use of surface electromyography (SEMG) in vibration studies is problematic since motion artifacts occupy the same frequency band with the SEMG signal containing information on synchronous motor unit activity. We hypothesize that using a harsher, 80-500 Hz band-pass filter and using rectification can help eliminate motion artifacts and provide a way to observe synchronous motor unit activity that is phase locked to vibration using SEMG recordings only. Multi Motor Unit (MMU) action potentials using intramuscular electrodes along with SEMG were recorded from the gastrocnemius medialis (GM) of six healthy male volunteers. Data were collected during whole body vibration, using vibration frequencies of 30 Hz, 35 Hz, 40 Hz or 50 Hz. A computer simulation was used to investigate the efficacy of filtering under different scenarios: with or without artifacts and/or motor unit synchronization. Our findings indicate that motor unit synchronization took place during WBV as verified by MMU recordings. A harsh filtering regimen along with rectification proved successful in demonstrating motor unit synchronization in SEMG recordings. Our findings were further supported by the results from the computer simulation, which indicated that filtering and rectification was efficient in discriminating motion artifacts from motor unit synchronization. We suggest that the proposed signal processing technique may provide a new methodology to evaluate the effects of vibration treatments using only SEMG. This is a major advantage, as this non-intrusive method is able to overcome movement artifacts and also indicate the synchronization of underlying motor units. PMID:23098913

  5. An exploration of the estrogen receptor transcription activity of capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays.

    PubMed

    Li, Juan; Ma, Duo; Lin, Yuan; Fu, Jianjie; Zhang, Aiqian

    2014-06-16

    Capsaicin has been considered as an alternative template of dichlorodiphenyl trichloroethane (DDT) in antifouling paint. However, information regarding the estrogenic activity of capsaicin analogues is rather limited in comparison to that of DDT analogues and their metabolites. We here explore the ER transcription activity of selected capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays. Molecular simulation and the agonist/antagonist differential-docking screening identified 6-iodonordihydrocapsaicin (6-I-CPS) as a weak ERα agonist, while anti-estrogenicity was expected for N-arachidonoyldopamine, capsazepine, dihydrocapsaicin, trichostatin A, and capsaicin. On the contrary, the large volume of analogues, such as phorbol 12-phenylacetate 13-acetate 20-homovanillate and phorbol 12,13-dinonanoate 20-homovanillate, cannot fit well with the ER cavity. The result of MVLN assay was in accord with the in silico prediction. 6-I-CPS was demonstrated to induce luciferase gene expression, while the other analogues of relatively small molecular volume reduced luciferase gene expression in MVLN cells, both in the absence and presence of estradiol. This finding suggested that the ER transcription activity of capsaicin analogues is generated at least partly through the ERα-mediated pathway. Moreover, receptor polymorphism analysis indicated that capsaicin analogues may exhibit diverse species selectivity for human beings and marine species. PMID:24747365

  6. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  7. A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.

    PubMed

    Bachovchin, Daniel A; Koblan, Luke W; Wu, Wengen; Liu, Yuxin; Li, Youhua; Zhao, Peng; Woznica, Iwona; Shu, Ying; Lai, Jack H; Poplawski, Sarah E; Kiritsy, Christopher P; Healey, Sarah E; DiMare, Matthew; Sanford, David G; Munford, Robert S; Bachovchin, William W; Golub, Todd R

    2014-08-01

    The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery. PMID:24997602

  8. Melanin-Like Nanoquencher on Graphitic Carbon Nitride Nanosheets for Tyrosinase Activity and Inhibitor Assay.

    PubMed

    Liu, Jin-Wen; Wang, Yu-Min; Xu, Liu; Duan, Lu-Ying; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-09-01

    Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer. PMID:27417635

  9. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna.

    PubMed

    Ørsted, Michael; Roslev, Peter

    2015-08-01

    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, the authors investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2 Cr2 O7 or the herbicide formulation Roundup®. Toxicant-induced changes in hydrolytic enzyme activity were compared with changes in mobility (International Organization for Standardization standard 6341). The results showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna and the fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup resulted in loss of whole body enzyme activity and release of cell constituents, including enzymes and DNA. Roundup caused comparable inhibition of mobility and alkaline phosphatase activity with median effective concentration values at 20 °C of 8.7 mg active ingredient (a.i.)/L to 11.7 mg a.i./L. Inhibition of alkaline phosphatase activity by Roundup was lowest at 14 °C and greater at 20 °C and 26 °C. The results suggest that the fluorescence-based hydrolytic enzyme activity assay (FLEA assay) can be used as an index of D. magna stress. Combining enzyme activity with fluorescence measurements may be applied as a simple and quantitative supplement for toxicity testing with D. magna. PMID:25809520

  10. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in Hiv-positive adults

    PubMed Central

    Shah, Maunank; Hanrahan, Colleen; Wang, Zhuo Yu; Dendukuri, Nandini; Lawn, Stephen D; Denkinger, Claudia M; Steingart, Karen R

    2016-01-01

    Background Rapid detection of tuberculosis (TB) among people living with human immunodeficiency virus (HIV) is a global health priority. HIV-associated TB may have different clinical presentations and is challenging to diagnose. Conventional sputum tests have reduced sensitivity in HIV-positive individuals, who have higher rates of extrapulmonary TB compared with HIV-negative individuals. The lateral flow urine lipoarabinomannan assay (LF-LAM) is a new, commercially available point-of-care test that detects lipoarabinomannan (LAM), a lipopolysaccharide present in mycobacterial cell walls, in people with active TB disease. Objectives To assess the accuracy of LF-LAM for the diagnosis of active TB disease in HIV-positive adults who have signs and symptoms suggestive of TB (TB diagnosis).To assess the accuracy of LF-LAM as a screening test for active TB disease in HIV-positive adults irrespective of signs and symptoms suggestive of TB (TB screening). Search methods We searched the following databases without language restriction on 5 February 2015: the Cochrane Infectious Diseases Group Specialized Register; MEDLINE (PubMed,1966); EMBASE (OVID, from 1980); Science Citation Index Expanded (SCI-EXPANDED, from 1900), Conference Proceedings Citation Index-Science (CPCI-S, from 1900), and BIOSIS Previews (from 1926) (all three using the Web of Science platform; MEDION; LILACS (BIREME, from 1982); SCOPUS (from 1995); the metaRegister of Controlled Trials (mRCT); the search portal of the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP); and ProQuest Dissertations & Theses A&l (from 1861). Selection criteria Eligible study types included randomized controlled trials, cross-sectional studies, and cohort studies that determined LF-LAM accuracy for TB against a microbiological reference standard (culture or nucleic acid amplification test from any body site). A higher quality reference standard was one in which two or more specimen types were

  11. U.S. Department of Energy -- Advanced Vehicle Testing Activity: Plug-in Hybrid Electric Vehicle Testing and Demonstration Activities

    SciTech Connect

    James E. Francfort; Donald Karner; John G. Smart

    2009-05-01

    The U.S. Department of Energy’s (DOE) Advanced Vehicle Testing Activity (AVTA) tests plug-in hybrid electric vehicles (PHEV) in closed track, dynamometer and onroad testing environments. The onroad testing includes the use of dedicated drivers on repeated urban and highway driving cycles that range from 10 to 200 miles, with recharging between each loop. Fleet demonstrations with onboard data collectors are also ongoing with PHEVs operating in several dozen states and Canadian Provinces, during which trips- and miles-per-charge, charging demand and energy profiles, and miles-per-gallon and miles-per-kilowatt-hour fuel use results are all documented, allowing an understanding of fuel use when vehicles are operated in charge depleting, charge sustaining, and mixed charge modes. The intent of the PHEV testing includes documenting the petroleum reduction potential of the PHEV concept, the infrastructure requirements, and operator recharging influences and profiles. As of May 2008, the AVTA has conducted track and dynamometer testing on six PHEV conversion models and fleet testing on 70 PHEVs representing nine PHEV conversion models. A total of 150 PHEVs will be in fleet testing by the end of 2008, all with onboard data loggers. The onroad testing to date has demonstrated 100+ miles per gallon results in mostly urban applications for approximately the first 40 miles of PHEV operations. The primary goal of the AVTA is to provide advanced technology vehicle performance benchmark data for technology modelers, research and development programs, and technology goal setters. The AVTA testing results also assist fleet managers in making informed vehicle purchase, deployment and operating decisions. The AVTA is part of DOE’s Vehicle Technologies Program. These AVTA testing activities are conducted by the Idaho National Laboratory and Electric Transportation Engineering Corporation, with Argonne National Laboratory providing dynamometer testing support. The proposed paper

  12. Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates.

    PubMed

    Serveau-Avesque, Carole; Verger, Robert; Rodriguez, Jorge A; Abousalham, Abdelkarim

    2013-09-21

    We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains α-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples. PMID:23851449

  13. Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

    PubMed

    Isbrucker, R; Daas, A; Wagner, L; Costanzo, A

    2016-01-01

    Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work. PMID:27506252

  14. Compounds Derived from the Bhutanese Daisy, Ajania nubigena, Demonstrate Dual Anthelmintic Activity against Schistosoma mansoni and Trichuris muris

    PubMed Central

    Pearson, Mark S.; Giacomin, Paul R.; Becker, Luke; Sotillo, Javier; Pickering, Darren

    2016-01-01

    Background Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties. Methodology/Principal Findings Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds—luteolin (3) and (3R,6R)-linalool oxide acetate (1)—showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8–36.9 μg/mL) and T. muris (IC50 range = 9.7–20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice. Conclusions/Significance Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths—T. muris and S. mansoni—and was

  15. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    PubMed

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest. PMID:27241123

  16. High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid, 96-well microtiter assays were compared to conventional assays for quantifying total phenolic content, flavonoid content, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in sorghum grain. The 96-well assays exhibited a correlation of >0.9 to the conventional assays. The 96-well assays allowed for ...

  17. Inhibition of human immunodeficiency virus type 1 activity by purified human breast milk mucin (MUC1) in an inhibition assay.

    PubMed

    Habte, Habtom H; de Beer, Corena; Lotz, Zoë E; Tyler, Marilyn G; Kahn, Delawir; Mall, Anwar S

    2008-01-01

    It has been reported that breast-feeding is responsible for approximately 40% of the HIV transmissions from HIV-positive mothers to children. Human breast milk, however, is known to contain numerous biologically active components which protect breast-fed infants against bacteria, viruses, and toxins. The purpose of this study was to purify and characterize breast milk mucin and to determine its anti-HIV-1 activity in an HIV inhibition assay. Sepharose CL-4B column chromatography and caesium chloride isopycnic density gradient purification were used to isolate and purify the mucin. Following Western blotting and amino acid analysis, an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of crude breast milk and purified milk mucin (MUC1) by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). SDS-PAGE analysis of the mucin, together with its amino acid composition and Western blotting, suggested that this purified mucin from human breast milk was MUC1. The HIV inhibition assay revealed that while the purified milk mucin (MUC1) inhibited the HIV-1 activity by approximately 97%, there was no inhibition of the HIV-1 activity by crude breast milk. Although the reason for this is not clear, it is likely that because the MUC1 in crude milk is enclosed by fat globules, there may not be any physical contact between the mucin and the virus in the crude breast milk. Thus, there is a need to free the mucin from the fat globules for it to be effective against the virus. PMID:17878743

  18. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    PubMed

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system. PMID:25563179

  19. Simplification of the DPPH assay for estimating the antioxidant activity of wine and wine by-products.

    PubMed

    Carmona-Jiménez, Yolanda; García-Moreno, M Valme; Igartuburu, Jose M; Garcia Barroso, Carmelo

    2014-12-15

    The DPPH assay is one of the most commonly employed methods for measuring antioxidant activity. Even though this method is considered very simple and efficient, it does present various limitations which make it complicated to perform. The range of linearity between the DPPH inhibition percentage and sample concentration has been studied with a view to simplifying the method for characterising samples of wine origin. It has been concluded that all the samples are linear in a range of inhibition below 40%, which allows the analysis to be simplified. A new parameter more appropriate for the simplification, the EC20, has been proposed to express the assay results. Additionally, the reaction time was analysed with the object of avoiding the need for kinetic studies in the method. The simplifications considered offer a more functional method, without significant errors, which could be used for routine analysis. PMID:25038667

  20. Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

    USGS Publications Warehouse

    House, M.L.; Kim, C.H.; Reno, P.W.

    1998-01-01

    Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

  1. Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

    PubMed

    Kwon, Keehwan; Nagarajan, Rajesh; Stivers, James T

    2004-11-30

    Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover. PMID:15554707

  2. Strong Nonadditivity as a Key Structure–Activity Relationship Feature: Distinguishing Structural Changes from Assay Artifacts

    PubMed Central

    2015-01-01

    Nonadditivity in protein–ligand affinity data represents highly instructive structure–activity relationship (SAR) features that indicate structural changes and have the potential to guide rational drug design. At the same time, nonadditivity is a challenge for both basic SAR analysis as well as many ligand-based data analysis techniques such as Free-Wilson Analysis and Matched Molecular Pair analysis, since linear substituent contribution models inherently assume additivity and thus do not work in such cases. While structural causes for nonadditivity have been analyzed anecdotally, no systematic approaches to interpret and use nonadditivity prospectively have been developed yet. In this contribution, we lay the statistical framework for systematic analysis of nonadditivity in a SAR series. First, we develop a general metric to quantify nonadditivity. Then, we demonstrate the non-negligible impact of experimental uncertainty that creates apparent nonadditivity, and we introduce techniques to handle experimental uncertainty. Finally, we analyze public SAR data sets for strong nonadditivity and use recourse to the original publications and available X-ray structures to find structural explanations for the nonadditivity observed. We find that all cases of strong nonadditivity (ΔΔpKi and ΔΔpIC50 > 2.0 log units) with sufficient structural information to generate reasonable hypothesis involve changes in binding mode. With the appropriate statistical basis, nonadditivity analysis offers a variety of new attempts for various areas in computer-aided drug design, including the validation of scoring functions and free energy perturbation approaches, binding pocket classification, and novel features in SAR analysis tools. PMID:25760829

  3. Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells.

    PubMed

    Wu, Q; Delpire, E; Hebert, S C; Strange, K

    1998-12-01

    The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood. PMID:9843718

  4. Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles.

    PubMed

    Meijer, A E; Stegehuis, F

    1980-01-01

    A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate D-fructose-6-phosphate to D-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and D-glyceral-dehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-D-glycerate. Concomitantly the electrons are transported via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes. PMID:6446532

  5. Exploring potential contributors to endocrine disrupting activities in Taiwan's surface waters using yeast assays and chemical analysis.

    PubMed

    Chou, Pei-Hsin; Lin, Yi-Ling; Liu, Tong-Cun; Chen, Kuang-Yu

    2015-11-01

    Surface waters serve as sinks for anthropogenic contaminants, including naturally occurring hormones and a variety of synthetic endocrine active substances. To investigate the presence of endocrine active contaminants in the aquatic environment in Taiwan, river water and suspended solids were analyzed by yeast assays to examine the distribution of estrogenic, androgenic, and aryl hydrocarbon receptor agonist activities. The results showed that dry-season river samples exhibited strong estrogenic and aryl hydrocarbon receptor agonist activities, but no androgenic activity was detected. Owing to the ubiquitous detection of estrogenic activities in Taiwan's surface waters, samples were further subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for quantification of selected estrogenic compounds. LC-MS/MS results indicated that natural estrogens, such as estrone and 17β-estradiol were often the most contributing compounds for the bioassay-derived estrogenic activities due to their strong estrogenic potencies and high detection frequencies, whereas high concentrations of bisphenol A and nonylphenol also posed a threat to the aquatic ecosystems in Taiwan. Water samples eliciting strong estrogenic activities were further fractionated using high performance liquid chromatography, and significant estrogenic activities were detected in fractions containing estrone, 17β-estradiol, 17α-ethynylestradiol, and bisphenol A. Also, the presence of unidentified estrogenic compounds was found in few river water samples. Further identification of unknown endocrine active substances is necessary to better protect the aquatic environment in Taiwan. PMID:26295540

  6. A fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon quantum dots through host-guest recognition.

    PubMed

    Tang, Cong; Qian, Zhaosheng; Huang, Yuanyuan; Xu, Jiamin; Ao, Hang; Zhao, Meizhi; Zhou, Jin; Chen, Jianrong; Feng, Hui

    2016-09-15

    A convenient, reliable and highly sensitive assay for alkaline phosphatase (ALP) activity in the real-time manner is developed based on β-cyclodextrin-modified carbon quantum dots (β-CD-CQDs) nanoprobe through specific host-guest recognition. Carbon quantum dots were first functionalized with 3-aminophenyl boronic acid to produce boronic acid-functionalized CQDs, and then further modified with hydropropyl β-cyclodextrins (β-CD) through B-O bonds to form β-CD-CQDs nanoprobe. p-Nitrophenol phosphate disodium salt is used as the substrate of ALP, and can hydrolyze to p-nitrophenol under the catalysis of ALP. The resulting p-nitrophenol can enter the cavity of β-CD moiety in the nanoprobe due to their specific host-guest recognition, where photoinduced electron transfer process between p-nitrophenol and CQDs takes place to efficiently quench the fluorescence of the probe. The correlation between quenched fluorescence and ALP level can be used to establish quantitative evaluation of ALP activity in a broad range from 3.4 to 100.0U/L with the detection limit of 0.9U/L. This assay shows a high sensitivity to ALP even in the presence of a very high concentration of glucose. This study demonstrates a good electron donor/acceptor pair, which can be used to design general detection strategy through PET process, and also broadens the application of host-guest recognition for enzymes detection in clinical practice. PMID:27132001

  7. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    PubMed Central

    Elnager, Abuzar; Hassan, Rosline; Idris, Zamzuri; Mustafa, Zulkifli; Wan-Arfah, Nadiah; Sulaiman, S. A.; Gan, Siew Hua; Abdullah, Wan Zaidah

    2015-01-01

    Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted. PMID:25664321

  8. Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements

    PubMed Central

    FORBES, ALAINA M.; LIN, HUIMIN; MEADOWS, GARY G.; MEIER, G. PATRICK

    2014-01-01

    Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability. PMID:24859601

  9. Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani.

    PubMed

    Schmidt-Arras, Dirk; Leclercq, Olivier; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Faigle, Wolfgang; Loew, Damarys; Späth, Gerald F

    2011-08-24

    The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase-substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development. PMID:21443974

  10. Binary classification of a large collection of environmental chemicals from estrogen receptor assays by quantitative structure-activity relationship and machine learning methods.

    PubMed

    Zang, Qingda; Rotroff, Daniel M; Judson, Richard S

    2013-12-23

    There are thousands of environmental chemicals subject to regulatory decisions for endocrine disrupting potential. The ToxCast and Tox21 programs have tested ∼8200 chemicals in a broad screening panel of in vitro high-throughput screening (HTS) assays for estrogen receptor (ER) agonist and antagonist activity. The present work uses this large data set to develop in silico quantitative structure-activity relationship (QSAR) models using machine learning (ML) methods and a novel approach to manage the imbalanced data distribution. Training compounds from the ToxCast project were categorized as active or inactive (binding or nonbinding) classes based on a composite ER Interaction Score derived from a collection of 13 ER in vitro assays. A total of 1537 chemicals from ToxCast were used to derive and optimize the binary classification models while 5073 additional chemicals from the Tox21 project, evaluated in 2 of the 13 in vitro assays, were used to externally validate the model performance. In order to handle the imbalanced distribution of active and inactive chemicals, we developed a cluster-selection strategy to minimize information loss and increase predictive performance and compared this strategy to three currently popular techniques: cost-sensitive learning, oversampling of the minority class, and undersampling of the majority class. QSAR classification models were built to relate the molecular structures of chemicals to their ER activities using linear discriminant analysis (LDA), classification and regression trees (CART), and support vector machines (SVM) with 51 molecular descriptors from QikProp and 4328 bits of structural fingerprints as explanatory variables. A random forest (RF) feature selection method was employed to extract the structural features most relevant to the ER activity. The best model was obtained using SVM in combination with a subset of descriptors identified from a large set via the RF algorithm, which recognized the active and

  11. ASSAY OF CHICKEN BRAIN NEUROTOXIC ESTERASE ACTIVITY USING LEPTOPHOSOXON AS THE SELECTIVE NEUROTOXIC INHIBITOR

    EPA Science Inventory

    Hen brain microsomal preparation has phenyl valeratehydrolyzing activity associated with neurotoxic esterase activity. Part of that activity is due to paraoxon-insensitive esterases and a sub-part of this is sensitive to neurotoxic organophosphates, i.e., mipafox and leptophosoxo...

  12. Nondestructive assay of fluorine in geological and other materials by instrumental photon activation analysis with a microtron

    NASA Astrophysics Data System (ADS)

    Krausová, Ivana; Mizera, Jiří; Řanda, Zdeněk; Chvátil, David; Krist, Pavel

    2015-01-01

    Reliable determination of low concentrations of fluorine in geological and coal samples is difficult. It usually requires tedious decomposition and dissolution of the sample followed by chemical conversion of fluorine into its anionic form. The present paper examines possibilities of non-destructive determination of fluorine, mainly in minerals, rocks and coal, by instrumental photon activation analysis (IPAA) using the MT-25 microtron. The fluorine assay consists of counting the positron-electron annihilation line of 18F at 511 keV, which is a product of the photonuclear reaction 19F(γ, n)18F and a pure positron emitter. The assay is complicated by the simultaneous formation of other positron emitters. The main contributors to interference in geological samples are from 45Ti and 34mCl, whereas those from 44Sc and 89Zr are minor. Optimizing beam energy and irradiation-decay-counting times, together with using interfering element calibration standards, allowed reliable IPAA determination of fluorine in selected USGS and CRPG geochemical reference materials, NIST coal reference materials, and NIST RM 8414 Bovine Muscle. In agreement with the published data obtained by PIGE, the results of the F assay by IPAA have revealed erroneous reference values provided for the NIST reference materials SRM 1632 Bituminous Coal and RM 8414 Bovine Muscle. The detection limits in rock and coal samples are in the range of 10-100 μg g-1.

  13. An electrochemical one-step system for assaying methyltransferase activity based on transport of a quantum dot signaling tracer.

    PubMed

    Baek, Songyi; Won, Byoung Yeon; Park, Ki Soo; Park, Hyun Gyu

    2013-11-15

    A one-step, electrochemical method for assaying methyltransferase (MTase) activity, based on the convective transport of a quantum dot (QD) signaling tracer, has been developed. The assay chip used in this system was prepared by modifying a gold matrix with CdSe/ZnS QD-tagged dsDNA, which contains a specific methylation site (5'-GATC-3') recognized by MTase. Treatment of the chip with DNA adenine methylation (Dam) MTase, generates a methylated sequence (5'-GAmTC-3') within the dsDNA. The methylated dsDNA is then subjected to a cleavage reaction, induced by DpnI, which leads to release from the gold matrix of a DNA fragment tethered to a QD. Detection of the released QD, using square wave anodic stripping voltammetry (SWASV) on a glassy carbon (GC) electrode, enables the reliable quantitation of the methylated DNA. Because it is accomplished in a simple and convenient one step and does not require any complicated secondary or tedious washing steps, the new assay method holds great promise for epigenetic analysis in facility-limited environments or point-of-care testing (POCT) applications. PMID:23777705

  14. A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

    PubMed

    Do, Viet Ha; Tran, Phuong Lan; Ni, Li; Park, Kwan Hwa

    2016-01-01

    A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glcn-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis. PMID:26403601

  15. Determination of fission neutron transmission through waste matrix material using neutron signal correlation from active assay of {sup 239}Pu

    SciTech Connect

    Hollas, C.L.; Arnone, G.; Brunson, G.; Coop, K.

    1996-09-01

    The accuracy of TRU (transuranic) waste assay using the differential die-away technique depends upon significant corrections to compensate for the effects of the matrix material in which the TRU waste is located. The authors have used a new instrument, the Combined Thermal/Epithermal Neutron (CTEN) instrument for the assay of TRU waste, to develop methods to improve the accuracy of these corrections. Neutrons from a pulsed 14-MeV neutron generator are moderated in the walls of the CTEN cavity and induce fission in the TRU material. The prompt neutrons from these fission events are detected in cadmium-wrapped {sup 3}He neutron detectors. They report new methods of data acquisition and analysis to extract correlation in the neutron signals resulting form fission during active interrogation. They use the correlation information in conjunction with the total number of neutrons to determine the fraction of fission neutrons transmitted through the matrix material into the {sup 3}He detectors. This determination allows them to cleanly separate the matrix effects into two processes: matrix modification upon the neutron interrogating flux and matrix modification upon the fraction of fission neutrons transmitted to the neutron detectors. This transmission information is also directly applied in a neutron multiplicity analysis in the passive assay of {sup 240}Pu.

  16. ACTIVITY OF 1, 1, 1- AND 1, 1, 3-TRICHLOROACETONES IN A CHROMOSOMAL ABERRATION ASSAY IN CHO CELLS AND THE MICRONUCLEUS AND SPERMHEAD ABNORMALITY ASSAYS IN MICE

    EPA Science Inventory

    1,1,1- and 1,1,3-trichloroacetones (TCA) result from the disinfection of municipal water supplies with chlorine, and are direct-acting mutagens in the Ames/Salmonella assay. The objective of this study was to further investigate the genotoxicity of these compounds in mammalian ce...

  17. Complexes between tissue-type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay

    SciTech Connect

    Rijken, D.C.; Juhan-Vague, I.; Collen, D.

    1983-02-01

    Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with M/sub r/ approx. =70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with M/sub r/ approx. =140,000 without plasminogen activator activity. Overnight incubation at 37/sup 0/C of postexercise plasma revealed a shift of the M/sub r/ approx. =70,000 peak to the M/sub r/ approx. =140,000 position, suggesting that the M/sub r/ approx. =140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). ..cap alpha../sub 2/-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma and is probably responsible for the generation of the M/sub r/ approx. =140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of /sup 125/I-labeled extrinsic plasminogen activator in ..cap alpha../sub 2/-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-..cap alpha../sub 2/-antiplasmin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with ..cap alpha../sub 2/-antiplasmin and ..cap alpha../sub 1/-antitrypsin in plasma was obtained from two-site immunoradiometric assays. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with ..cap alpha../sub 2/-antiplasmin and ..cap alpha../sub 1/-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH/sub 2/Cl, at rest and after exercise and are therefore assumed to circulate in vivo. (JMT)

  18. Bacillus subtilis GSY 1057 assay for aflatoxin B activation by rainbow trout (Salmo gairdneri).

    PubMed

    Schoenhard, G L; Bishop, P E; Lee, D J; Sinnhuber, R O

    1975-09-01

    A rapid and sensitive microbial assay was developed to detect lethal products of aflatoxin B metabolism by rainbow trout (Salmon gairdneri) Mt. Shasta strain. Bacillus subtilis GSY 1057 (hisA1, uvr-1, metB4), a DNA repair deficient strain, was incubated for 20 min in the 20,000 times g supernate from trout liver homogenates which had been preincubated for 10 min with various levels of aflatoxin B. Serial dilutions of the incubation mixture were plated in triplicate on tryptose blood agar base plates and colonies were counted after 12 hr at 37 degrees C. One mumole aflatoxin B in 3.2 ml incubation mixture reduced viability 60%. PMID:808527

  19. A 32,000-molecular weight protein from bovine placenta with placental lactogen-like activity in radioreceptor assays

    SciTech Connect

    Eakle, K.A.; Arima, Y.; Swanson, P.; Grimek, H.; Bremel, R.D.

    1982-05-01

    Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated with a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones.

  20. A 32,000-molecular weight protein from bovine placenta with placental lactogen-like activity in radioreceptor assays.

    PubMed

    Eakle, K A; Arima, Y; Swanson, P; Grimek, H; Bremel, R D

    1982-05-01

    Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated which a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones. PMID:7075536

  1. Hanford Low-Activity Waste Processing: Demonstration of the Off-Gas Recycle Flowsheet - 13443

    SciTech Connect

    Ramsey, William G.; Esparza, Brian P.

    2013-07-01

    Vitrification of Hanford Low-Activity Waste (LAW) is nominally the thermal conversion and incorporation of sodium salts and radionuclides into borosilicate glass. One key radionuclide present in LAW is technetium-99. Technetium-99 is a low energy, long-lived beta emitting radionuclide present in the waste feed in concentrations on the order of 1-10 ppm. The long half-life combined with a high solubility in groundwater results in technetium-99 having considerable impact on performance modeling (as potential release to the environment) of both the waste glass and associated secondary waste products. The current Hanford Tank Waste Treatment and Immobilization Plant (WTP) process flowsheet calls for the recycle of vitrification process off-gas condensates to maximize the portion of technetium ultimately immobilized in the waste glass. This is required as technetium acts as a semi-volatile specie, i.e. considerable loss of the radionuclide to the process off-gas stream can occur during the vitrification process. To test the process flowsheet assumptions, a prototypic off-gas system with recycle capability was added to a laboratory melter (on the order of 1/200 scale) and testing performed. Key test goals included determination of the process mass balance for technetium, a non-radioactive surrogate (rhenium), and other soluble species (sulfate, halides, etc.) which are concentrated by recycling off-gas condensates. The studies performed are the initial demonstrations of process recycle for this type of liquid-fed melter system. This paper describes the process recycle system, the waste feeds processed, and experimental results. Comparisons between data gathered using process recycle and previous single pass melter testing as well as mathematical modeling simulations are also provided. (authors)

  2. Demonstration of a Spoken Dialogue Interface for Planning Activities of a Semi-autonomous Robot

    NASA Technical Reports Server (NTRS)

    Dowding, John; Frank, Jeremy; Hockey, Beth Ann; Jonsson, Ari; Aist, Gregory

    2002-01-01

    Planning and scheduling in the face of uncertainty and change pushes the capabilities of both planning and dialogue technologies by requiring complex negotiation to arrive at a workable plan. Planning for use of semi-autonomous robots involves negotiation among multiple participants with competing scientific and engineering goals to co-construct a complex plan. In NASA applications this plan construction is done under severe time pressure so having a dialogue interface to the plan construction tools can aid rapid completion of the process. But, this will put significant demands on spoken dialogue technology, particularly in the areas of dialogue management and generation. The dialogue interface will need to be able to handle the complex dialogue strategies that occur in negotiation dialogues, including hypotheticals and revisions, and the generation component will require an ability to summarize complex plans. This demonstration will describe a work in progress towards building a spoken dialogue interface to the EUROPA planner for the purposes of planning and scheduling the activities of a semi-autonomous robot. A prototype interface has been built for planning the schedule of the Personal Satellite Assistant (PSA), a mobile robot designed for micro-gravity environments that is intended for use on the Space Shuttle and International Space Station. The spoken dialogue interface gives the user the capability to ask for a description of the plan, ask specific questions about the plan, and update or modify the plan. We anticipate that a spoken dialogue interface to the planner will provide a natural augmentation or alternative to the visualization interface, in situations in which the user needs very targeted information about the plan, in situations where natural language can express complex ideas more concisely than GUI actions, or in situations in which a graphical user interface is not appropriate.

  3. Deactivation of the inferior colliculus by cooling demonstrates intercollicular modulation of neuronal activity

    PubMed Central

    Orton, Llwyd D.; Poon, Paul W. F.; Rees, Adrian

    2012-01-01

    The auditory pathways coursing through the brainstem are organized bilaterally in mirror image about the midline and at several levels the two sides are interconnected. One of the most prominent points of interconnection is the commissure of the inferior colliculus (CoIC). Anatomical studies have revealed that these fibers make reciprocal connections which follow the tonotopic organization of the inferior colliculus (IC), and that the commissure contains both excitatory and, albeit fewer, inhibitory fibers. The role of these connections in sound processing is largely unknown. Here we describe a method to address this question in the anaesthetized guinea pig. We used a cryoloop placed on one IC to produce reversible deactivation while recording electrophysiological responses to sounds in both ICs. We recorded single units, multi-unit clusters and local field potentials (LFPs) before, during and after cooling. The degree and spread of cooling was measured with a thermocouple placed in the IC and other auditory structures. Cooling sufficient to eliminate firing was restricted to the IC contacted by the cryoloop. The temperature of other auditory brainstem structures, including the contralateral IC and the cochlea were minimally affected. Cooling below 20°C reduced or eliminated the firing of action potentials in frequency laminae at depths corresponding to characteristic frequencies up to ~8 kHz. Modulation of neural activity also occurred in the un-cooled IC with changes in single unit firing and LFPs. Components of LFPs signaling lemniscal afferent input to the IC showed little change in amplitude or latency with cooling, whereas the later components, which likely reflect inter- and intra-collicular processing, showed marked changes in form and amplitude. We conclude that the cryoloop is an effective method of selectively deactivating one IC in guinea pig, and demonstrate that auditory processing in the IC is strongly influenced by the other. PMID:23248587

  4. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase.

    PubMed

    Loose, Jennifer S M; Forsberg, Zarah; Fraaije, Marco W; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2014-09-17

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing. PMID:25109775

  5. A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride.

    PubMed

    Li, Xue; Zhou, Yunlei; Xu, Yan; Xu, Huijie; Wang, Minghui; Yin, Huanshun; Ai, Shiyun

    2016-08-31

    Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity. PMID:27506341

  6. Evidence from a carbohydrate incorporation assay for direct activation of bone marrow myelopoietic precursor cells by bacterial cell wall constitutents.

    PubMed Central

    Monner, D A; Gmeiner, J; Mühlradt, P F

    1981-01-01

    The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event. PMID:7014467

  7. Structure-activity relationships of aromatic diamines in the Ames Salmonella typhimurium assay. Part II.

    PubMed

    Kalopissis, G

    1992-09-01

    Structure-activity relationships in the case of aromatic monoamines, diversely substituted on the ring, using the mutagenic activity in the Ames test were studied in part I. This part II is based on the same general principles but applied to phenylene diamines (ortho, para and meta) diversely substituted on the ring. PMID:1381475

  8. 20 CFR 641.630 - What pilot, demonstration, and evaluation project activities are allowable under § 502(e)?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 3 2012-04-01 2012-04-01 false What pilot, demonstration, and evaluation project activities are allowable under § 502(e)? 641.630 Section 641.630 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROVISIONS GOVERNING THE SENIOR COMMUNITY SERVICE EMPLOYMENT PROGRAM Pilot, Demonstration,...

  9. In Situ Sediment Treatment Using Activated Carbon: A Demonstrated Sediment Cleanup Technology

    PubMed Central

    Patmont, Clayton R; Ghosh, Upal; LaRosa, Paul; Menzie, Charles A; Luthy, Richard G; Greenberg, Marc S; Cornelissen, Gerard; Eek, Espen; Collins, John; Hull, John; Hjartland, Tore; Glaza, Edward; Bleiler, John; Quadrini, James

    2015-01-01

    This paper reviews general approaches for applying activated carbon (AC) amendments as an in situ sediment treatment remedy. In situ sediment treatment involves targeted placement of amendments using installation options that fall into two general approaches: 1) directly applying a thin layer of amendments (which potentially incorporates weighting or binding materials) to surface sediment, with or without initial mixing; and 2) incorporating amendments into a premixed, blended cover material of clean sand or sediment, which is also applied to the sediment surface. Over the past decade, pilot- or full-scale field sediment treatment projects using AC—globally recognized as one of the most effective sorbents for organic contaminants—were completed or were underway at more than 25 field sites in the United States, Norway, and the Netherlands. Collectively, these field projects (along with numerous laboratory experiments) have demonstrated the efficacy of AC for in situ treatment in a range of contaminated sediment conditions. Results from experimental studies and field applications indicate that in situ sequestration and immobilization treatment of hydrophobic organic compounds using either installation approach can reduce porewater concentrations and biouptake significantly, often becoming more effective over time due to progressive mass transfer. Certain conditions, such as use in unstable sediment environments, should be taken into account to maximize AC effectiveness over long time periods. In situ treatment is generally less disruptive and less expensive than traditional sediment cleanup technologies such as dredging or isolation capping. Proper site-specific balancing of the potential benefits, risks, ecological effects, and costs of in situ treatment