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Sample records for activity assays showed

  1. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    PubMed Central

    Kusch, Peter; Deininger, Susanne; Specht, Sabine; Maniako, Rudeka; Haubrich, Stefanie; Pommerening, Tanja; Lin, Paul Kong Thoo; Hoerauf, Achim; Kaiser, Annette

    2011-01-01

    Balanites aegyptiaca (Balanitaceae) is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 μg/μL ± 3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H)-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a Ki of 2.35 μg/μL and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a Ki of 4.8 μg/μL. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 μM ± 3 and 47.82 μM ± 2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed. PMID:21687598

  2. Arches showing UV flaring activity

    NASA Technical Reports Server (NTRS)

    Fontenla, J. M.

    1988-01-01

    The UVSP data obtained in the previous maximum activity cycle show the frequent appearance of flaring events in the UV. In many cases these flaring events are characterized by at least two footpoints which show compact impulsive non-simultaneous brightenings and a fainter but clearly observed arch developes between the footpoints. These arches and footpoints are observed in line corresponding to different temperatures, as Lyman alpha, N V, and C IV, and when observed above the limb display large Doppler shifts at some stages. The size of the arches can be larger than 20 arcsec.

  3. 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

  4. 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

  5. DNA Methyltransferase Activity Assays: Advances and Challenges.

    PubMed

    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.

  6. A microtiter assay shows effectivness of fungicides for control of Colletotrichum spp from strawberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The anthracnose pathogens, Colletotrichum acutatum, C. gloeosporioides and C. fragariae, have developed resistance to some fungicides. We used a micro-dilution broth assay to test in vitro the activity of 16 chemicals against ten isolates of Colletotrichum spp. using a dose-response format. At a c...

  7. Assay and Inhibition of Diacylglycerol Lipase Activity

    PubMed Central

    Johnston, Meghan; Bhatt, Shachi R.; Sikka, Surina; Mercier, Richard W.; West, Jay M.; Makriyannis, Alexandros; Gatley, S. John; Duclos, Richard I.

    2012-01-01

    A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-14C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-14C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-14C]arachidonic acid. PMID:22738638

  8. Sirt5 Deacylation Activities Show Differential Sensitivities to Nicotinamide Inhibition

    PubMed Central

    Suenkel, Benjamin; Lakshminarasimhan, Mahadevan; Schutkowski, Mike; Steegborn, Clemens

    2012-01-01

    Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD+ by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry–based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD+ affinity as well as the apparent NAD+ affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitive Sirtuin deacetylase family. The same Arg was recently reported to render Sirt5 a preferential desuccinylase, and we find that this Sirt5 activity is highly sensitive to nicotinamide inhibition. Analysis of Sirt5 structures and activity data suggest that an Arg/succinate interaction is the molecular basis of the differential nicotinamide sensitivities of the two Sirt5 activities. Our results thus indicate a Sirtuin subfamily with nicotinamide-insensitive deacetylase activity and suggest that the molecular features determining nicotinamide sensitivity overlap with those dominating deacylation specificity, possibly suggesting that other subfamily members might also prefer other acylations than acetylations. PMID:23028781

  9. Serving Up Activities for TV Cooking Shows.

    ERIC Educational Resources Information Center

    Katchen, Johanna E.

    This paper documents a presentation given on the use of English-language television cooking shows in English-as-a-Second-Language (ESL) and English-as-a-Foreign-Language (EFL) classrooms in Taiwan. Such shows can be ideal for classroom use, since they have a predictable structure consisting of short segments, are of interest to most students,…

  10. Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.

    PubMed

    Sanghera, Jasbinder; Li, Rick; Yan, Jun

    2009-12-01

    Many assay technologies have been developed and utilized to efficiently assay and screen against protein kinase targets. The radiometric assay format for assaying the protein kinase targets has been considered the "Gold Standard" format since it allows the direct readout of kinase functional activity and is a universal assay that is highly sensitive. However, the hazardous nature of the radiometric assay together with the regulatory hurdles has led to the development of alternative assay formats for assessing protein kinase activity measurements. The luminescent ADP-Glo assay has been developed as an alternative to radiometric format for assaying protein kinase targets. This assay allows the measurement of the ADP product formed during the kinase reaction. Therefore, the luminescent ADP-Glo assay is similar to the radiometric format in that it measures the direct product of the protein kinase reaction. Furthermore, since the ADP product is generated by all protein kinase reactions, this is a universal format that can be used for assaying any given protein kinase target. Analysis of data generated with multiple protein kinase targets and the luminescent ADP-Glo technology shows comparable results to the radiometric assay format. Therefore, the luminescent ADP-Glo assay is a robust new technology for evaluating catalytic function of protein kinases as well as other ATPases.

  11. A novel nucleic acid analogue shows strong angiogenic activity

    SciTech Connect

    Tsukamoto, Ikuko; Sakakibara, Norikazu; Maruyama, Tokumi; Igarashi, Junsuke; Kosaka, Hiroaki; Kubota, Yasuo; Tokuda, Masaaki; Ashino, Hiromi; Hattori, Kenichi; Tanaka, Shinji; Kawata, Mitsuhiro; Konishi, Ryoji

    2010-09-03

    Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

  12. 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

  13. 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.

  14. Rho family and Rap GTPase activation assays.

    PubMed

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  15. A passive-active neutron device for assaying remote-handled transuranic waste

    SciTech Connect

    Estep, R.J.; Coop, K.L.; Deane, T.M.; Lujan, J.E.

    1989-01-01

    A combined passive-active neutron assay device was constructed for assaying remote-handled transuranic waste. A study of matrix and source position effects in active assays showed that a knowledge of the source position alone is not sufficient to correct for position-related errors in highly moderating or absorbing matrices. An alternate function for the active assay of solid fuel pellets was derived, although the efficacy of this approach remains to be established. 4 refs., 7 figs., 1 tab.

  16. 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.

  17. 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

  18. 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)

  19. A calibration curve for immobilized dihydrofolate reductase activity assay.

    PubMed

    Singh, Priyanka; Morris, Holly; Tivanski, Alexei V; Kohen, Amnon

    2015-09-01

    An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate (k cat) of an immobilized dihydrofolate reductase model system (Singh et al., (2015), Anal. Biochem). This data article contains a calibration plot for the developed assay. In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship. The concentration of immobilized enzyme was varied by using 5 different size mica chips. The dsDNA concentration was the same for all chips, assuming that the surface area of the mica chip dictates the resulting amount of bound enzyme (i.e. larger sized chip would have more bound DHFR). The activity and concentration of each chip was measured.

  20. 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.

  1. 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.

  2. Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer

    PubMed Central

    Bhattacharyya, Sanchari; Yu, Yiting; Suzuki, Masako; Campbell, Nathaniel; Mazdo, Jozef; Vasanthakumar, Aparna; Bhagat, Tushar D.; Nischal, Sangeeta; Christopeit, Maximilian; Parekh, Samir; Steidl, Ulrich; Godley, Lucy; Maitra, Anirban; Greally, John M.; Verma, Amit

    2013-01-01

    5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with β-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis. PMID:23861445

  3. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    PubMed

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  4. Assay of nitrogenase activity in intact plant systems.

    PubMed

    Jain, M K; Vlassak, K

    1975-01-01

    Nitrogenase activity was assayed in intact system of Cichorium intybus, a non-leguminous commercially cultivated crop, Dahlia pinnata and Helianthus annus, and Taraxacum officinale, a common weed plant. The assay was made in fabricated cylinders which could accomodate pot with plants. In such kind of assay along with rhizosphere microflora, the nitrogen fixed by phyllosphere nitrogen fixing microflora could also be accounted, which otherwise was difficult to be accounted for. PMID:1211718

  5. Secukinumab, a novel anti-IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity.

    PubMed

    Karle, Anette; Spindeldreher, Sebastian; Kolbinger, Frank

    2016-01-01

    Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics--including monoclonal antibodies (mAbs)--can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex-associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR-associated biotherapeutic-derived peptides, representing potential T-cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4(+) T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence. PMID:26817498

  6. Secukinumab, a novel anti–IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

    PubMed Central

    Karle, Anette; Spindeldreher, Sebastian; Kolbinger, Frank

    2016-01-01

    ABSTRACT Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence. PMID:26817498

  7. Nanochannel-based electrochemical assay for transglutaminase activity.

    PubMed

    Fernández, Iñigo; Sánchez, Alfredo; Díez, Paula; Martínez-Ruiz, Paloma; Di Pierro, Prospero; Porta, Raffaele; Villalonga, Reynaldo; Pingarrón, José M

    2014-11-11

    A novel electrochemical assay to quantify transglutaminase activity is reported. The assay is based on the enzyme-controlled diffusion of Fe(CN)6(3-/4-) through amino-functionalized nanochannels of a mesoporous silica thin film on a Au surface in the presence of N-benzyloxycarbonyl-L-glutaminylglycine.

  8. Psychopaths Show Enhanced Amygdala Activation during Fear Conditioning

    PubMed Central

    Schultz, Douglas H.; Balderston, Nicholas L.; Baskin-Sommers, Arielle R.; Larson, Christine L.; Helmstetter, Fred J.

    2016-01-01

    Psychopathy is a personality disorder characterized by emotional deficits and a failure to inhibit impulsive behavior and is often subdivided into “primary” and “secondary” psychopathic subtypes. The maladaptive behavior related to primary psychopathy is thought to reflect constitutional “fearlessness,” while the problematic behavior related to secondary psychopathy is motivated by other factors. The fearlessness observed in psychopathy has often been interpreted as reflecting a fundamental deficit in amygdala function, and previous studies have provided support for a low-fear model of psychopathy. However, many of these studies fail to use appropriate screening procedures, use liberal inclusion criteria, or have used unconventional approaches to assay amygdala function. We measured brain activity with BOLD imaging in primary and secondary psychopaths and non-psychopathic control subjects during Pavlovian fear conditioning. In contrast to the low-fear model, we observed normal fear expression in primary psychopaths. Psychopaths also displayed greater differential BOLD activity in the amygdala relative to matched controls. Inverse patterns of activity were observed in the anterior cingulate cortex (ACC) for primary versus secondary psychopaths. Primary psychopaths exhibited a pattern of activity in the dorsal and ventral ACC consistent with enhanced fear expression, while secondary psychopaths exhibited a pattern of activity in these regions consistent with fear inhibition. These results contradict the low-fear model of psychopathy and suggest that the low fear observed for psychopaths in previous studies may be specific to secondary psychopaths. PMID:27014154

  9. Psychopaths Show Enhanced Amygdala Activation during Fear Conditioning.

    PubMed

    Schultz, Douglas H; Balderston, Nicholas L; Baskin-Sommers, Arielle R; Larson, Christine L; Helmstetter, Fred J

    2016-01-01

    Psychopathy is a personality disorder characterized by emotional deficits and a failure to inhibit impulsive behavior and is often subdivided into "primary" and "secondary" psychopathic subtypes. The maladaptive behavior related to primary psychopathy is thought to reflect constitutional "fearlessness," while the problematic behavior related to secondary psychopathy is motivated by other factors. The fearlessness observed in psychopathy has often been interpreted as reflecting a fundamental deficit in amygdala function, and previous studies have provided support for a low-fear model of psychopathy. However, many of these studies fail to use appropriate screening procedures, use liberal inclusion criteria, or have used unconventional approaches to assay amygdala function. We measured brain activity with BOLD imaging in primary and secondary psychopaths and non-psychopathic control subjects during Pavlovian fear conditioning. In contrast to the low-fear model, we observed normal fear expression in primary psychopaths. Psychopaths also displayed greater differential BOLD activity in the amygdala relative to matched controls. Inverse patterns of activity were observed in the anterior cingulate cortex (ACC) for primary versus secondary psychopaths. Primary psychopaths exhibited a pattern of activity in the dorsal and ventral ACC consistent with enhanced fear expression, while secondary psychopaths exhibited a pattern of activity in these regions consistent with fear inhibition. These results contradict the low-fear model of psychopathy and suggest that the low fear observed for psychopaths in previous studies may be specific to secondary psychopaths.

  10. Psychopaths Show Enhanced Amygdala Activation during Fear Conditioning.

    PubMed

    Schultz, Douglas H; Balderston, Nicholas L; Baskin-Sommers, Arielle R; Larson, Christine L; Helmstetter, Fred J

    2016-01-01

    Psychopathy is a personality disorder characterized by emotional deficits and a failure to inhibit impulsive behavior and is often subdivided into "primary" and "secondary" psychopathic subtypes. The maladaptive behavior related to primary psychopathy is thought to reflect constitutional "fearlessness," while the problematic behavior related to secondary psychopathy is motivated by other factors. The fearlessness observed in psychopathy has often been interpreted as reflecting a fundamental deficit in amygdala function, and previous studies have provided support for a low-fear model of psychopathy. However, many of these studies fail to use appropriate screening procedures, use liberal inclusion criteria, or have used unconventional approaches to assay amygdala function. We measured brain activity with BOLD imaging in primary and secondary psychopaths and non-psychopathic control subjects during Pavlovian fear conditioning. In contrast to the low-fear model, we observed normal fear expression in primary psychopaths. Psychopaths also displayed greater differential BOLD activity in the amygdala relative to matched controls. Inverse patterns of activity were observed in the anterior cingulate cortex (ACC) for primary versus secondary psychopaths. Primary psychopaths exhibited a pattern of activity in the dorsal and ventral ACC consistent with enhanced fear expression, while secondary psychopaths exhibited a pattern of activity in these regions consistent with fear inhibition. These results contradict the low-fear model of psychopathy and suggest that the low fear observed for psychopaths in previous studies may be specific to secondary psychopaths. PMID:27014154

  11. Diced electrophoresis gel assay for screening enzymes with specified activities.

    PubMed

    Komatsu, Toru; Hanaoka, Kenjiro; Adibekian, Alexander; Yoshioka, Kentaro; Terai, Takuya; Ueno, Tasuku; Kawaguchi, Mitsuyasu; Cravatt, Benjamin F; Nagano, Tetsuo

    2013-04-24

    We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.

  12. Measuring MAP kinase activity in immune complex assays.

    PubMed

    Cherkasova, Vera A

    2006-11-01

    I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins. PMID:16890454

  13. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  14. 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

  15. Synthesis and evaluation of dioleoyl glyceric acids showing antitrypsin activity.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Sato, Shun; Kitamoto, Dai; Sakaki, Keiji

    2011-01-01

    Previously, Lešová et al. reported the isolation and identification of metabolite OR-1, showing antitrypsin activity, produced during fermentation by Penicillium funiculosum. The structure of OR-1 was a mixture of glyceric acid (GA), esterified with C(14)-C(18) fatty acids, and oleic acid (C18:1) as the most predominant fatty acid (Folia Microbiol. 46, 21-23, 2001). In this study, dioleoyl D-GA and dioleoyl L-GA were synthesized via diesterification with oleoyl chloride, and their antitrypsin activities were evaluated using both a disk diffusion method and spectral absorption measurements. The results show that both compounds and their equivalent mixtures possess antitrypsin activities; however, their IC(50) values (approximately 2 mM) are much higher than that of OR-1 (4.25 µM), suggesting that dioleoyl GA does not play a major role in the OR-1 antitrypsin activity. PMID:21606621

  16. 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

  17. Pitfalls in the assay of carboxymethylcellulase activity. [Sclerotium rolfsii

    SciTech Connect

    Lindner, W.A.; Dennison, C.; Quicke, G.V.

    1983-02-01

    A purified endocellulase from Sclerotium rolfsii and a crude cellulase preparation from Trichoderma reesei are used to illustrate several pitfalls associated with the assay of carboxymethylcellulase activity and the subsequent attainment of linear enzyme dilution curves. It is shown that the nature of both the enzymes and the substrate make the assay unsuitable for use in the calculation of enzyme recovery and purity. (Refs. 16).

  18. Your Library--Greatest Show of All! Activity Manual.

    ERIC Educational Resources Information Center

    Jackson, Mary, Ed.

    Intended as a companion volume for librarians planning a children's summer reading program, this activity manual provides ideas for games, puzzles, puppet shows, story reading, and more, all based on the theme of "Circus Summer." The manual suggests ways to promote the program, such as visits to schools, and provides directions for registering…

  19. Croton grewioides Baill. (Euphorbiaceae) Shows Antidiarrheal Activity in Mice

    PubMed Central

    da Silva, Anne Dayse Soares; de Melo e Silva, Karoline; Neto, José Clementino; Costa, Vicente Carlos de Oliveira; Pessôa, Hilzeth de Luna F.; Tavares, Josean Fechine; da Silva, Marcelo Sobral; Cavalcante, Fabiana de Andrade

    2016-01-01

    Based on chemotaxonomy, we decided to investigate the possible antidiarrheal activity in mice of a crude ethanolic extract obtained from aerial parts of Croton grewioides (CG-EtOH). We tested for any possible toxicity in rat erythrocytes and acute toxicity in mice. Antidiarrheal activity was assessed by determining the effect of CG-EtOH on defecation frequency, liquid stool, intestinal motility and intestinal fluid accumulation. CG-EtOH showed no in vitro cytotoxicity and was not orally lethal. In contrast, the extract given intraperitoneally (at 2000 mg/kg) was lethal, but only in females. CG-EtOH produced a significant and equipotent antidiarrheal activity, both in defecation frequency (ED50 = 106.0 ± 8.1 mg/kg) and liquid stools (ED50 = 105.0 ± 9.2 mg/kg). However, CG-EtOH (125 mg/kg) decreased intestinal motility by only 22.7% ± 4.4%. Moreover, extract markedly inhibited the castor oil-induced intestinal contents (ED50 = 34.6 ± 5.4 mg/kg). We thus conclude that CG-EtOH is not orally lethal and contains active principles with antidiarrheal activity, and this effect seems to involve mostly changes in intestinal secretion. SUMMARY CG-EtOH showed no in vitro cytotoxicity and was not orally lethal. In contrast, the extract given intraperitoneally (at 2000 mg/kg) was lethal, but only in females.CG-EtOH probably contains active metabolites with antidiarrheal activity.CG-EtOH reduced the frequency and number of liquid stools.Metabolites presents in the CG-EtOH act mainly by reducing intestinal fluid and, to a lesser extent, reducing intestinal motility. Abbreviations Used: CG-EtOH: crude ethanolic extract obtained from the aerial parts of C. grewioides; WHO: World Health Organization; ED50: dose of a drug that produces 50% of its maximum effect; Emax: maximum effect PMID:27365990

  20. 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

  1. Urolithins display both antioxidant and pro-oxidant activities depending on assay system and conditions.

    PubMed

    Kallio, Tuija; Kallio, Johanna; Jaakkola, Mari; Mäki, Marianne; Kilpeläinen, Pekka; Virtanen, Vesa

    2013-11-13

    The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

  2. High-Throughput FRET Assay Yields Allosteric SERCA Activators

    PubMed Central

    Cornea, Razvan L.; Lockamy, Elizabeth L.; Gruber, Simon J.; Muretta, Joseph M.; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M.; Gillispie, Gregory D.; Thomas, David D.

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT. PMID:22923787

  3. High-throughput FRET assay yields allosteric SERCA activators.

    PubMed

    Cornea, Razvan L; Gruber, Simon J; Lockamy, Elizabeth L; Muretta, Joseph M; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M; Gillispie, Gregory D; Thomas, David D

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

  4. Arginase Activity in Mitochondria - an Interfering Factor in Nitric Oxide Synthase Activity Assays

    PubMed Central

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

    2009-01-01

    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 [14C]-L-arginine to [14C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent [1]. 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 [14C]-L-arginine to [14C]-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. (Supported by ES 011982 & 2G12RR008124 to RTM & UTEP, respectively). PMID:19896461

  5. 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.

  6. 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

  7. GTP-specific fab fragment-based GTPase activity assay.

    PubMed

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  8. Tetrahdroxysqualene from Rhus taitensis Shows Antimycobacterial Activity Against Mycobacterium tuberculosis

    PubMed Central

    Noro, Jeffrey C.; Barrows, Louis R.; Gideon, Osia G.; Ireland, Chris M.; Koch, Michael; Matainaho, Teatulohi; Piskaut, Pius; Pond, Christopher D.; Bugni, Tim S.

    2010-01-01

    Tuberculosis has become a major health problem, in particular with the emergence of extremely drug resistant tuberculosis (XDRTB). In our search for new therapeutic leads against TB, we isolated a new triterpene (1) from the plant Rhus taitensis collected in Papua New Guinea. Tetrahydroxysqualene (1) was isolated using bioassay-guided fractionation of the methanolic extract of R. taitensis leaves and twigs. The structure of tetrahydroxysqualene (1) was elucidated on the basis of HRESIMS and 1D and 2D NMR spectra. Tetrahydroxysqualene (1) exhibited anti–tuberculosis activity with an MIC of 10.0 μg/mL while showing only modest cytotoxicity. PMID:18710283

  9. 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.

  10. 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

  11. The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei

    PubMed Central

    Carvalho, Luis; Martínez-García, Marta; Pérez-Victoria, Ignacio; Manzano, José Ignacio; Yardley, Vanessa

    2015-01-01

    The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca2+, and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei. PMID:26195527

  12. The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei.

    PubMed

    Carvalho, Luis; Martínez-García, Marta; Pérez-Victoria, Ignacio; Manzano, José Ignacio; Yardley, Vanessa; Gamarro, Francisco; Pérez-Victoria, José M

    2015-10-01

    The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei. PMID:26195527

  13. A new assay system for guinea pig interferon biological activity.

    PubMed

    Yamamoto, Toshiko; Jeevan, Amminikutty; Ohishi, Kazue; Nojima, Yasuhiro; Umemori, Kiyoko; Yamamoto, Saburo; McMurray, David N

    2002-07-01

    We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

  14. Measurement of factor v activity in human plasma using a microplate coagulation assay.

    PubMed

    Tilley, Derek; Levit, Irina; Samis, John A

    2012-09-09

    above the pre-existing pathology (15). The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP). The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

  15. High-Resolution Observations of a Filament showing Activated Barb

    NASA Astrophysics Data System (ADS)

    Joshi, Anand; Martin, Sara F.; Mathew, Shibu; Srivastava, Nandita

    2012-07-01

    Analysis of a filament showing an activated barb using observations from the Dutch Open Telescope (DOT) on 2010 August 20 are presented. The DOT takes Doppler images in Hα, among other wavelengths, in a region about 110 × 110 arcsec^{2} in area, at a cadence of 30~seconds. The offline image restoration technique of speckle reconstruction is applied to obtain diffraction limited images. The filament developed a new barb in 10~minutes, which disappeared within the next 35~minutes. Such a rapid formation and disappearance of a filament barb is unusual, and has not been reported earlier. Line-of-sight velocity maps were constructed from the Doppler images of the target filament. We observe flows in the filament spine towards the barb location prior to its formation, and flows in the barb towards the spine during its disappearance. Photospheric magnetograms from Heliospheric Magnetic Imager on board the Solar Dynamics Observatory, at a cadence of 45~seconds, were used to determine the changes in magnetic flux in the region surrounding the barb location. The variation of magnetic flux in this duration supports the view that barbs are rooted in minor magnetic polarity. Our analysis shows that barbs can be short-lived and formation and disappearance of the barb was associated with cancellation of magnetic flux.

  16. Heavy metal impurities impair the spectrophotometric assay of ribulose bisphosphate carboxylase activity.

    PubMed

    Walbot, V

    1977-01-01

    An inverse relationship between the concentration of ribose 5-phosphate and apparent ribulose bisphosphate carboxylase activity was observed. The Lilley-Walker assay spectrophotometric assay, in which the 3-phosphoglyceric acid-dependent oxidation of reduced pyridine nucleotide is measured, is shown to be highly sensitive to inhibition by heavy metals. Analysis of the purity of reagents showed that ribose 5-phosphate is often contaminated with lead in sufficient quantity to impair the assay. This noncompetitive inhibition by ribose 5-phosphate is independent of the competitive inhibition of this substrate as an ATP sink as described by Slabas and Walker. A method for checking reagent purity and removing heavy metal contaminants is described.

  17. 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

  18. A molecular beacon assay for measuring base excision repair activities.

    PubMed

    Maksimenko, Andrei; Ishchenko, Alexander A; Sanz, Guenhaël; Laval, Jacques; Elder, Rhoderick H; Saparbaev, Murat K

    2004-06-18

    The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.

  19. 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.

  20. Synthesis and Assay of SIRT1-Activating Compounds.

    PubMed

    Dai, H; Ellis, J L; Sinclair, D A; Hubbard, B P

    2016-01-01

    The NAD(+)-dependent deacetylase SIRT1 plays key roles in numerous cellular processes including DNA repair, gene transcription, cell differentiation, and metabolism. Overexpression of SIRT1 protects against a number of age-related diseases including diabetes, cancer, and Alzheimer's disease. Moreover, overexpression of SIRT1 in the murine brain extends lifespan. A number of small-molecule sirtuin-activating compounds (STACs) that increase SIRT1 activity in vitro and in cells have been developed. While the mechanism for how these compounds act on SIRT1 was once controversial, it is becoming increasingly clear that they directly interact with SIRT1 and enhance its activity through an allosteric mechanism. Here, we present detailed chemical syntheses for four STACs, each from a distinct structural class. Also, we provide a general protocol for purifying active SIRT1 enzyme and outline two complementary enzymatic assays for characterizing the effects of STACs and similar compounds on SIRT1 activity. PMID:27423864

  1. A novel prothrombin time assay for assessing the anticoagulant activity of oral factor Xa inhibitors.

    PubMed

    Barrett, Yu Chen; Wang, Zhaoqing; Knabb, Robert M

    2013-09-01

    Conventional prothrombin time (PT) assays have limited sensitivity and dynamic range in monitoring the anticoagulant activity of direct factor Xa inhibitors. Hence, new assays are needed. We modified a PT assay by adding calcium chloride (CaCl2) to the thromboplastin reagent to increase assay dynamic range and improve sensitivity. Effects of calcium and sodium ion concentrations, and sample handling, were evaluated to optimize assay performance. Increasing concentrations of calcium ions produced progressive increases in PT across the factor Xa inhibitor concentrations of 0 to 2500 nmol/L for razaxaban and apixaban. The greatest effect was seen when the thromboplastin reagent was diluted 1:2.25 with 100 mmol/L CaCl2 (thus selected for routine use). The optimized assay showed an interassay precision of 1.5 to 9.3 percentage coefficient of variation (%CV) for razaxaban and 3.1 to 4.6 %CV for apixaban. We conclude that the modified PT assay is likely to be suitable as a pharmacodynamic marker for activity at therapeutic concentrations of factor Xa inhibitors.

  2. 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.

  3. 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

  4. Fluorescence assay for evaluating microbicidal activity of hand antiseptics.

    PubMed

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

    2015-11-01

    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 (R(2) = 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.

  5. Fluorescence assay for evaluating microbicidal activity of hand antiseptics.

    PubMed

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

    2015-11-01

    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 (R(2) = 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

  6. 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

  7. NASA's Fermi Shows How Active Galaxies Can Be

    NASA Video Gallery

    Active galaxies called blazars make up the largest class of objects detected by Fermi's Large Area Telescope (LAT). Massive black holes in the hearts of these galaxies fire particle jets in our dir...

  8. Ultraviolet imaging spectroscopy shows an active saturnian system.

    PubMed

    Esposito, Larry W; Colwell, Joshua E; Larsen, Kristopher; McClintock, William E; Stewart, A Ian F; Hallett, Janet Tew; Shemansky, Donald E; Ajello, Joseph M; Hansen, Candice J; Hendrix, Amanda R; West, Robert A; Keller, H Uwe; Korth, Axel; Pryor, Wayne R; Reulke, Ralf; Yung, Yuk L

    2005-02-25

    Neutral oxygen in the saturnian system shows variability, and the total number of oxygen atoms peaks at 4 x 10(34). Saturn's aurora brightens in response to solar-wind forcing, and the auroral spectrum resembles Jupiter's. Phoebe's surface shows variable water-ice content, and the data indicate it originated in the outer solar system. Saturn's rings also show variable water abundance, with the purest ice in the outermost A ring. This radial variation is consistent with initially pure water ice bombarded by meteors, but smaller radial structures may indicate collisional transport and recent renewal events in the past 10(7) to 10(8) years.

  9. 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

  10. 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.

  11. Automated conductimetric assay of human serum cholinesterase activity.

    PubMed

    Duffy, P; Wallach, J M

    1989-01-01

    Serum cholinesterase activity was determined by conductimetry using samples in the microliter range. Butyrylcholine iodide was demonstrated to be a convenient substrate for the conductimetric assay. Validation of the microassay was made by using either purified enzyme or control serum. In the range of 0-60 U/l, a linear relationship was demonstrated. Correlation with a reference spectrophotometric method was obtained with a slope of 1.18. An explanation of this value is proposed, as different hydrolysis rates were obtained with human sera, depending on the substrate used (butyrylthio- or butyryl-choline ester).

  12. 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.

  13. Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase

    PubMed Central

    2012-01-01

    Background Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7. Methods The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane) and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase) assays in MCF-7 cells. Results Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml). Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase. Conclusions Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide dismutase in the treated cells

  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. Assaying the Kinase Activity of LRRK2 in vitro

    PubMed Central

    Lewis, Patrick A.

    2012-01-01

    Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate. A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Daniëls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different

  16. A barium based coordination polymer for the activity assay of deoxyribonuclease I.

    PubMed

    Song, Chan; Wang, Guan-Yao; Wang, Ya-Ling; Kong, De-Ming; Wang, Yong-Jian; Li, Yue; Ruan, Wen-Juan

    2014-10-01

    A new coordination polymer which shows an unusual 2D inorganic connectivity was constructed. This compound exhibits distinct fluorescence quenching ability to the dye-labeled single-stranded DNA probes with different lengths, based on which an analytical method was developed for the activity assay of deoxyribonuclease I.

  17. Insurance Applications of Active Fault Maps Showing Epistemic Uncertainty

    NASA Astrophysics Data System (ADS)

    Woo, G.

    2005-12-01

    Insurance loss modeling for earthquakes utilizes available maps of active faulting produced by geoscientists. All such maps are subject to uncertainty, arising from lack of knowledge of fault geometry and rupture history. Field work to undertake geological fault investigations drains human and monetary resources, and this inevitably limits the resolution of fault parameters. Some areas are more accessible than others; some may be of greater social or economic importance than others; some areas may be investigated more rapidly or diligently than others; or funding restrictions may have curtailed the extent of the fault mapping program. In contrast with the aleatory uncertainty associated with the inherent variability in the dynamics of earthquake fault rupture, uncertainty associated with lack of knowledge of fault geometry and rupture history is epistemic. The extent of this epistemic uncertainty may vary substantially from one regional or national fault map to another. However aware the local cartographer may be, this uncertainty is generally not conveyed in detail to the international map user. For example, an area may be left blank for a variety of reasons, ranging from lack of sufficient investigation of a fault to lack of convincing evidence of activity. Epistemic uncertainty in fault parameters is of concern in any probabilistic assessment of seismic hazard, not least in insurance earthquake risk applications. A logic-tree framework is appropriate for incorporating epistemic uncertainty. Some insurance contracts cover specific high-value properties or transport infrastructure, and therefore are extremely sensitive to the geometry of active faulting. Alternative Risk Transfer (ART) to the capital markets may also be considered. In order for such insurance or ART contracts to be properly priced, uncertainty should be taken into account. Accordingly, an estimate is needed for the likelihood of surface rupture capable of causing severe damage. Especially where a

  18. Phenolic compounds from leaves of Casimiroa edulis showed adipogenesis activity.

    PubMed

    Nagai, Hiroyuki; Tanaka, Toshiyuki; Goto, Tsuyoshi; Kusudo, Tatsuya; Takahashi, Nobuyuki; Kawada, Teruo

    2014-01-01

    Casimiroa edulis is known as cochitzapotl, and it belongs to a species of tropical fruiting tree in the family Rutaceae, native to eastern Mexico and Central America south to Costa Rica. In this study, we isolated two furocoumarins and two polymethoxyflavones from leaves of C. edulis and evaluated the functions of glucose and lipid metabolism activity with 3T3-L1 adipocytes. We discovered that the addition of furocoumarins increased glucose uptake and lipid accumulation in 3T3-L1 adipocyte. These results suggest that furocoumarin compounds can be used as functional food-derived compounds, to regulate adipocyte functioning for the management of metabolic syndrome, which is associated with dysfunctions of glucose and lipid metabolism. PMID:25036684

  19. Automated filter paper assay for determination of cellulase activity.

    PubMed

    Decker, Stephen R; Adney, William S; Jennings, Edward; Vinzant, Todd B; Himmel, Michael E

    2003-01-01

    Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in beta-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and beta-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.

  20. 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.

  1. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    PubMed

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was < 10%. Therefore, these chemicals were classified as moderate skin sensitizers in the LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE.

  2. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    PubMed

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was < 10%. Therefore, these chemicals were classified as moderate skin sensitizers in the LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE. PMID:26558466

  3. 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.

  4. Evaluation of immunostimulatory activity of Chyawanprash using in vitro assays.

    PubMed

    Madaan, Alka; Kanjilal, Satyajyoti; Gupta, Arun; Sastry, J L N; Verma, Ritu; Singh, Anu T; Jaggi, Manu

    2015-03-01

    Chyawanprash is an ayurvedic formulation used in Indian traditional medicinal system for its beneficial effect on human health. We investigated the immunostimulatory effects of Chyawanprash (CHY) using in vitro assays evaluating the secretion of cytokines such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1beta (IL-1β) and Macrophage Inflammatory Protein-1-alpha (MIP-1-α) from murine bone marrow derived Dendritic Cells (DC) which play pivotal role in immunostimulation. The effects of CHY on phagocytosis in murine macrophages (RAW264.7) and Natural Killer (NK) cell activity were also investigated. At non-cytotoxic concentrations (20-500 μg/ml), CHY enhanced the secretion of all the three cytokines from DC. CHY also stimulated both, macrophage (RAW264.7) as well as NK cell activity, in vitro. In conclusion, the data substantiates the immunoprotective role of CHY at cellular level mediated by immunostimulation in key immune cells viz. dendritic Cells, macrophages and NK cells.

  5. Trajectory Hunting: Analysis of UARS Measurements showing Rapid Chlorine Activation

    NASA Technical Reports Server (NTRS)

    Danilin, M.Y.; Santee, M. L.; Rodriquez, J. M.; Ko, M. K. W.; Mergenthaler, J. M.; Kumer, J. B.; Tabazadeh, A.

    1998-01-01

    Trajectory hunting (i.e., a technique to find air parcels sampled at least twice over the course of a few days) is applied to analyze Upper Atmosphere Research Satellite (UARS) measurements in conjunction with the AER photochemical box model. In this study, we investigate rapid chlorine activation in the Arctic lower stratosphere on 29 Dec 1992 associated with a polar stratospheric cloud (PSC) event. Six air parcels that have been sampled twice were followed along 5-day trajectories at the 465 K (approximately 46 mb) and 585 K (approximately 22 mb) levels. A detailed sensitivity study with the AER. photochemical box model along these trajectories leads to the following conclusions for the episode considered: (1) model results are in better agreement with UARS measurements at these levels if the UKMO temperature is decreased by at least 1-2 K; (2) the NAT (nitric acid trihydrate) PSC formation scheme produces results in better agreement with observations than the STS (supercooled ternary solution) scheme; (3) the model can explain the UARS measurements at 585 K, but under-estimates the ClO abundance at 465 K, suggesting some inconsistency between the UARS measurements at this level.

  6. Trajectory Hunting: Analysis of UARS Measurements Showing Rapid Chlorine Activation

    NASA Technical Reports Server (NTRS)

    Danilin, M. Y.; Santee, M. L.; Rodriquez, J. M.; Ko, M. K. W.; Mergenthaler, J. M.; Kumer, J. B.; Tabazadeh, A.

    1998-01-01

    Trajectory hunting (i.e., a technique to find air parcels sampled at least twice over the course of a few days) is applied to analyze Upper Atmosphere Research Satellite (UARS) measurements in conjunction with the AER photochemical box model. In this study, we investigate rapid chlorine activation in the Arctic lower stratosphere on 29 Dec. 1992 associated with a polar stratospheric cloud (PSC) event. Six air parcels that have been sampled twice were followed along 5-day trajectories at the 465 K (approx. 46 mb) and 585 K (approxi. 22 mb) levels. A detailed sensitivity study with the AER photochemical box model along these trajectories leads to the following conclusions for the episode considered: 1) model results are in better agreement with UARS measurements at these levels if the U.K. Meteorological Office (UKMO) temperature is decreased by at least 1-2 K; 2) the NAT (nitric acid trihydrate) PSC formation scheme produces results in better agreement with observations than the STS (supercooled ternary solution) scheme; 3) the model can explain the UARS measurements at 585 K, but under-estimates the ClO abundance at 465 K, suggesting some inconsistency between the UARS measurements at this level.

  7. DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae

    PubMed Central

    Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie

    2016-01-01

    Background Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. Methodology/Principal Findings To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8- and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone–mediated cleavage assay showed that potencies of those drugs were at most 13.5- and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. Conclusions/Significance The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans. PMID:27681932

  8. Sulfur dioxide - Episodic injection shows evidence for active Venus volcanism

    NASA Astrophysics Data System (ADS)

    Esposito, L. W.

    1984-03-01

    Pioneer Venus ultraviolet spectra from the first 5 years of operation show a decline (by more than a factor of 10) in sulfur dioxide abundance at the cloud tops and in the amount of submicron haze above the clouds. At the time of the Pioneer Venus encounter, the values for both parameters greatly exceeded earlier upper limits. However, Venus had a similar appearance in the late 1950's, implying the episodic injection of sulfur dioxide possibly caused by episodic volcanism. The amount of haze in the Venus middle atmosphere is about ten times that found in earth's stratosphere after the most recent major volcanic eruptions, and the thermal energy required for this injection on Venus is greater by about an order of magnitude than the largest of these recent earth eruptions and about as large as the Krakatoa eruption of 1883. The episodic behavior of sulfur dioxide implies that steady-state models of the chemistry and dynamics of cloud-top regions may be of limited use.

  9. Sulfur dioxide: episodic injection shows evidence for active venus volcanism.

    PubMed

    Esposito, L W

    1984-03-01

    Pioneer Venus ultraviolet spectra from the first 5 years of operation show a decline (by more than a factor of 10) in sulfur dioxide abundance at the cloud tops and in the amount of submicron haze above the clouds. At the time of the Pioneer Venus encounter, the values for both parameters greatly exceeded earlier upper limits. However, Venus had a similar appearance in the late 1950's, implying the episodic injection of sulfur dioxide possibly caused by episodic volcanism. The amount of haze in the Venus middle atmosphere is about ten times that found in Earth's stratosphere after the most recent major volcanic eruptions, and the thermal energy required for this injection on Venus is greater by about an order of magnitude than the largest of these recent Earth eruptions and about as large as the Krakatoa eruption of 1883. The episodic behavior of sulfur dioxide implies that steady-state models of the chemistry and dynamics of cloud-top regions may be of limited use.

  10. A Fluorescence-based Assay of Phospholipid Scramblase Activity.

    PubMed

    Ploier, Birgit; Menon, Anant K

    2016-01-01

    Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin's scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin's scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins. PMID:27684510

  11. 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

  12. Cell-based flow cytometry assay to measure cytotoxic activity.

    PubMed

    Noto, Alessandra; Ngauv, Pearline; Trautmann, Lydie

    2013-12-17

    Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

  13. Quantitative detection of RT activity by PERT assay: feasibility and limits to a standardized screening assay for human vaccines.

    PubMed

    André, M; Morgeaux, S; Fuchs, F

    2000-06-01

    The detection of adventitious retroviruses has always been critical for assessing the safety concerns associated with viral vaccines. Assays for the enzymatic activity of reverse transcriptase (RT) are used as general methods for the detection of both known and unknown retroviruses. Several studies using newly-developed ultrasensitive PCR-based RT assays reported RT activity in viral vaccines grown in chicken cells. Here, we have assessed the performances of such a PCR-based RT assay--PERT assay--for the quantitative detection of RT activity in vaccines. Sensitivity, linearity and reproducibility of the method were studied on purified RT and viral vaccines treated to release RT from potentially contaminant retroviruses. The level of RT activity detected in chicken cell-derived vaccines was higher for live attenuated vaccines compared to inactivated ones. Contrary to other studies, RT activity was found in some mammalian cell-derived vaccines. AZT-TP sensitivity of RT activities detected in these vaccines and discrimination between retroviral and RT-like activities was further investigated. Feasibility and limits of PERT assay as a broad-spectrum retroviruses detection method in vaccines are discussed.

  14. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.

    PubMed

    Auld, Douglas S; Thorne, Natasha; Maguire, William F; Inglese, James

    2009-03-01

    High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

  15. The three catalases in Deinococcus radiodurans: Only two show catalase activity.

    PubMed

    Jeong, Sun-Wook; Jung, Jong-Hyun; Kim, Min-Kyu; Seo, Ho Seong; Lim, Heon-Man; Lim, Sangyong

    2016-01-15

    Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas ΔdrA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress.

  16. Sterols from Mytilidae show anti-aging and neuroprotective effects via anti-oxidative activity.

    PubMed

    Sun, Yujuan; Lin, Yanfei; Cao, Xueli; Xiang, Lan; Qi, Jianhua

    2014-11-25

    For screening anti-aging samples from marine natural products, K6001 yeast strain was employed as a bioassay system. The active mussel extract was separated to give an active sterol fraction (SF). SF was further purified, and four sterol compounds were obtained. Their structures were determined to be cholesterol (CHOL), brassicasterol, crinosterol, and 24-methylenecholesterol. All compounds showed similar anti-aging activity. To understand the action mechanism involved, anti-oxidative experiments, reactive oxygen species (ROS) assays, and malondialdehyde (MDA) tests were performed on the most abundant compound, CHOL. Results indicated that treatment with CHOL increases the survival rate of yeast under oxidative stress and decreases ROS and MDA levels. In addition, mutations of uth1, skn7, sod1, and sod2, which feature a K6001 background, were employed and the lifespans of the mutations were not affected by CHOL. These results demonstrate that CHOL exerts anti-aging effects via anti-oxidative stress. Based on the connection between neuroprotection and anti-aging, neuroprotective experiments were performed in PC12 cells. Paraquat was used to induce oxidative stress and the results showed that the CHOL and SF protect the PC12 cells from the injury induced by paraquat. In addition, these substance exhibited nerve growth factor (NGF) mimic activities again confirmed their neuroprotective function.

  17. The three catalases in Deinococcus radiodurans: Only two show catalase activity.

    PubMed

    Jeong, Sun-Wook; Jung, Jong-Hyun; Kim, Min-Kyu; Seo, Ho Seong; Lim, Heon-Man; Lim, Sangyong

    2016-01-15

    Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas ΔdrA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress. PMID:26692481

  18. Perfluorooctanoic acid (PFOA) but not perfluorooctane sulfonate (PFOS) showed DNA damage in comet assay on Paramecium caudatum.

    PubMed

    Kawamoto, Kosuke; Oashi, Takahiro; Oami, Kazunori; Liu, Wei; Jin, Yihe; Saito, Norimitsu; Sato, Itaru; Tsuda, Shuji

    2010-12-01

    Persistent perfluorinated organic compounds such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are distributed widely in the global environment including wildlife and human. In this study, we investigated the genotoxicity of PFOS and PFOA using the novel in vivo comet assay developed for Paramecium caudatum. For the comet assay, large nuclei squeezed out of the paramecia with 0.25 M sucrose containing 0.6% Triton X-100 were embedded in a layer of agarose gel placed over the slide glass. N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) and 2-aminoanthracene (2-AA) were successfully used for positive controls. Productions of 8-hydroxydeoxyguanosine (8-OH-dG) and intracellular reactive oxygen species (ROS) were also measured in paramecia. PFOS did not cause DNA damage on any conditions examined. On the other hand, 12 and 24 hr exposure to PFOA (100 µM) increased DNA migration in electrophoresis condition at pH 13, but not at pH 12.1, suggesting that the DNA damage may be alkali labile site (such as apurinic/apyrimidinic (AP) site). Exposure of paramecia to 100 µM PFOA for 1, 3 and 24 hr and to 10 µM PFOA for 24 hr significantly increased intracellular ROS. Under the same condition, however, 8-OH-dG level was not affected by PFOA. The PFOA-induced DNA damage was not abolished by the application of 100 µM GSH which completely inhibited the increase of intracellular ROS. In conclusion, the PFOA-induced in vivo DNA damage was first shown in paramecia, and the DNA damage might not be directly attributable to increase in intracellular ROS.

  19. 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

  20. Immunomodulatory assays to study structure-activity relationships of thalidomide.

    PubMed

    Shannon, E J; Morales, M J; Sandoval, F

    1997-01-01

    Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

  1. Mutagenic activity of isoxazolylnaphthoquinoneimines assayed by micronucleus bone marrow test.

    PubMed

    Sicardi, S M; Ferrato, E

    1995-05-01

    Studies were undertaken to evaluate the ability of various quinoneimines to induce micronuclei in bone marrow cells as a measure of their genotoxicity. Accordingly, 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I), its 2-acetyl derivative (II) and 2-[(5-methyl-3-isoxazolyl)amino]-N-(5-methyl-3-isoxazolyl)-1 ,4- naphthoquinone-4-imine (III), as well as two of their precursors, 2-hydroxynaphthoquinone (NQ-2-OH) and 3,4-dimethyl-5-aminoisoxazole (DMAI) were given by intraperitoneal injection at 5, 50, 100 and 200 mg/Kg doses to S.J.L. Swiss mice with 24 h sampling time. Compounds I and II displayed highly significant differences at 50, 100 and 200 mg/kg doses (p < 0.01) and their mutagenic dose response curves correlated closely with an inverted U-shaped form whose interpretation is still the subject of controversy. NQ-2-OH only produced a significant increase in micronucleus frequency at 50 mg/kg, whereas no mutagenic activity was found for compound III and DMAI at the doses assayed. At 50 mg/kg the order of relative mutagenic potencies was I > II > NQ-2-OH. Mechanisms advanced to explain loss of drug activity at high doses include capture saturation, enzymatic induction during metabolism and participation of an independent defense system. PMID:7753107

  2. Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems.

    PubMed

    Luckert, Claudia; Hessel, Stefanie; Lampen, Alfonso; Braeuning, Albert

    2015-10-15

    Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals.

  3. 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

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

    PubMed

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

    2015-03-11

    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.

  6. Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay.

    PubMed

    McDowell, Arlene; Thompson, Scott; Stark, Mirjam; Ou, Zong-Quan; Gould, Kevin S

    2011-12-01

    There is considerable interest in antioxidant dietary components that can be protective against degenerative diseases in humans. Puha (Sonchus oleraceus L.) is a rich source of polyphenols, and exhibits strong antioxidant activity as measured by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. However, the potential of puha to protect against degenerative diseases requires that low molecular weight antioxidants (LMWA) are absorbed by, and active in, human cells. The cellular antioxidant activity (CAA) assay was used to investigate the antioxidant activity of puha leaf extracts. Preparation methods of freezing and freeze-drying reduced the total polyphenolic content compared with fresh puha, but did not affect the LMWA potential as determined by the DPPH assay. The IC(50) values were 0.012 ± 0.003 mg/mL and 0.010 ± 0.005 mg/mL for freeze-dried and fresh puha leaves, respectively. Using the CAA assay, it was shown that LMWAs from foliar extracts of puha were effectively absorbed into HepG2 cells, and exerted antioxidant activity at levels comparable to those of extracts from blueberry fruits, the much-touted antioxidant superfood. Methylene blue staining of HepG2 cells indicated that puha extracts were not cytotoxic at concentrations below 100 mg DW/mL. The data indicate the potential of puha as a nutraceutical supplement for human health.

  7. Total alkaloids of Rubus alceifolius Poir shows anti-angiogenic activity in vivo and in vitro.

    PubMed

    Zhao, Jinyan; Lin, Wei; Zhuang, Qunchuan; Zhong, Xiaoyong; Cao, Zhiyun; Hong, Zhenfeng; Peng, Jun

    2014-11-01

    Total alkaloids is an active ingredient of the natural plant Rubus alceifolius Poir, commonly used for the treatment of various cancers. Antitumor effects may be mediated through anti-angiogenic mechanisms. As such, the goal of the present study was to investigate and evaluate the effect of total alkaloids in Rubus alceifolius Poir (TARAP) on tumor angiogenesis and investigate the underlying molecular mechanisms of TARAP action in vivo and in vitro. A chick embryo chorioallantoic membrane (CAM) assay was used to assess angiogenesis in vivo. An MTT assay was performed to determine the viability of human umbilical vein endothelial cells (HUVECs) with and without treatment. Cell cycle progression of HUVECs was examined by FACS analysis with propidium iodide staining. HUVEC migration was determined using a scratch wound method. Tube formation of HUVECs was assessed with an ECMatrix gel system, and mRNA and protein expression of VEGF-A in both HUVECs and HepG2 human hepatocellular carcinoma cells were examined by RT-PCR and ELISA, respectively. Our results showed that TARAP inhibited angiogenesis in the CAM model in vivo and inhibited HUVEC proliferation via blocking cell cycle G1 to S progression in a dose- and time-dependent manners in vitro. Moreover, TARAP inhibited HUVEC migration and tube formation and downregulated mRNA and protein expression of VEGF-A in both HepG2 cells and HUVECs. Our findings suggest that the anti-angiogenic activity of TARAP may partly contribute to its antitumor properties and may be valuable for the treatment of diseases involving pathologic angiogenesis such as cancer.

  8. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  9. 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

  10. Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity

    PubMed Central

    de Beer, Abré; Vivier, Melané A

    2008-01-01

    Background Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors. Results A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal

  11. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications

    NASA Astrophysics Data System (ADS)

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.

    2013-04-01

    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  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.

  13. 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

  14. Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro.

    PubMed

    Fung, King-Leung; Zhao, Kai-Jun; He, Zhu-Mei; Chye, Mee-Len

    2002-09-01

    We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009-1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic Ro plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 degrees C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size. PMID:12175020

  15. Satellite microglia show spontaneous electrical activity that is uncorrelated with activity of the attached neuron.

    PubMed

    Wogram, Emile; Wendt, Stefan; Matyash, Marina; Pivneva, Tatyana; Draguhn, Andreas; Kettenmann, Helmut

    2016-06-01

    Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact, and biocytin injection into satellite microglia showed no dye-coupling with the apposed neurons or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations that were not correlated with neuronal activity. These events could be divided into fast-rising and slow-rising depolarizations, which showed different characteristics in satellite and non-satellite microglia. Fast-rising and slow-rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast-rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.

  16. 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

  17. 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

  18. [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

  19. "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.

  20. "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

  1. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    PubMed

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  2. A continuous assay for foot-and-mouth disease virus 3C protease activity.

    PubMed

    Jaulent, Agnès M; Fahy, Aodhnait S; Knox, Stephen R; Birtley, James R; Roqué-Rosell, Núria; Curry, Stephen; Leatherbarrow, Robin J

    2007-09-15

    Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3C(pro), carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k(cat)/K(M) of 57.6+/-2.0M(-1) s(-1), was used in inhibition assays to further characterize the protease's activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3C(pro).

  3. Comparison of in vitro hormone activities of selected phthalates using reporter gene assays.

    PubMed

    Shen, Ouxi; Du, Guizhen; Sun, Hong; Wu, Wei; Jiang, Yi; Song, Ling; Wang, Xinru

    2009-12-01

    Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC(50) value of 1.05x10(-6), 1.22x10(-7)M and exceeding 1x10(-4)M respectively, but also showed the androgenic activity with EC(50) value of 6.17x10(-6), 1.13x10(-5)M and exceeding 1x10(-4)M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC(50) of 1.31x10(-5), 2.77x10(-6)M and exceeding 1x10(-4)M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0x10(-4)M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health.

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

    PubMed

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud'homme, Marie-Pascale; van der Graaff, Eric; 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.

  6. 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

  7. Syzyguim guineense Extracts Show Antioxidant Activities and Beneficial Activities on Oxidative Stress Induced by Ferric Chloride in the Liver Homogenate

    PubMed Central

    Pieme, Constant Anatole; Ngoupayo, Joseph; Khou-Kouz Nkoulou, Claude Herve; Moukette Moukette, Bruno; Njinkio Nono, Borgia Legrand; Ama Moor, Vicky Jocelyne; Ze Minkande, Jacqueline; Yonkeu Ngogang, Jeanne

    2014-01-01

    The aim of this study was to determine the in vitro antioxidant activity, free radical scavenging property and the beneficial effects of extracts of various parts of Syzygium guineense in reducing oxidative stress damage in the liver. The effects of extracts on free radicals were determined on radicals DPPH, ABTS, NO and OH followed by the antioxidant properties using Ferric Reducing Antioxidant Power assay (FRAP) and hosphomolybdenum (PPMB). The phytochemical screening of these extracts was performed by determination of the phenolic content. The oxidative damage inhibition in the liver was determined by measuring malondialdehyde (MDA) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase. Overall, the bark extract of the ethanol/water or methanol showed the highest radical scavenging activities against DPPH, ABTS and OH radicals compared to the other extracts. This extract also contained the highest phenolic content implying the potential contribution of phenolic compounds towards the antioxidant activities. However, the methanol extract of the root demonstrated the highest protective effects of SOD and CAT against ferric chloride while the hydro-ethanol extract of the leaves exhibited the highest inhibitory effects on lipid peroxidation. These findings suggest that antioxidant properties of S. guineense extracts could be attributed to phenolic compounds revealed by phytochemical studies. Thus, the present results indicate clearly that the extracts of S. guineense possess antioxidant properties and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. The antioxidant properties of the bark extract may thus sustain its various biological activities. PMID:26785075

  8. A microsystem to assay lysosomal enzyme activities in cultured retinal pigment epithelial cells.

    PubMed

    Cabral, L; Unger, W; Boulton, M; Marshall, J

    1988-11-01

    A microsystem to assay the activity of lysosomal enzymes in a small number of cultured RPE cells is described. The activities of acid phosphatase, a-mannosidase, B-glucuronidase and N-acetyl-B-glucosaminidase were estimated in different human RPE cultures of varying passages. Some biochemical characteristics for each of the enzyme assays were studied including the effect of pH, the saturating concentrations of the appropriate substrates and the relationship between the enzyme activity and the number of cells assayed. The method presented is straightforward, avoids complicated tissue fractionation procedures and is able to estimate enzyme activities in as few as 10(4) cells. PMID:3243083

  9. 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

  10. A high-throughput assay for modulators of NNT activity in permeabilized yeast cells.

    PubMed

    Meadows, Nicholas A; Saxty, Barbara; Albury, Mary S; Kettleborough, Catherine A; Ashcroft, Frances M; Moore, Anthony L; Cox, Roger D

    2011-08-01

    Nicotinamide nucleotide transhydrogenase (NNT) mutant mice show glucose intolerance with impaired insulin secretion during glucose tolerance tests. Uncoupling of the β cell mitochondrial metabolism due to such mutations makes NNT a novel target for therapeutics in the treatment of pathologies such as type 2 diabetes. The authors propose that increasing NNT activity would help reduce deleterious buildup of reactive oxygen species in the inner mitochondrial matrix. They have expressed human Nnt cDNA for the first time in Saccharomyces cerevisiae, and transhydrogenase activity in mitochondria isolated from these cells is six times greater than is seen in wild-type mitochondria. The same mitochondria have partially uncoupled respiration, and the cells have slower growth rates compared to cells that do not express NNT. The authors have used NNT's role as a redox-driven proton pump to develop a robust fluorimetric assay in permeabilized yeast. Screening in parallel a library of known pharmacologically active compounds (National Institute of Neurological Disorders and Stroke collection) against NNT ± cells, they demonstrate a robust and reproducible assay suitable for expansion into larger and more diverse compound sets. The identification of NNT activators may help in the elucidation of the role of NNT in mammalian cells and assessing its potential as a therapeutic target for insulin secretion disorders.

  11. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  12. Advantages and limitations of different p62-based assays for estimating autophagic activity in Drosophila.

    PubMed

    Pircs, Karolina; Nagy, Peter; Varga, Agnes; Venkei, Zsolt; Erdi, Balazs; Hegedus, Krisztina; Juhasz, Gabor

    2012-01-01

    Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy. PMID:22952930

  13. Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arylesterase.

    PubMed

    Furlong, C E; Richter, R J; Seidel, S L; Costa, L G; Motulsky, A G

    1989-08-01

    Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.

  14. 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

  15. Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations.

    PubMed

    Neis, J M; Yap, S H; van Gemert, P J; Roelofs, H M; Bos, R P; Henderson, P T

    1986-02-01

    The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.

  16. 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.

  17. Assays to measure the activation of membrane tyrosine kinase receptors: focus on cellular methods.

    PubMed

    Minor, Lisa K

    2003-09-01

    Many methods have been explored as means to measure the activation and inhibition of tyrosine kinase receptors, in vitro using the isolated kinase domain, and in living cells. Kinase activity has been measured in enzyme assays using a peptide substrate, but with different detection systems. These include the radioactive FlashPlate assay, the fluorescent resonance energy transfer (FRET) assay, the dissociation-enhance lanthanide fluorescence immunoassay (DELFIA) and other formats. These methods have successfully identified inhibitors of receptor activity. Cell-based assays have recently emerged to measure receptor activation and inhibition. When membrane tyrosine kinase receptors become activated, they increase their state of phosphorylation. This phosphorylation may lead to an increase in tyrosine kinase-specific activity. Methods have been developed that take advantage of these properties. These include measuring the ligand-stimulated total tyrosine phosphorylation of the receptor using a DELFIA or an ELISA assay, measuring ligand-stimulated enzyme activation of the receptor by quantifying enzyme activity, and dimerization of the activated receptor using bioluminescence resonance energy transfer (BRET). Although cell-based assays are still in their infancy, these techniques may prove a valuable addition to the receptor screening strategy.

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

    PubMed

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

    2014-08-01

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

  19. [Methods of hygromycin B phosphotransferase activity assay in transgenic plant].

    PubMed

    Zhuo, Qin; Yang, Xiaoguang

    2004-07-01

    Hygromycin B phosphotransferase (HPT) is a widely used selectable marker protein of transgenic plant. Detection of its activity is critical to studies on the development of various transgenic plants, silence of inserted gene, marker-free system development and safety assessment of transgenic food. In this paper, several methods for detecting the activity of this enzyme were reviewed.

  20. Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays.

    PubMed

    Inami, Keiko; Mochizuki, Masataka

    2002-08-26

    DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism. We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F(5)P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction. In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens. We activated 2-aminofluorene (AF), benzo[a]pyrene (B[a]P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F(5)P)Cl plus an oxidant-t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)-and we noted the effect of three solvents-acetonitrile (CH(3)CN),1,4-dioxane, and N,N-dimethylformamide (DMF)-on AF activation. All the promutagens became mutagenic in the presence of Fe(F(5)P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical. Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA. All the promutagens were mutagenic in the presence of Fe(F(5)P)Cl plus MPPT. For AF activation, the order of effectiveness of the solvents was CH(3)CN>1,4-dioxane>DMF. The results suggested that these systems would serve as

  1. 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.

  2. One novel quinoxaline derivative as a potent human cyclophilin A inhibitor shows highly inhibitory activity against mouse spleen cell proliferation.

    PubMed

    Li, Jian; Chen, Jing; Zhang, Li; Wang, Feng; Gui, Chunshan; Zhang, Li; Qin, Yu; Xu, Qiang; Liu, Hong; Nan, Fajun; Shen, Jingkang; Bai, Donglu; Chen, Kaixian; Shen, Xu; Jiang, Hualiang

    2006-08-15

    Cyclophilin A (CypA) is a ubiquitous cellular enzyme playing critical roles in many biological processes, and its inhibitor has been reported to have potential immunosuppressive activity. In this work, we reported a novel quinoxaline derivative, 2,3-di(furan-2-yl)-6-(3-N,N-diethylcarbamoyl-piperidino)carbonylamino quinoxaline (DC838, 3), which was confirmed to be a potent inhibitor against human CypA. By using the surface plasmon resonance (SPR) and fluorescence titration techniques, the kinetic analysis of CypA/DC838 interaction was quantitatively performed. CypA peptidyl prolyl cis-trans isomerase (PPIase) activity inhibition assay showed that DC838 demonstrated highly CypA PPIase inhibitory activity. In vivo assay results showed that DC838 could inhibit mouse spleen cell proliferation induced by concanavalin A (Con A). Molecular docking simulation further elucidated the specific DC838 binding to CypA at the atomic level. The current work should provide useful information in the discovery of immunosuppressor based on CypA inhibitor.

  3. Enzyme activity assays within microstructured optical fibers enabled by automated alignment

    PubMed Central

    Warren-Smith, Stephen C.; Nie, Guiying; Schartner, Erik P.; Salamonsen, Lois A.; Monro, Tanya M.

    2012-01-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women’s health. PMID:23243579

  4. 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

  5. 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.

  6. 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

  7. 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

  8. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

    PubMed

    Evandri, M G; Battinelli, L; Daniele, C; Mastrangelo, S; Bolle, P; Mazzanti, G

    2005-09-01

    Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare. PMID:15907354

  9. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

    PubMed

    Evandri, M G; Battinelli, L; Daniele, C; Mastrangelo, S; Bolle, P; Mazzanti, G

    2005-09-01

    Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare.

  10. A novel solubility-enhanced curcumin formulation showing stability and maintenance of anticancer activity.

    PubMed

    Zhang, Fang; Koh, Gar Yee; Jeansonne, Duane P; Hollingsworth, Javoris; Russo, Paul S; Vicente, Graca; Stout, Rhett W; Liu, Zhijun

    2011-07-01

    Curcumin (CUR) is an active food compound, but its insolubility and instability in water contributes to low bioavailability. In this study, the solubility of CUR was enhanced by utilizing the solubilizing properties of rubusoside (RUB). The solubility of CUR in water increased linearly from 61 μg/mL to 2.318 mg/mL in the presence of RUB ranging from 1% to 10% (w/v). Dynamic light scattering and transmission electron microscopy studies found that CUR and RUB formed CUR-RUB nanoparticle (∼8 nm) complexes. The RUB-solubilized CUR was stable in physiological conditions and did not precipitate when diluted or degrade when spray-dried to a completely reconstitutable powder. Furthermore, cell viability assays demonstrated the efficacy of RUB-solubilized CUR against human colon, breast, and pancreatic cancer cell lines. The development of this new solubilized, stable, and biologically active CUR formulation lays the foundation for future bioavailability improvement.

  11. Echinomycin, a potential binder of FKBP12, shows minor effect on calcineurin activity.

    PubMed

    Singh, Vikramjeet; Nand, Amita; Chen, Caixia; Li, ZhiPeng; Li, Sheng-Jie; Wang, Songbai; Yang, Mo; Merino, Alejandro; Zhang, Lixin; Zhu, Jingsong

    2014-10-01

    Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor-1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was -10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.

  12. A sensitive and facile assay for the measurement of activated protein C activity levels in vivo.

    PubMed

    Orthner, C L; Kolen, B; Drohan, W N

    1993-05-01

    Activated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va: APC is inhibited by several members of the serpin family as well a by alpha 2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 microliters of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate. The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. 26 CFR 1.513-3 - Qualified convention and trade show activity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... segment thereof) of the members of the qualifying organization; and (iii) The show is designed to achieve...— (i) Activities designed to attract to the show members of the sponsoring organization, members of an... interest in, and demand for such products or services; (ii) Activities designed to educate persons in...

  14. 26 CFR 1.513-3 - Qualified convention and trade show activity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... segment thereof) of the members of the qualifying organization; and (iii) The show is designed to achieve...— (i) Activities designed to attract to the show members of the sponsoring organization, members of an... interest in, and demand for such products or services; (ii) Activities designed to educate persons in...

  15. 26 CFR 1.513-3 - Qualified convention and trade show activity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... segment thereof) of the members of the qualifying organization; and (iii) The show is designed to achieve...— (i) Activities designed to attract to the show members of the sponsoring organization, members of an... interest in, and demand for such products or services; (ii) Activities designed to educate persons in...

  16. 26 CFR 1.513-3 - Qualified convention and trade show activity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... segment thereof) of the members of the qualifying organization; and (iii) The show is designed to achieve...— (i) Activities designed to attract to the show members of the sponsoring organization, members of an... interest in, and demand for such products or services; (ii) Activities designed to educate persons in...

  17. 26 CFR 1.513-3 - Qualified convention and trade show activity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... segment thereof) of the members of the qualifying organization; and (iii) The show is designed to achieve...— (i) Activities designed to attract to the show members of the sponsoring organization, members of an... interest in, and demand for such products or services; (ii) Activities designed to educate persons in...

  18. Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity

    PubMed Central

    2014-01-01

    Background The arthropod-borne Mayaro virus (MAYV) causes ‘Mayaro fever’, a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Methods Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. Results The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. Conclusions B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus. PMID:24678592

  19. Assessing Kinase Activity in Plants with In-Gel Kinase Assays.

    PubMed

    Wang, Pengcheng; Zhu, Jian-Kang

    2016-01-01

    The in-gel protein kinase assay is a powerful method to measure the protein phosphorylation activity of specific protein kinases. Any protein substrate can be embedded in polyacrylamide gels where they can be phosphorylated by protein kinases that are separated in the gel under denaturing conditions and then renatured. The kinase activity can be visualized in situ in the gels by autoradiography. This method has been used to compare the activities of protein kinases in parallel samples or to identify their potential substrates. Here, we describe in detail an in-gel kinase assay to measure the activity of some protein kinases in plants.

  20. Assays of physical stability and antioxidant activity of a topical formulation added with different plant extracts.

    PubMed

    Di Mambro, Valéria M; Fonseca, Maria J V

    2005-02-23

    In the present investigation the changes on physical stability (pH, viscosity, flow index and tixotropy) of topical formulations were evaluated following inclusion of different plant extracts containing flavonoids. Also, the antioxidant effect of these plant extracts alone and after addition in the formulation was evaluated using chemiluminescence and the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH(.-)) assays, as well as the inhibition of lipid peroxidation. Formulation added with dl-alpha-tocopherol was used to compare the physical stability and antioxidant activity. Formulations with plant extracts showed pseudoplastic behavior with decreasing on viscosity and tixotropy. The Glycyrrhiza glabra (GG) and Ginkgo biloba (GB) extracts alone and the formulations containing these extracts showed great antioxidant and free radical scavenging activities while the other extracts studied (mixture of Glycyrrhiza glabra, Symphytum officinale L and Arctium majus root, Nelumbium speciosum and soybean) showed lower activity. The results suggest that GG and GB extracts may be used in topical formulations in order to protect skin against damage caused by free radical and reactive oxygen species. PMID:15708669

  1. Antimutagenic and mutagenic activities of some terpenes in the bacterial reverse mutation assay.

    PubMed

    Di Sotto, Antonella; Evandri, Maria Grazia; Mazzanti, Gabriela

    2008-05-31

    The mutagenic and antimutagenic effects of linalool, linalyl acetate and beta-caryophyllene were evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA 98 and TA 100, and on Escherichia coli WP2uvrA strains. Neither linalool nor beta-caryophyllene showed mutagenicity, but linalyl acetate induced a statistically significant increase in the number of revertant colonies in WP2uvrA, both with and without S9 mixture. Linalool was devoid of antimutagenic activity against 2-nitrofluorene (2NF), sodium azide (SA), methyl methane sulfonate (MMS) and 2-aminoanthracene (2AA). In contrast, beta-caryophyllene showed a strong antimutagenic activity against 2NF: at the maximum concentration tested (6.40mg/plate) the number of 2NF-induced revertant colonies was reduced by 83.9%. beta-Caryophyllene also showed to counteract the mutagenicity of SA (in TA 100), MMS and 2AA (in WP2uvrA): the effect was weak against SA (inhibition lower than 25%) and moderate against MMS and 2AA (up to 30.5%). The antimutagenic activity of beta-caryophyllene observed here suggests further studies to evaluate its possible chemopreventive properties. PMID:18514567

  2. Antimutagenic activity of extracts of natural substances in the Salmonella/microsome assay.

    PubMed

    Horn, Rubem Cesar; Vargas, Vera Maria Ferrão

    2003-03-01

    Scientific information regarding plants used in folk medicine in the form of teas and their effect on human health or on genetic material has been the subject of many different types of investigation. The antimutagenic activity of two plants Maytenus ilicifolia and Peltastes peltatus, both rich in compounds of the flavonoid and tannin groups and frequently employed in folk medicine, was studied. Antimutagenicity was determined against known mutagenic substances (4-oxide-1-nitroquinoline, sodium azide, 2-nitrofluorene, aflatoxin B(1), 2-aminofluorene and 2-aminoanthracene), using the Salmonella/microsome assay. Infusions of P.peltatus showed high cytotoxicity and a co-mutagenic effect for induction of base pair substitution mutations with 4-oxide-1-nitroquinoline (-S9 mix). Infusions of M.ilicifolia produced similar effects for frameshift and base pair substitution mutations. With the mutagens 2-nitrofluorene (TA98) and sodium azide (TA100) no significant enhancement effects (co-mutagenic effects) were observed and inhibition of mutagenic activity and cytotoxicity were also diminished. In assays evaluating antimutagenic activity in the presence of metabolic activation utilizing S9 mix, high and significant inhibition of aflatoxin B(1)-, 2-aminofluorene- and 2-aminoanthracene-induced mutagenicity was observed in the presence of the infusions using both TA98 and TA100 and employing doses ranging from 25 to 500 mg/plate. Seventy-five percent of the doses tested exhibited a significant or suggestive decrease in induced mutagenicity with the infusion of M.ilicifolia. With the infusion of P.peltatus significant or suggestive antimutagenic responses were observed with 50% of the doses evaluated. Complexity was clearly noted in the responses observed in the interaction of aqueous extracts of M.ilicifolia and P.peltastes with the genetic material and metabolites generated by the S9 mix played an important role in the protection of DNA. PMID:12621065

  3. 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.

  4. 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

  5. Inhibition of Cathepsin Activity in a Cell-Based Assay by a Light-Activated Ruthenium Compound

    PubMed Central

    Respondek, Tomasz; Sharma, Rajgopal; Herroon, Mackenzie K.; Garner, Robert N.; Knoll, Jessica D.; Cueny, Eric; Turro, Claudia; Podgorski, Izabela; Kodanko, Jeremy J.

    2014-01-01

    Light-activated inhibition of cathepsin activity was demonstrated with in a cell-based assay. Inhibitors of cathepsin K, Cbz-Leu-NHCH2CN (2) and Cbz-Leu-Ser(OBn)-CN (3), were caged within the complexes cis-[Ru(bpy)2(2)2]Cl2 (4) and cis-[Ru(bpy)2(3)2](BF4)2 (5), where bpy = 2,2′-bipyridine, as 1:1 mixtures of Δ- and Λ stereoisomers. Complexes 4 and 5 were characterized by 1H NMR, IR and UV-vis spectroscopies and electrospray mass spectrometry. Photochemical experiments confirm that 4 releases two molecules of 2 upon exposure to visible light for 15 min, whereas release of 3 by 5 requires longer irradiation times. IC50 determinations against purified cathepsin K under light and dark conditions with 4 and 5 confirm that inhibition is enhanced from 35 to 88-fold, respectively, upon irradiation with visible light. No apparent toxicity was observed for 4 in the absence or presence of irradiation in bone marrow macrophage (BMM) or PC-3 cells, as judged by the MTT assay, at concentrations up to 10 μM. Compound 5 is well tolerated at lower concentrations (<1 μM) but does show growth inhibitory effects at higher concentrations. Confocal microscopy experiments show that 4 reduces intracellular cathepsin activity in osteoclasts with light activation. These results support further development of caged nitrile-based inhibitors as chemical tools for investigating spatial aspects of proteolysis within living systems. PMID:24729544

  6. AHTN and HHCB show weak estrogenic--but no uterotrophic activity.

    PubMed

    Seinen, W; Lemmen, J G; Pieters, R H; Verbruggen, E M; van der Burg, B

    1999-12-20

    The ubiquitous presence of the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-gamma-2-b enzopyreen) in surface waters and their identification in human milk fat together with their polycyclic nature, which makes them potential candidates for interference with estrogen receptors, prompted us to assess these compounds for their potential estrogenic effects. We therefore investigated the effects of AHTN and HHCB in ERalpha- and ERbeta-dependent gene transcription assays with Human Embryonal Kidney 293 (HEK293) cells, which have proven to be very suitable to estimate the estrogenic activity of compounds with low binding activity (Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252-4264). Both AHTN and HHCB were found to induce a slight but dose-dependent stimulation of transcriptional activity in the transiently ERalpha transfected HEK293 cells. This weak estrogenic response was not observed in the ERbeta transiently transfected cells. However, these cells were less responsive to estradiol than the ERalpha transfected HEK293 cells. Also, no significant increase in transcriptional activity was observed in HEK293 cell lines, permanently expressing the same estrogen-responsive reporter gene construct and either ERalpha or ERbeta. In the classical uterine weight assay performed in juvenile Balb/c mice, no uterotrophic activity of AHTN and HHCB was noted at relatively high dietary exposure levels up to 50 and 300 ppm, respectively, at which levels an increase in liver weight was evident. Also the vitellogenin production by carp hepatocytes, a sensitive marker of estrogenic activity, was not affected by these two fragrance materials (Smeets, J.M.W., Rouhani Rankouhi, T., Nichols, K.M., Komen, H., Kaminsky, N

  7. New immunocapture enzyme (ICE) assay for quantification of cancer procoagulant activity: studies of inhibitors.

    PubMed

    Mielicki, W P; Tagawa, M; Gordon, S G

    1994-04-01

    A new, sensitive and specific immunocapture enzyme (ICE) assay for quantitation of the enzymatic activity of cancer procoagulant (CP) has been developed. The assay had good reproducibility (inter- and intra-assay CV were 6.4% and 5.7% respectively) and was linear for concentrations of CP from 0.5 microgram/ml to 10 micrograms/ml (r2 = 0.995). Using this assay the inhibition of CP by iodoacetamide, mercuric chloride, E-64, leupeptin and antipain was demonstrated. There was no significant effect of cystatin and natural plasma proteinase inhibitors alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and antithrombin-III/heparin, on the activity of the CP.

  8. 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

  9. 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.

  10. 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

  11. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    PubMed

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also

  12. Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

    PubMed

    Jacqz-Aigrain, E; Bessa, E; Medard, Y; Mircheva, Y; Vilmer, E

    1994-07-01

    1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively).

  13. Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

    PubMed Central

    Jacqz-Aigrain, E; Bessa, E; Medard, Y; Mircheva, Y; Vilmer, E

    1994-01-01

    1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively). PMID:7946931

  14. Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

    PubMed

    Loper, J C; Lang, D R; Schoeny, R S; Richmond, B B; Gallagher, P M; Smith, C C

    1978-01-01

    Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.

  15. 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

    ca. 20 mmol H2 cm-3 d-1 close to the sediment-water interface to 0.5 mmol H2 cm-3 d-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-3 d-1 down to a depth of 1.60 m. At all sites the SRR profile followed the H2ase activity profile until SRR declined to values close to the minimum detection limit (~10 pmol cm-3 d-1). H2ase activity increased again after SRR declined, indicating that other microbial processes are becoming quantitatively more important. The H2ase and SRR data show that our assay has a potential to become a valuable tool to measure total subsurface microbial activity.

  16. 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

  17. 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…

  18. 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

  19. 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.

  20. Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method.

    PubMed

    Kondakçı, Esin; Özyürek, Mustafa; Güçlü, Kubilay; Apak, Reşat

    2013-10-15

    In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid > epicatechin > quercetin ≈ catechin > α-tocopherol > rosmarinic acid > trolox > caffeic acid > ascorbic acid.

  1. Determination of Pulmozyme (dornase alpha) stability using a kinetic colorimetric DNase I activity assay.

    PubMed

    Lichtinghagen, Ralf

    2006-07-01

    An enzymatic activity assay was developed for the determination of dornase alpha human recombinant desoxyribonuclease (DNase I) stability. The method was adapted from a colorimetric endpoint enzyme activity assay for DNase I based on the degradation of a DNA/methyl green complex. With the described modifications the kinetic measurement of enzyme activity is feasible on an automated analyzer system within a rather short time. The development of this assay was based on the need for reliable detection of a possible loss of enzyme activity after transferring the commercial therapeutic agent into sealed glass vials required for a placebo-controlled study. The measuring range of this stability test was from 0 to 3000 U/L corresponding to 0-120% of the original enzyme activity; CV values of control solutions inside the measuring range were between 3% and 5%. The enzyme activity decreased less than 15% during the observation period of 180 days. In conclusion the current kinetic assay is a reliable method for a simple time-saving determination of DNase I activity to test Pulmozyme stability as required for quality control. As dornase alpha is used for inhalation, this method also proved its reliability in testing DNase stability during aerosolization with new inhalation devices (e-flow). PMID:16682175

  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. 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.

  5. An improved 96-well turbidity assay for T4 lysozyme activity.

    PubMed

    Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

  6. Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus.

    PubMed

    Padilla, Marina Aiello; Rodrigues, Rodney Alexandre Ferreira; Bastos, Juliana Cristina Santiago; Martini, Matheus Cavalheiro; Barnabé, Ana Caroline de Souza; Kohn, Luciana Konecny; Uetanabaro, Ana Paula Trovatti; Bomfim, Getúlio Freitas; Afonso, Rafael Sanches; Fantinatti-Garboggini, Fabiana; Arns, Clarice Weis

    2015-01-01

    Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV). Two bacterial strains were identified as active, with percentages of inhibition (IP) equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s) responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s) that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection.

  7. Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus

    PubMed Central

    Padilla, Marina Aiello; Rodrigues, Rodney Alexandre Ferreira; Bastos, Juliana Cristina Santiago; Martini, Matheus Cavalheiro; Barnabé, Ana Caroline de Souza; Kohn, Luciana Konecny; Uetanabaro, Ana Paula Trovatti; Bomfim, Getúlio Freitas; Afonso, Rafael Sanches; Fantinatti-Garboggini, Fabiana; Arns, Clarice Weis

    2015-01-01

    Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV). Two bacterial strains were identified as active, with percentages of inhibition (IP) equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s) responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s) that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection. PMID:26579205

  8. Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus.

    PubMed

    Padilla, Marina Aiello; Rodrigues, Rodney Alexandre Ferreira; Bastos, Juliana Cristina Santiago; Martini, Matheus Cavalheiro; Barnabé, Ana Caroline de Souza; Kohn, Luciana Konecny; Uetanabaro, Ana Paula Trovatti; Bomfim, Getúlio Freitas; Afonso, Rafael Sanches; Fantinatti-Garboggini, Fabiana; Arns, Clarice Weis

    2015-01-01

    Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV). Two bacterial strains were identified as active, with percentages of inhibition (IP) equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s) responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s) that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection. PMID:26579205

  9. Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes.

    PubMed

    Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

    2014-04-01

    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

  10. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    PubMed Central

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  11. A yeast-based assay identifies drugs active against human mitochondrial disorders.

    PubMed

    Couplan, Elodie; Aiyar, Raeka S; Kucharczyk, Roza; Kabala, Anna; Ezkurdia, Nahia; Gagneur, Julien; St Onge, Robert P; Salin, Bénédicte; Soubigou, Flavie; Le Cann, Marie; Steinmetz, Lars M; di Rago, Jean-Paul; Blondel, Marc

    2011-07-19

    Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.

  12. A yeast-based assay identifies drugs active against human mitochondrial disorders

    PubMed Central

    Couplan, Elodie; Aiyar, Raeka S.; Kucharczyk, Roza; Kabala, Anna; Ezkurdia, Nahia; Gagneur, Julien; St. Onge, Robert P.; Salin, Bénédicte; Soubigou, Flavie; Le Cann, Marie; Steinmetz, Lars M.; di Rago, Jean-Paul; Blondel, Marc

    2011-01-01

    Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases. PMID:21715656

  13. 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

  14. 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

  15. FRET-based protein-DNA binding assay for detection of active NF-kappa B

    SciTech Connect

    Giannetti, Ambra; Baldini, Francesco; Wabuyele, Musundi B; Vo Dinh, Tuan

    2006-01-01

    A novel method to detect the active form of NF-{kappa}B, a transcription factor regulating a battery of inflammatory genes and playing a fundamental role in the development of numerous pathological states, has been developed. In the present work, we used fluorescence resonance energy transfer (FRET) to study DNA-protein binding interaction taking place between double-strand (ds) DNA immobilized in a glass capillary wall and p50 proteins. For this purpose, we developed a regenerable FRET-based system comprising of a single-strand (ss) DNA with auto-complementary sequence that is end-labeled with Cy5 dye and is highly specific for p50 proteins. The proteins were labeled with a Black Hole Quencher (BHQ-3) to be used as FRET pair. The interaction of p50/p50 homodimer active form with its DNA binding site was demonstrated by both electrophoretic mobility shift assays and FRET studies. These preliminary results demonstrated the feasibility of the FRET-based DNA technique to detect the active form of NF-{kappa}B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.

  16. Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity.

    PubMed

    Rizzo, J; Gifford, L K; Zhang, X; Gewirtz, A M; Lu, P

    2002-08-01

    Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming. Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids. An RNA-DNA chimeric beacon assay for RNase H enzymatic activity was developed. The substrate is a single-stranded RNA-DNA chimeric oligonucleotide labeled with a 5'-fluorescein and a 3'-DABCYL. The fluorophore (fluorescein) of the probe is held in close proximity to the quencher (DABCYL) by the RNA:DNA stem-loop structure. When the RNA sequence of the RNA:DNA hybrid stem is cleaved, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Chimeric beacons with different stem lengths and sequences have been surveyed for this assay with E. coli RNase H. We found that the beacon kinetic parameters are in qualitative agreement with previously reported values using more cumbersome assays. This method permits real-time detection of RNase H activity and a convenient approach to RNase H kinetic and mechanistic study.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

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

    PubMed Central

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

    2008-01-01

    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 β-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-β-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced γ-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. PMID:18319341

  3. [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.

  4. 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

  5. Image-based ELISA on an activated polypropylene microtest plate--a spectrophotometer-free low cost assay technique.

    PubMed

    Parween, Shahila; Nahar, Pradip

    2013-10-15

    In this communication, we report ELISA technique on an activated polypropylene microtest plate (APPµTP) as an illustrative example of a low cost diagnostic assay. Activated test zone in APPµTP binds a capture biomolecule through covalent linkage thereby, eliminating non-specific binding often prevalent in absorption based techniques. Efficacy of APPµTP is demonstrated by detecting human immunoglobulin G (IgG), human immunoglobulin E (IgE) and Aspergillus fumigatus antibody in patient's sera. Detection is done by taking the image of the assay solution by a desktop scanner and analyzing the color of the image. Human IgE quantification by color saturation in the image-based assay shows excellent correlation with absorbance-based assay (Pearson correlation coefficient, r=0.992). Significance of the relationship is seen from its p value which is 4.087e-11. Performance of APPµTP is also checked with respect to microtiter plate and paper-based ELISA. APPµTP can quantify an analyte as precisely as in microtiter plate with insignificant non-specific binding, a necessary prerequisite for ELISA assay. In contrast, paper-ELISA shows high non-specific binding in control sera (false positive). Finally, we have carried out ELISA steps on APPµTP by ultrasound waves on a sonicator bath and the results show that even in 8 min, it can convincingly differentiate a test sample from a control sample. In short, spectrophotometer-free image-based miniaturized ELISA on APPµTP is precise, reliable, rapid, and sensitive and could be a good substitute for conventional immunoassay procedures widely used in clinical and research laboratories.

  6. An evaluation of a genotoxicity assay with liver s9 for activation and luminescent bacteria for detection

    USGS Publications Warehouse

    Johnson, B. Thomas

    1992-01-01

    A new short-term in vitro genotoxicity assay with marine bioluminescent bacteria was evaluated for sensitivity and cost. Known under the trade name of Mutatox™, this assay is a simple and rapid screening tool that detects DNA-damaging substances (genotoxins) by measuring light output from an isolated dark mutant strain of the luminescent bacterium Photobacterium phosphoreum. A positive response indicates the ability of the test chemical to restore the luminescent state in the dark mutant strain; the degree of light increase indicates the relative genotoxicity of the sample. In this study, the Mutatox assay with rat hepatic fractions (S9) as an exogenous metabolic activation system detected genotoxic activity with known progenotoxins: 2-acetamidofluorene, aflatoxin B1, 2-aminoanthracene, 2-aminofluorene, 2-aminonaphthalene, benzo[a]pyrene, 3-methyl-cholanthrene, and pyrene. Each chemical clearly demonstrated a dose response between 5.0 and 0.6 μg per tube. Known nongenotoxic controls carbofuran, di-2-ethylhexyl phthalate, malathion, simazine, and permethrin showed no genotoxic responses. The optimum assay conditions were determined to be rat S9 concentration of 0.4 mg/ml, preincubation at 37°C for 30 min, and 18 h incubation at 23°C. Genotoxicity data were obtained in <24 h. The Mutatox assay compared favorably in sensitivity with the Ames test; it was easier and more rapid to perform and, as a result, cost less. The sensitivity, specificity, and predictive value suggested that the Mutatox assay could be a valuable screening tool to monitor complex environmental samples for genotoxins.

  7. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    SciTech Connect

    Bradbury, Andrew M; Velappan, Nileena; Schmidt, Jurgen G

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  8. A piperidine amide extracted from Piper longum L. fruit shows activity against Aedes aegypti mosquito larvae.

    PubMed

    Yang, Young-Cheol; Lee, Sang-Guei; Lee, Hee-Kwon; Kim, Moo-Key; Lee, Sang-Hyun; Lee, Hoi-Seon

    2002-06-19

    Mosquito larvicidal activity of Piper longum fruit-derived materials against the fourth-instar larvae of Aedes aegypti was examined. A crude methanol extract of P. longum fruits was found to be active against the larvae, and the hexane fraction of the methanol extract showed a strong larvicidal activity of 100% mortality. The biologically active component of P. longum fruits was characterized as pipernonaline by spectroscopic analyses. The LC(50) value of pipernonaline was 0.25 mg/L. The toxicity of pipernonaline is comparable to that of pirimiphos-methyl as a mosquito larvicide. In tests with available components derived from P. longum, no activity was observed with piperettine, piperine, or piperlongumine.

  9. A chemiluminescent microtiter plate assay for sensitive detection of protein kinase activity.

    PubMed

    Lehel, C; Daniel-Issakani, S; Brasseur, M; Strulovici, B

    1997-01-15

    A chemiluminescent protein kinase assay using biotinylated substrate peptides captured on a streptavidin-coated microtiter plate and monoclonal antibodies to detect their phosphorylation is described. Assay conditions were optimized and validated for sensitive measurement of protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II (CAM-KII), receptor interacting protein, and src activities. The newly developed chemiluminescent assay has several advantages over currently used radioactive or colorimetric methods. It is highly sensitive at low enzyme and substrate concentrations and high, close to physiological ATP levels. It is fast, simple to perform and amenable to automation and high-throughput drug screening. The assay is also robust, exhibiting minimum interference from solvents and test substances from various sources. Overall, among the presently available methods for the detection of protein kinase activity, chemiluminescence was found to provide the highest sensitivity under conditions most closely mimicking the intracellular environment. This assay is expected to be useful in both academic and industrial laboratories, especially in identifying novel classes of protein kinase inhibitors.

  10. A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells.

    PubMed

    Rinne, Andreas; Blatter, Lothar A

    2010-09-01

    Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

  11. 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

  12. 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...

  13. A chip-based assay for botulinum neurotoxin A activity in pharmaceutical preparations.

    PubMed

    Lévêque, Christian; Ferracci, Géraldine; Maulet, Yves; Grand-Masson, Chloé; Seagar, Michael; El Far, Oussama

    2015-05-01

    The production of botulinum neurotoxin A (BoNT/A) for therapeutic and cosmetic applications requires precise determination of batch potency, and the enzymatic activity of BoNT/A light chain is a crucial index that can be measured in vitro. We previously established a SNAP-25 chip-based assay using surface plasmon resonance (SPR) that is more sensitive than the standard mouse bioassay for the quantification of BoNT/A activity. We have now adapted this procedure for pharmaceutical preparations. The optimized SPR assay allowed multiple measurements on a single chip, including the kinetics of substrate cleavage. The activity of five different batches of a pharmaceutical BoNT/A preparation was determined in a blind study by SPR and found to be in agreement with data from the in vivo mouse lethality assay. Biosensor detection of specific proteolytic products has the potential to accurately monitor the activity of pharmaceutical BoNT/A preparations, and a single chip can be used to assay more than 100 samples.

  14. A specific mechanism for nonspecific activation in reporter-gene assays.

    PubMed

    Auld, Douglas S; Thorne, Natasha; Nguyen, Dac-Trung; Inglese, James

    2008-08-15

    The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.

  15. 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

  16. Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge.

    PubMed

    Sibag, Mark; Kim, Seung Hwan; Kim, Choah; Kim, Hee Jun; Cho, Jinwoo

    2015-06-01

    ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209). We observed a high degree of interference (90%) in the presence of 100 mg/L silica nanoparticles and a low level of ATP being measured (0.01 μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used. PMID:25892589

  17. Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge.

    PubMed

    Sibag, Mark; Kim, Seung Hwan; Kim, Choah; Kim, Hee Jun; Cho, Jinwoo

    2015-06-01

    ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209). We observed a high degree of interference (90%) in the presence of 100 mg/L silica nanoparticles and a low level of ATP being measured (0.01 μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used.

  18. 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.

  19. Distributive Education--Fashion Show. Kit No. 88. Instructor's Manual [and] Student Learning Activity Guide.

    ERIC Educational Resources Information Center

    Walters, Brenda B.

    An instructor's manual and student activity guide on fashion shows are provided in this set of prevocational education materials which focuses on the vocational area of distributive education. (This set of materials is one of ninety-two prevocational education sets arranged around a cluster of seven vocational offerings: agriculture, home…

  20. HPLC-MTT assay: anticancer activity of aqueous garlic extract is from allicin.

    PubMed

    Lee, Jenny; Gupta, Shalini; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Bao-Shiang

    2013-05-15

    A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

  1. 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

  2. Estimation of specific activity of 177Lu by 'saturation assay' principle using DOTA as ligand.

    PubMed

    Pillai, Ambikalmajan M R; Chakraborty, Sudipta; Das, Tapas

    2015-01-01

    Lutetium-177 is a widely used therapeutic radionuclide in targeted therapy and it is important to know its specific activity at the time of radiopharmaceutical preparation, especially for radiolabeling peptides. However, there are no direct methods for the experimental determination of the specific activity which can be readily applied in a hospital radiopharmacy. A new technique based on the 'saturation assay' principle using DOTA as the binding agent for the estimation of specific activity of (177)Lu is reported. The studies demonstrate the proof of principle of this new assay technique. The method is general and can be modified and applied for the estimation of specific activity of other metallic radionuclides by using DOTA or other suitable chelating agents.

  3. Capability and limitation study of the DDT passive-active neutron waste assay instrument

    SciTech Connect

    Nicholas, N.J.; Coop, K.L.; Estep, R.J.

    1992-05-01

    The differential-dieaway-technique passive-active neutron assay system is widely used by transuranic waste generators to certify their drummed waste for eventual shipment to the Waste Isolation Pilot Plant (WIPP). Stricter criteria being established for waste emplacement at the WIPP site has led to a renewed interest in improvements to and a better understanding of current nondestructive assay (NDA) techniques. Our study includes the effects of source position, extreme matrices, high neutron backgrounds, and source self-shielding to explore the system`s capabilities and limitations and to establish a basis for comparison with other NDA systems. 11 refs.

  4. Capability and limitation study of the DDT passive-active neutron waste assay instrument

    SciTech Connect

    Nicholas, N.J.; Coop, K.L.; Estep, R.J.

    1992-05-01

    The differential-dieaway-technique passive-active neutron assay system is widely used by transuranic waste generators to certify their drummed waste for eventual shipment to the Waste Isolation Pilot Plant (WIPP). Stricter criteria being established for waste emplacement at the WIPP site has led to a renewed interest in improvements to and a better understanding of current nondestructive assay (NDA) techniques. Our study includes the effects of source position, extreme matrices, high neutron backgrounds, and source self-shielding to explore the system's capabilities and limitations and to establish a basis for comparison with other NDA systems. 11 refs.

  5. 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

  6. 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

  7. Isolation of Lactic Acid Bacteria Showing Antioxidative and Probiotic Activities from Kimchi and Infant Feces.

    PubMed

    Ji, Keunho; Jang, Na Young; Kim, Young Tae

    2015-09-01

    The purpose of this study was to investigate lactic acid bacteria with antioxidative and probiotic activities isolated from Korean healthy infant feces and kimchi. Isolates A1, A2, S1, S2, and S3 were assigned to Lactobacillus sp. and isolates A3, A4, E1, E2, E3, and E4 were assigned to Leuconostoc sp. on the basis of their physiological properties and 16S ribosomal DNA sequence analysis. Most strains were confirmed as safe bioresources through nonhemolytic activities and non-production of harmful enzymes such as β-glucosidase, β- glucuronidase and tryptophanase. The 11 isolates showed different resistance to acid and bile acids. In addition, they exhibited antibacterial activity against foodborne bacteria, especially Bacillus cereus, Listeria monocytogenes, and Escherichia coli. Furthermore, all strains showed significantly high levels of hydrophobicity. The antioxidant effects of culture filtrates of the 11 strains included 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging capacity, 2.2'- azino-bis (2-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity, and superoxide dismutase activity. The results revealed that most of the culture filtrates have effective scavenging activity for DPPH and ABTS radicals. All strains appeared to have effective superoxide dismutase activity. In conclusion, the isolated strains A1, A3, S1, and S3 have significant probiotic activities applicable to the development of functional foods and health-related products. These strains might also contribute to preventing and controlling several diseases associated with oxidative stress, when used as probiotics.

  8. Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay.

    PubMed

    Gomes-Carneiro, M R; Dias, Daniela M M; De-Oliveira, A C A X; Paumgartten, Francisco J R

    2005-08-01

    alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens. PMID:15936245

  9. Development and validation of an assay for urinary tissue factor activity.

    PubMed Central

    Lwaleed, B A; Chisholm, M; Francis, J L

    1999-01-01

    BACKGROUND: Activation of blood coagulation is a common complication of cancer and inflammation in both humans and experimental animals. Increased production of tissue factor--the principal initiator of the coagulation process--by endothelial cells, monocytes, and macrophages has been implicated in these conditions. AIM: To investigate whether urinary tissue factor (uTF) might reflect the state of monocyte/macrophage activation and be a useful diagnostic test. METHODS: Urine was centrifuged at 51,000 g to sediment tissue factor containing membrane vesicles. The tissue factor was then solubilised in beta-octyl-glucopyranoside and assayed in a specific chromogenic assay adapted for use in microtitre plates. RESULTS: The assay proved to be sensitive, specific, and reproducible. The normal range of uTF was relatively narrow and unaffected by age, sex, or cigarette smoking. Levels were not significantly influenced by storage of urine samples before assay or by the presence of fresh blood in the urine sample. CONCLUSIONS: This method may have diagnostic application in the study of haemostatic activation in patients with cancer and other disease states. Images PMID:10450183

  10. Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions.

    PubMed

    Mani, Helen; Hesse, Christian; Stratmann, Gertrud; Lindhoff-Last, Edelgard

    2013-01-01

    Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

  11. An optical assay of the transport activity of ClC-7

    PubMed Central

    Zanardi, Ilaria; Zifarelli, Giovanni; Pusch, Michael

    2013-01-01

    Osteoporosis, characterized by excessive osteoclast mediated bone resorption, affects millions of people worldwide representing a major public health problem. ClC-7 is a chloride-proton exchanger localized in lysosomes and in the resorption lacuna in osteoclasts where it is essential for bone resorption. Thus, drugs targeted at ClC-7 have been proposed for ameliorating osteoporosis. However, functional assays suited for high throughput screening (HTS) of ClC-7 function are lacking. Here we describe two complementary variants of purely optical assays of the transport activity of ClC-7, redirected to the plasma membrane employing a genetically encoded fluorescent Cl−/pH indicator fused to the ClC-7 protein. These simple and robust functional assays of ClC-7 transport are well-suited to be applied in HTS of small-molecule inhibitors and may help to develop drugs suited for the treatment of osteoporosis. PMID:23390581

  12. In vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening

    PubMed Central

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Lysine acetylation is a post-translational modification that is carried out by acetyltransferases. The MYST proteins form the largest and most diverse family of acetyltransferases, which regulate gene expression, DNA repair, and cell cycle homeostasis, among other activities, by acetylating both histone and non-histone proteins. This chapter will describe methods for the preparation and biochemical characterization of MYST family acetyltransferases, including protocols for the preparation of recombinant protein, enzyme assays for measuring steady state parameters and binding assays to measure cofactor and inhibitor binding. We also provide details on adapting these assays for high throughput screening for small molecule MYST inhibitors. This chapter seeks to prepare researchers for some hurdles that they may encounter when studying the MYST proteins so that there may be better opportunity to plan appropriate controls and obtain high quality data. PMID:27372752

  13. 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

  14. Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity.

    PubMed

    Hsu, Chia-Wen Amy; Zhao, Jinghua; Xia, Menghang

    2016-01-01

    The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations. PMID:27518622

  15. 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

  16. Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis.

    PubMed

    Pascual, P; Martinez-Lara, E; Bárcena, J A; López-Barea, J; Toribio, F

    1992-10-01

    A fast, sensitive and direct method has been developed for the determination of glutathione peroxidase activity (both selenium- and non-selenium-dependent) in cell-free preparations. The assay is based on the separation and quantitation of reduced and oxidized glutathione by capillary electrophoresis. The electrophoretic separation buffer was 100 mM sodium tetraborate (pH 8.2) containing 100 mM sodium dodecylsulphate. A micellar electrokinetic mechanism took place under these conditions, and a total mass recovery was observed for both peptides. The reproducibility of migration times was excellent (less than 3% variability). A linear detector response range was observed in the range 5-50 U/ml, and both the reproducibility and accuracy were satisfied. Samples out of this linear range could be analysed by either increasing the reaction time or diluting the enzyme preparation. The results obtained with the new direct capillary electrophoresis assay were compared with those derived from a reversed phase high-performance liquid chromatographic and spectrophotometric coupled assay. A very good agreement was found between the two direct assay methods in all samples. Capillary electrophoresis is a versatile technique that allows the automation of the glutathione peroxidase assay in a reproducible manner and within a relatively short time with sufficient accuracy and precision. PMID:1430007

  17. Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity.

    PubMed

    Hsu, Chia-Wen Amy; Zhao, Jinghua; Xia, Menghang

    2016-01-01

    The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations.

  18. 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. PMID:27610220

  19. 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

  20. 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.

  1. Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity

    PubMed Central

    Dorjsuren, Dorjbal; Kim, Daemyung; Maloney, David J.; Wilson, David M.; Simeonov, Anton

    2011-01-01

    FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC50. The assays were optimized to a low 4 µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC50 of 0.59 and 0.93 µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target. PMID:21062821

  2. 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.

  3. Bipolar I disorder and major depressive disorder show similar brain activation during depression

    PubMed Central

    Cerullo, Michael A; Eliassen, James C; Smith, Christopher T; Fleck, David E; Nelson, Erik B; Strawn, Jeffrey R; Lamy, Martine; DelBello, Melissa P; Adler, Caleb M; Strakowski, Stephen M

    2014-01-01

    Objectives Despite different treatments and course of illness, depressive symptoms appear similar in major depressive disorder (MDD) and bipolar I disorder (BP-I). This similarity of depressive symptoms suggests significant overlap in brain pathways underlying neurovegetative, mood, and cognitive symptoms of depression. These shared brain regions might be expected to exhibit similar activation in individuals with MDD and BP-I during functional magnetic resonance imaging (fMRI). Methods fMRI was used to compare regional brain activation in participants with BP-I (n = 25) and MDD (n = 25) during a depressive episode as well as 25 healthy comparison (HC) participants. During the scans, participants performed an attentional task that incorporated emotional pictures. Results During the viewing of emotional images, subjects with BP-I showed decreased activation in the middle occipital gyrus, lingual gyrus, and middle temporal gyrus compared to both subjects with MDD and HC participants. During attentional processing, participants with MDD had increased activation in the parahippocampus, parietal lobe, and postcentral gyrus. However, among these regions, only the postcentral gyrus also showed differences between MDD and HC participants. Conclusions No differences in cortico-limbic regions were found between participants with BP-I and MDD during depression. Instead, the major differences occurred in primary and secondary visual processing regions with decreased activation in these regions in BP-I compared to major depression. These differences were driven by abnormal decreases in activation seen in the participants with BP-I. Posterior activation changes are a common finding in studies across mood states in participants with BP-I. PMID:24990479

  4. Solid-phase assay of lectin activity using HRP-conjugated glycoproteins.

    PubMed

    Kojima-Aikawa, Kyoko

    2014-01-01

    Various enzyme-conjugated probes have been widely used for detection of specific interactions between biomolecules. In the case of glycan-protein interaction, horseradish peroxidase (HRP)-conjugated glycoproteins (HRP-GPs) are useful for the detection of carbohydrate-binding activity of plant and animal lectins. In this chapter, a typical solid-phase assay of the carbohydrate-binding activity of Sophora japonica agglutinin I, a Gal/GalNAc-specific lectin, using HRP-conjugated asialofetuin is described. HRP-GPs are versatile tools for probing lectin activities in crude extracts, screening many samples at one time, and applicable not only for solid-phase binding assays but also samples which are dot- or Western-blotted onto the membrane. PMID:25117228

  5. Considerations for an active and passive scanner to assay nuclear waste drums

    SciTech Connect

    Martz, H.E.; Azevedo, S.G.; Roberson, G.P.; Schneberk, D.J.; Koenig, Z.M.; Camp, D.C. )

    1990-06-08

    Radioactive wastes are generated at many DOE laboratories, military facilities, fuel fabrication and enrichment plants, reactors, hospitals, and university research facilities. At all of these sites, wastes must be separated, packaged, categorized, and packed into some sort of container--usually 208-L (55-gal) drums--for shipment to waste-storage sites. Prior to shipment, the containers must be labeled, assayed, and certified; the assay value determines the ultimate disposition of the waste containers. An accurate nondestructive assay (NDA) method would identify all the radioisotopes present and provide a quantitative measurement of their activity in the drum. In this way, waste containers could be routed in the most cost-effective manner and without having to reopen them. Currently, the most common gamma-ray method used to assay nuclear waste drums is segmented gamma-ray scanning (SGS) spectrometer that crudely measures only the amount of {sup 235}U or {sup 239}Pu present in the drum. This method uses a spatially-averaged, integrated, emitted gamma-ray-intensity value. The emitted intensity value is corrected by an assumed constant-attenuation value determined by a spatially-averaged, transmission (or active) measurement. Unfortunately, this typically results in an inaccurate determination of the radioactive activities within a waste drum because this measurement technique is valid only for homogeneous-attenuation or known drum matrices. However, since homogeneous-attenuation matrices are not common and may be unknown, other NDA techniques based on active and Passive CT (A PCT) are under development. The active measurement (ACT) yields a better attenuation matrix for the drum, while the passive measurement (PCT) more accurately determines the identity of the radioisotopes present and their activities. 9 refs., 2 figs.

  6. Task Control Signals in Pediatric Tourette Syndrome Show Evidence of Immature and Anomalous Functional Activity

    PubMed Central

    Church, Jessica A.; Wenger, Kristin K.; Dosenbach, Nico U. F.; Miezin, Francis M.; Petersen, Steven E.; Schlaggar, Bradley L.

    2009-01-01

    Tourette Syndrome (TS) is a pediatric movement disorder that may affect control signaling in the brain. Previous work has proposed a dual-networks architecture of control processing involving a task-maintenance network and an adaptive control network (Dosenbach et al., 2008). A prior resting-state functional connectivity MRI (rs-fcMRI) analysis in TS has revealed functional immaturity in both putative control networks, with “anomalous” correlations (i.e., correlations outside the typical developmental range) limited to the adaptive control network (Church et al., 2009). The present study used functional MRI (fMRI) to study brain activity related to adaptive control (by studying start-cues signals), and to task-maintenance (by studying signals sustained across a task set). Two hypotheses from the previous rs-fcMRI results were tested. First, adaptive control (i.e., start-cue) activity will be altered in TS, including activity inconsistent with typical development (“anomalous”). Second, group differences found in task-maintenance (i.e., sustained) activity will be consistent with functional immaturity in TS. We examined regions found through a direct comparison of adolescents with and without TS, as well as regions derived from a previous investigation that showed differences between unaffected children and adults. The TS group showed decreased start-cue signal magnitude in regions where start-cue activity is unchanged over typical development, consistent with anomalous adaptive control. The TS group also had higher magnitude sustained signals in frontal cortex regions that overlapped with regions showing differences over typical development, consistent with immature task-maintenance in TS. The results demonstrate task-related fMRI signal differences anticipated by the atypical functional connectivity found previously in adolescents with TS, strengthening the evidence for functional immaturity and anomalous signaling in control networks in adolescents with TS

  7. Functional characterization of a Plagiochasma appendiculatum flavone synthase I showing flavanone 2-hydroxylase activity.

    PubMed

    Han, Xiao-Juan; Wu, Yi-Feng; Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Lou, Hong-Xiang; Cheng, Ai-Xia

    2014-06-27

    FNS I is a 2-oxoglutarate dependent dioxygenase (2-ODD) found mainly in species of the Apiaceae family. Here, an FNS I cDNA sequence was isolated from the liverwort Plagiochasma appendiculatum (Aytoniaceae) and characterized. The recombinant protein exhibited high FNS I activity catalyzing the conversion of naringenin to apigenin and 2-hydroxynaringenin. The critical residue for flavanone-2-hydroxylation activity was Tyr240, as identified from homology modeling and site-directed mutagenesis. The recombinant protein also showed some flavonol synthase activity, as it can convert dihydrokaempferol to kaempferol. When the Leu311 residue was mutated to Phe, the enzyme's capacity to convert dihydrokaempferol to kaempferol was substantially increased. PaFNS I represents a 2-ODD in which a hydrophobic π-stacking interaction between the key residue and the naringenin A-ring determines 2-hydroxyflavanone formation.

  8. Meropenem-clavulanic acid shows activity against Mycobacterium tuberculosis in vivo.

    PubMed

    England, Kathleen; Boshoff, Helena I M; Arora, Kriti; Weiner, Danielle; Dayao, Emmanuel; Schimel, Daniel; Via, Laura E; Barry, Clifton E

    2012-06-01

    The carbapenems imipenem and meropenem in combination with clavulanic acid reduced the bacterial burden in Mycobacterium tuberculosis-infected macrophages by 2 logs over 6 days. Despite poor stability in solution and a short half-life in rodents, treatment of chronically infected mice revealed significant reductions of bacterial burden in the lungs and spleens. Our results show that meropenem has activity in two in vivo systems, but stability and pharmacokinetics of long-term administration will offer significant challenges to clinical evaluation.

  9. Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay.

    PubMed

    Ganiere-Monteil, C; Pineau, A; Kergueris, M F; Azoulay, C; Bourin, M

    1999-04-30

    A new liquid-liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH4Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5-250 ng ml(-1) (r> or =0.997, slope=1.497, intercept=-0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml(-1), respectively. The coefficients of variation were < or =6.1% for within-day assay (n=6) and < or =9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (7 =70). The results ranged from 7.8 to 27.8 nmol h(-1) ml(-1) packed red blood cells and the frequency distribution histogram is similar to that previously published.

  10. 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.

  11. A novel thrombelastograph tissue factor/kaolin assay of activated clotting times for monitoring heparin anticoagulation during cardiopulmonary bypass.

    PubMed

    Chavez, Jack J; Foley, Donald E; Snider, Carolyn C; Howell, James C; Cohen, Eli; Muenchen, Robert A; Carroll, Roger C

    2004-11-01

    We used a thrombelastograph (TEG) assay with tissue factor and kaolin (TEG TF/K) to measure activated clotting time (ACT) in 31 patients during cardiopulmonary bypass. For comparison, ACTs were also determined by a Hemochron Jr. Signature and a Hepcon HMS. The TEG TF/K correlated with both the Hepcon (r(2) = 0.789) and Hemochron (r(2) = 0.743) ACTs. The average ACT after heparin was 319 +/- 119 s (mean +/- sd) for the TEG TF/K compared with 624 +/- 118 s for the Hepcon instrument. To evaluate the effects of hemodilution on TEG TF/K and Hemochron assays, ACT assays were performed on blood diluted to 50% and titrated with heparin from 0 to 6 U/mL. Both instruments showed significant (P < 0.01) changes in the ACT-versus-heparin slope, but the 0 heparin intercept for the TEG TF/K ACTs was not significantly changed (P = 0.292), in contrast to that for the Hemochron device (P = 0.041). Both instruments also indicated the same 1.3:1 ratio of protamine to heparin for optimum heparin neutralization, with increasing ACTs at ratios >2.6:1. The TEG TF/K ACT assay rapidly monitors heparin anticoagulation, in addition to the capabilities of this instrument to monitor platelet function, clotting factors, and fibrinolysis.

  12. Sapphyrins induce apoptosis in hematopoietic tumor-derived cell lines and show in vivo antitumor activity.

    PubMed

    Naumovski, Louie; Ramos, Jason; Sirisawad, Mint; Chen, Jun; Thiemann, Patti; Lecane, Philip; Magda, Darren; Wang, Zhong; Cortez, Cecilia; Boswell, Garry; Gyu Cho, Dong; Sessler, Jonathan; Miller, Richard

    2005-06-01

    Sapphyrins are pentapyrrolic, metal-free, expanded porphyrins. In the present study, the activity of sapphyrins as anticancer agents in hematopoietic-derived tumor cells was explored. It was found that a dihydroxylated water-soluble sapphyrin derivative (PCI-2000) is a potent inducer of apoptosis in a wide variety of tumor cell lines including lymphoma (Ramos, DHL-4, and HF-1), leukemia (Jurkat and HL-60), and myeloma (8226/S, 1-310, C2E3, and 1-414). PCI-2000 triggers an apoptotic pathway in these tumor cells as shown by release of cytochrome c from mitochondria; activation of caspases 9, 8, and 3; cleavage of the caspase substrate poly(ADP-ribose) polymerase; and Annexin V binding. Apoptosis can be partially inhibited by overexpression of the antiapoptotic protein Bcl-2 or treatment with benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone, a cell-permeable caspase inhibitor. Both PCI-2000 and PCI-2010, a tetrahydroxy bis-carbamate derivative of PCI-2000, result in increased levels of phosphorylated p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase phosphorylation resulted in a synergistic increase of PCI-2000 cytotoxicity. PCI-2010 showed less toxicity in mice than PCI-2000 and was active in slowing the growth of Ramos and HL-60 tumor xenografts in nude mice. These results provide preclinical rationale for the further study of sapphyrins for potential use in the treatment of hematopoietic-derived tumors.

  13. Commercial runs show TiO/sub 2/ Claus catalyst retains activity

    SciTech Connect

    Nougayrede, J.; Philippe, A.; Quesmerel, E.; Vermeersch, R.

    1987-08-10

    Commercial data are given for the CRS 31 Claus catalyst, jointly developed by Rhone Poulenc and Societe Nationale Elf Aquitane (Production) (SNEA). The data confirm laboratory results that show the catalyst's ability to deliver more complete hydrolysis in the first reactor of Claus sulfur recovery plants, and its ability to maintain activity in the second and third reactors. The most important losses in sulfur yields in Claus plants generally come from COS and CS/sub 2/ formed in the thermal steps of the process in the first reactor, and from the low Claus activity of alumina-based catalysts in the second and third reactors. In the first reactor, loaded with alumina-based catalyst, hydrolysis is only partial. Low Claus activity in second and third reactors is caused by the inevitable sulfation of the alumina. Catalytic-activity decrease of aluminas is caused particularly by the presence of oxygen, even in trace amounts, in the Claus gases. Therefore, in the first reactor, the CS/sub 2/ hydrolysis rate is lowered in the subsequent reactors, the H/sub 2/ + SO/sub 2/ conversion significantly drops. The chemical composition of CRS 31, TiO/sub 2/ only, in the same operating conditions, causes a significant improvement in activity because of its increased resistance to sulfation. The economics of the process are given.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens

    PubMed Central

    Yanasarn, Nijaporn; Sloat, Brian R.; Cui, Zhengrong

    2011-01-01

    Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome’s ability (i) to up-regulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. PMID:21615153

  20. [Detection of fungal metabolites showing toxic activity through Artemia salina bioassay].

    PubMed

    González, Ana María; Presa, Maximiliano; Latorre, María Gabriela; Lurá, María Cristina

    2007-03-01

    The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites. PMID:17592895

  1. Triterpenoids of sour jujube show pronounced inhibitory effect on human tumor cells and antioxidant activity.

    PubMed

    Qiao, Aimin; Wang, Yihai; Xiang, Limin; Zhang, Zhenxue; He, Xiangjiu

    2014-10-01

    Sour jujube is a common fruit and traditional medicine in China. Bioactivity-guided fractionation of sour jujube was used to determine the chemical identity of potent antiproliferative and antioxidant constituents. Four novel ursane-type triterpenoids, together with 8 known were isolated and identified. The new triterpenoids were elucidated to be 2α,3β,13β,23-tetrahydroxy-urs-11-en-28-oic acid (3), 2α,3β-dihydroxy-urs-20(30)-en-28-oic acid (9), 2α,3β,28-trihydroxy-urs-20(30)-ene (10), and 3β,12β,13β-trihydroxy-ursan-28-oic acid (11). Among the triterpenoids isolated, 2α,3β,19α-trihydroxy-urs-12-en-28-oic acid (7), 9 and 10 showed high potent inhibitory activity toward the proliferation of HepG2 cells, which the IC50 values were lower than 5 μM. Compounds 9 and 10 also exhibited pronounced activity against MCF-7 cells, with IC50 value of 0.8 ± 0.03 and 1.5 ± 0.1 μM, respectively. Compound 10 showed high antioxidant activity with an EC50 of 0.8 ± 0.02 μM, which was 18.9 times higher than ascorbic acid in antioxidant capacity.

  2. Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples.

    PubMed

    Feldman, B J; Gheller, S F; Bailey, G F; Newton, W E; Schultz, F A

    1990-02-15

    Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.

  3. Variegation of comet 67P/Churyumov-Gerasimenko in regions showing activity

    NASA Astrophysics Data System (ADS)

    Oklay, N.; Vincent, J.-B.; Fornasier, S.; Pajola, M.; Besse, S.; Davidsson, B. J. R.; Lara, L. M.; Mottola, S.; Naletto, G.; Sierks, H.; Barucci, A. M.; Scholten, F.; Preusker, F.; Pommerol, A.; Masoumzadeh, N.; Lazzarin, M.; Barbieri, C.; Lamy, P. L.; Rodrigo, R.; Koschny, D.; Rickman, H.; A'Hearn, M. F.; Bertaux, J.-L.; Bertini, I.; Bodewits, D.; Cremonese, G.; Da Deppo, V.; Debei, S.; De Cecco, M.; Fulle, M.; Groussin, O.; Gutiérrez, P. J.; Güttler, C.; Hall, I.; Hofmann, M.; Hviid, S. F.; Ip, W.-H.; Jorda, L.; Keller, H. U.; Knollenberg, J.; Kovacs, G.; Kramm, J.-R.; Kührt, E.; Küppers, M.; Lin, Z.-Y.; Lopez Moreno, J. J.; Marzari, F.; Moreno, F.; Shi, X.; Thomas, N.; Toth, I.; Tubiana, C.

    2016-02-01

    Aims.We carried out an investigation of the surface variegation of comet 67P/Churyumov-Gerasimenko, the detection of regions showing activity, the determination of active and inactive surface regions of the comet with spectral methods, and the detection of fallback material. Methods: We analyzed multispectral data generated with Optical, Spectroscopic, and Infrared Remote Imaging System (OSIRIS) narrow angle camera (NAC) observations via spectral techniques, reflectance ratios, and spectral slopes in order to study active regions. We applied clustering analysis to the results of the reflectance ratios, and introduced the new technique of activity thresholds to detect areas potentially enriched in volatiles. Results: Local color inhomogeneities are detected over the investigated surface regions. Active regions, such as Hapi, the active pits of Seth and Ma'at, the clustered and isolated bright features in Imhotep, the alcoves in Seth and Ma'at, and the large alcove in Anuket, have bluer spectra than the overall surface. The spectra generated with OSIRIS NAC observations are dominated by cometary emissions of around 700 nm to 750 nm as a result of the coma between the comet's surface and the camera. One of the two isolated bright features in the Imhotep region displays an absorption band of around 700 nm, which probably indicates the existence of hydrated silicates. An absorption band with a center between 800-900 nm is tentatively observed in some regions of the nucleus surface. This absorption band can be explained by the crystal field absorption of Fe2+, which is a common spectral feature seen in silicates.

  4. Side Chain Degradable Cationic-Amphiphilic Polymers with Tunable Hydrophobicity Show in Vivo Activity.

    PubMed

    Uppu, Divakara S S M; Samaddar, Sandip; Hoque, Jiaul; Konai, Mohini M; Krishnamoorthy, Paramanandham; Shome, Bibek R; Haldar, Jayanta

    2016-09-12

    Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions

  5. The novel AKT inhibitor afuresertib shows favorable safety, pharmacokinetics, and clinical activity in multiple myeloma

    PubMed Central

    Spencer, Andrew; Yoon, Sung-Soo; Harrison, Simon J.; Morris, Shannon R.; Smith, Deborah A.; Brigandi, Richard A.; Gauvin, Jennifer; Kumar, Rakesh; Opalinska, Joanna B.

    2014-01-01

    The PI3K/AKT pathway is constitutively active in hematologic malignancies, providing proliferative and antiapoptotic signals and possibly contributing to drug resistance. We conducted an open-label phase 1 study to evaluate the maximum tolerated dose (MTD), safety, pharmacokinetics, and clinical activity of afuresertib—an oral AKT inhibitor—in patients with advanced hematologic malignancies. Seventy-three patients were treated at doses ranging from 25 to 150 mg per day. The MTD was established at 125 mg per day because of 2 dose-limiting toxicities in the 150-mg cohort (liver function test abnormalities). The most frequent adverse events were nausea (35.6%), diarrhea (32.9%), and dyspepsia (24.7%). Maximum plasma concentrations and area under the plasma concentration-time curves from time 0 to 24 hours were generally dose proportional at >75-mg doses; the median time to peak plasma concentrations was 1.5 to 2.5 hours post dose, with a half-life of approximately 1.7 days. Three multiple myeloma patients attained partial responses; an additional 3 attained minimal responses. Clinical activity was also observed in non-Hodgkin lymphoma, Langerhan's cell histiocytosis, and Hodgkin disease. Single-agent afuresertib showed a favorable safety profile and demonstrated clinical activity against hematologic malignancies, including multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT00881946. PMID:25075128

  6. Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

    SciTech Connect

    Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.; Bolton, Harvey

    2011-02-01

    Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was added to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.

  7. Rapid parallel flow cytometry assays of active GTPases using effector beads

    PubMed Central

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-01-01

    We describe a rapid assay for measuring the cellular activity of small GTPases in response to a specific stimulus. Effector functionalized beads are used to quantify in parallel multiple, GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus. PMID:23928044

  8. Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity.

    PubMed

    Wang, Gang Lin; Luo, Hong Qun; Li, Nian Bing

    2014-09-21

    A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit of 0.25 U mL(-1), which might make this method a good candidate for monitoring DNA methylation in the future. PMID:25028809

  9. Plant vermicides of Haitian Vodou show in vitro activity against larval hookworm.

    PubMed

    Wolpert, Beverly J; Beauvoir, Max G; Wells, Elizabeth Fortson; Hawdon, John M

    2008-10-01

    Haitian Vodou priests (houngans) and priestesses (mambos) use plant remedies to treat many illnesses, including intestinal parasite infections. The present study screened 12 plants used in Vodou treatments for intestinal parasites to detect in vitro activity against infective-stage larvae of the hookworm Ancylostoma caninum. Water-soluble extracts of 4 of the 12 plants inhibited serum-stimulated feeding by larval A. caninum in a dose-dependent manner. All 4 plant extracts inhibited feeding induced by the muscarinic agonist arecoline, suggesting that these plant extracts may inhibit the insulin-like signaling pathway involved in the recovery and resumption of development of arrested A. caninum larvae. These results indicate that at least some of the plants used in traditional Haitian medicine as vermifuges show activity against nematode physiological processes.

  10. Two Analogues of Fenarimol Show Curative Activity in an Experimental Model of Chagas Disease

    PubMed Central

    2013-01-01

    Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is an increasing threat to global health. Available medicines were introduced over 40 years ago, have undesirable side effects, and give equivocal results of cure in the chronic stage of the disease. We report the development of two compounds, 6 and (S)-7, with PCR-confirmed curative activity in a mouse model of established T. cruzi infection after once daily oral dosing for 20 days at 20 mg/kg 6 and 10 mg/kg (S)-7. Compounds 6 and (S)-7 have potent in vitro activity, are noncytotoxic, show no adverse effects in vivo following repeat dosing, are prepared by a short synthetic route, and have druglike properties suitable for preclinical development. PMID:24304150

  11. Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances.

    PubMed

    Brychkova, Galina; Yarmolinsky, Dmitry; Sagi, Moshe

    2012-09-01

    Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.

  12. Assay for methylmalonyl coenzyme A mutase activity based on determination of succinyl coenzyme A by ultrahigh-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Gotoh, Kana; Nakajima, Yoko; Tajima, Go; Hotta, Yuji; Kataoka, Tomoya; Kawade, Yoshihiro; Sugiyama, Naruji; Ito, Tetsuya; Kimura, Kazunori; Maeda, Yasuhiro

    2015-07-01

    Methylmalonic acidemia (MMA) is an inherited metabolic disease. In this condition, metabolism from methylmalonyl coenzyme A (CoA) to succinyl-CoA is inhibited because of either low methylmalonyl-CoA mutase (MCM) activity or adenosylcobalamin deficiency owing to altered vitamin B12 metabolism. A high-precision assay for detecting MCM activity would facilitate not only MMA diagnosis but also the ability to determine the severity of MMA. We developed an MCM assay method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) that involves the determination of succinyl-CoA, which is formed in an enzyme reaction, using peripheral lymphocytes. Using 0.05, 0.5, and 5 μmol/L succinyl-CoA, the intra-assay coefficient of variation (CV) was less than 5.2% and the inter-assay CV was less than 8.7%. The MCM activities of five healthy individuals and four patients were investigated with this assay. The MCM activities of the patients were very low in relation to those of healthy individuals. Together, these results show that the UPLC-MS/MS method is useful for a detailed MCM activity assay.

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

    PubMed

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

    2015-02-10

    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.

  14. A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.

    PubMed

    Yu, Kai; Hu, Sheng; Huang, Jun; Mei, Le-He

    2011-08-10

    A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK(a) to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

  15. 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

  16. Development of a sensitive multi-well colorimetric assay for active NFκB

    PubMed Central

    Renard, Patricia; Ernest, Isabelle; Houbion, Andrée; Art, Muriel; Le Calvez, Hervé; Raes, Martine; Remacle, José

    2001-01-01

    The transcription factor nuclear factor κB (NFκB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFκB activation. Indeed, NFκB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFκB. The presence of the DNA-bound transcription factor is then detected by anti-NFκB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFκB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening. PMID:11160941

  17. 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

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

    PubMed Central

    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

  19. Meropenem-Clavulanic Acid Shows Activity against Mycobacterium tuberculosis In Vivo

    PubMed Central

    England, Kathleen; Boshoff, Helena I. M.; Arora, Kriti; Weiner, Danielle; Dayao, Emmanuel; Schimel, Daniel; Via, Laura E.

    2012-01-01

    The carbapenems imipenem and meropenem in combination with clavulanic acid reduced the bacterial burden in Mycobacterium tuberculosis-infected macrophages by 2 logs over 6 days. Despite poor stability in solution and a short half-life in rodents, treatment of chronically infected mice revealed significant reductions of bacterial burden in the lungs and spleens. Our results show that meropenem has activity in two in vivo systems, but stability and pharmacokinetics of long-term administration will offer significant challenges to clinical evaluation. PMID:22450968

  20. Endolymphatic Sac Tumor Showing Increased Activity on 68Ga DOTATATE PET/CT.

    PubMed

    Papadakis, Georgios Z; Millo, Corina; Sadowski, Samira M; Bagci, Ulas; Patronas, Nicholas J

    2016-10-01

    Endolymphatic sac tumors (ELSTs) are rare tumors arising from the epithelium of the endolymphatic sac and duct that can be either sporadic or associated with von Hippel-Lindau (VHL) disease. We report a case of a VHL patient with histologically proven residual ELST who underwent Ga DOTATATE PET/CT showing increased activity (SUVmax, 6.29) by the ELST. The presented case of a VHL-associated ELST with increased Ga DOTATATE uptake indicates cell-surface expression of somatostatin receptors by this tumor, suggesting the potential application of somatostatin receptor imaging using Ga DOTA-conjugated peptides in the workup and management of these patients. PMID:27454593

  1. 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.

  2. 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

  3. Honey, pollen, and propolis extracts show potent inhibitory activity against the zinc metalloenzyme carbonic anhydrase.

    PubMed

    Sahin, H; Aliyazicioglu, R; Yildiz, O; Kolayli, S; Innocenti, A; Supuran, C T

    2011-06-01

    Three different honey extracts from the endemic plant in the Black Sea region Rhododendron ponticum, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), more precisely the human (h) isoforms hCA I and hCA II. Hexane, methanol, ethanol, and water solid-phase extractions (SPEs) showed inhibitory activity towards the two CA isozymes which were related to the total phenolic content. The highest inhibitory effects (0.036-0.039 mg/mL) were those of propolis methanolic extract. Among the three different samples investigated here, the aqueous extracts showed lower inhibitory effects compared to the organic solvent SPE extracts (in the range of 1.150- 5.144 mg/mL). The studied honey extracts constitute an interesting source of phenolic derivatives that might serve to identify lead compounds, targeting the physiologically relevant enzymes CA I and CA II.

  4. An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays.

    PubMed

    Qureshi, Anjum; Roci, Irena; Gurbuz, Yasar; Niazi, Javed H

    2012-04-15

    An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.

  5. Youth at risk for obesity show greater activation of striatal and somatosensory regions to food.

    PubMed

    Stice, Eric; Yokum, Sonja; Burger, Kyle S; Epstein, Leonard H; Small, Dana M

    2011-03-23

    Obese humans, compared with normal-weight humans, have less striatal D2 receptors and striatal response to food intake; weaker striatal response to food predicts weight gain for individuals at genetic risk for reduced dopamine (DA) signaling, consistent with the reward-deficit theory of obesity. Yet these may not be initial vulnerability factors, as overeating reduces D2 receptor density, D2 sensitivity, reward sensitivity, and striatal response to food. Obese humans also show greater striatal, amygdalar, orbitofrontal cortex, and somatosensory region response to food images than normal-weight humans do, which predicts weight gain for those not at genetic risk for compromised dopamine signaling, consonant with the reward-surfeit theory of obesity. However, after pairings of palatable food intake and predictive cues, DA signaling increases in response to the cues, implying that eating palatable food contributes to increased responsivity. Using fMRI, we tested whether normal-weight adolescents at high- versus low-risk for obesity showed aberrant activation of reward circuitry in response to receipt and anticipated receipt of palatable food and monetary reward. High-risk youth showed greater activation in the caudate, parietal operculum, and frontal operculum in response to food intake and in the caudate, putamen, insula, thalamus, and orbitofrontal cortex in response to monetary reward. No differences emerged in response to anticipated food or monetary reward. Data indicate that youth at risk for obesity show elevated reward circuitry responsivity in general, coupled with elevated somatosensory region responsivity to food, which may lead to overeating that produces blunted dopamine signaling and elevated responsivity to food cues.

  6. Youth at Risk for Obesity Show Greater Activation of Striatal and Somatosensory Regions to Food

    PubMed Central

    Stice, E; Yokum, S; Burger, KS; Epstein, LH; Small, DM

    2011-01-01

    Obese versus normal-weight humans have less striatal D2 receptors and striatal response to food intake, and weaker striatal response to food predicts weight gain for individuals at genetic risk for reduced dopamine (DA) signaling, consistent with the reward deficit theory of obesity. Yet these may not be initial vulnerability factors, as overeating reduces D2 receptor density, D2 sensitivity, reward sensitivity, and striatal response to food. Obese versus normal-weight humans also show greater striatal, amygdalar, orbitofrontal cortex, and somatosensory region response to food images, which predicts weight gain for those not at genetic risk for compromised dopamine signaling, consonant with the reward surfeit theory of obesity. However, after pairings of palatable food intake and predictive cues, DA signaling increases in response to the cues, implying that eating palatable food contributes to increased responsivity. We tested whether normal-weight adolescents at high- versus low-risk for obesity showed aberrant activation of reward circuitry in response to receipt and anticipated receipt of palatable food and monetary reward using fMRI. High-risk youth showed greater activation in the caudate, parietal operculum, and frontal operculum in response to food intake and in the caudate, putamen, insula, thalamus, and orbitofrontal cortex in response to monetary reward. No differences emerged in response to anticipated food or monetary reward. Data indicate that youth at risk for obesity show elevated reward circuitry responsivity in general coupled with elevated somatosensory region responsivity to food, which may lead to overeating that produces blunted dopamine signaling and elevated responsivity to food cues. PMID:21430137

  7. Youth at risk for obesity show greater activation of striatal and somatosensory regions to food.

    PubMed

    Stice, Eric; Yokum, Sonja; Burger, Kyle S; Epstein, Leonard H; Small, Dana M

    2011-03-23

    Obese humans, compared with normal-weight humans, have less striatal D2 receptors and striatal response to food intake; weaker striatal response to food predicts weight gain for individuals at genetic risk for reduced dopamine (DA) signaling, consistent with the reward-deficit theory of obesity. Yet these may not be initial vulnerability factors, as overeating reduces D2 receptor density, D2 sensitivity, reward sensitivity, and striatal response to food. Obese humans also show greater striatal, amygdalar, orbitofrontal cortex, and somatosensory region response to food images than normal-weight humans do, which predicts weight gain for those not at genetic risk for compromised dopamine signaling, consonant with the reward-surfeit theory of obesity. However, after pairings of palatable food intake and predictive cues, DA signaling increases in response to the cues, implying that eating palatable food contributes to increased responsivity. Using fMRI, we tested whether normal-weight adolescents at high- versus low-risk for obesity showed aberrant activation of reward circuitry in response to receipt and anticipated receipt of palatable food and monetary reward. High-risk youth showed greater activation in the caudate, parietal operculum, and frontal operculum in response to food intake and in the caudate, putamen, insula, thalamus, and orbitofrontal cortex in response to monetary reward. No differences emerged in response to anticipated food or monetary reward. Data indicate that youth at risk for obesity show elevated reward circuitry responsivity in general, coupled with elevated somatosensory region responsivity to food, which may lead to overeating that produces blunted dopamine signaling and elevated responsivity to food cues. PMID:21430137

  8. 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

  9. 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.

  10. 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).

  11. Progress in high-throughput assays of MGMT and APE1 activities in cell extracts.

    PubMed

    Georgiadis, Panagiotis; Polychronaki, Nektaria; Kyrtopoulos, Soterios A

    2012-08-01

    DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6)-methylguanine-DNA-methyltransferase (MGMT), which repairs the O(6)-alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5' to the abasic sites. The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O(6) position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O(6)-benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG-biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate. A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a fluorescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been

  12. Development of a QPatch Automated Electrophysiology Assay for Identifying KCa3.1 Inhibitors and Activators

    PubMed Central

    Jenkins, David Paul; Yu, Weifeng; Brown, Brandon M.; Løjkner, Lars Damgaard

    2013-01-01

    Abstract The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay. PMID:24351043

  13. Isolation of natural compounds from Phlomis stewartii showing α-glucosidase inhibitory activity.

    PubMed

    Jabeen, Bushra; Riaz, Naheed; Saleem, Muhammad; Naveed, Muhammad Akram; Ashraf, Muhammad; Alam, Umber; Rafiq, Hafiza Mehwish; Tareen, Rasool Bakhsh; Jabbar, Abdul

    2013-12-01

    Stewartiiside (1), a phenylethanoid glycoside and three 28-nortriterpenoids: stewertiisins A-C [(17R)-19(18→17)-abeo-3α,18β,23,24-tetrahydroxy-28-norolean-12-ene, 2; (17R)-19(18→17)-abeo-2α,16β,18β,23,24-pentahydroxy-28-norolean-12-en-3-one, 3; (17R)-19(18→17)-abeo-2α,3α,23,24-tetrahydroxy-28-noroleane-11,13-diene, 4] together with eight known compounds: lunariifolioside (5), notohamosin A (6), phlomispentanol (7), isorhamnetin 3-(6-p-coumaroyl)-β-D-glucopyranoside (8), tiliroside (9), caffeic acid (10), p-hydrxybenzoic acid (11) and oleanolic acid (12) were isolated from the ethyl acetate soluble fraction of the methanolic extract of whole plant of Phlomis stewartii. The structures of these isolates (1-12) were elucidated by the combination of 1D ((1)H and (13)C NMR), 2D (HMQC, HMBC COSY, NOESY) NMR spectroscopy and mass spectrometry (EIMS, HREIMS, FABMS, HRFABMS) and in comparison with literature data of related compounds. All the isolates (1-12) showed α-glucosidase inhibitory activity with IC50 values ranging between 14.5 and 355.4 μM, whereas, compounds 1, 5, 9 and 10 showed promising α-glucosidase inhibitory activity with IC50 values below 30 μM.

  14. CpG oligodeoxynucleotides with double stem-loops show strong immunostimulatory activity.

    PubMed

    Yang, Liang; Wu, Xiuli; Wan, Min; Yu, Yue; Yu, Yongli; Wang, Liying

    2013-01-01

    Based on the current understanding of TLR9 recognition of CpG ODN, we have tried to design a series of CpG ODNs that display double stem-loops when being analyzed for their secondary structures using 'mfold web server'. Proliferation of human PBMC and bioassay for IFN production were used as technical platforms in primary screening. Interestingly, two of them, designated as DSL01 and D-SL03, belonging to B class CpG ODN and C class CpG ODN respectively, showed vigorous immunostimulatory activity and were chosen for further tests. Flow cytometry analysis showed that both of them could activate human B cells, NK cells, mononuclear cells and T cells and up-regulate expression of CD80, CD86 and HLA-DR on the surface of subsets in human PBMCs. Furthermore, we demonstrated that those two ODNs potently stimulated proliferation of PBMC/splenocytes obtained from diverse vertebrate species. Noticeably, both of them displayed anti-breast cancer effect in mice when administered by peritumoral injection. PMID:23142503

  15. Adolescent earthquake survivors' show increased prefrontal cortex activation to masked earthquake images as adults.

    PubMed

    Du, Xue; Wei, Dongtao; Ganzel, Barbara L; Kim, Pilyoung; Zhang, Qinglin; Qiu, Jiang

    2015-03-01

    The great Sichuan earthquake in China on May 12, 2008 was a traumatic event to many who live near the earthquake area. However, at present, there are few studies that explore the long-term impact of the adolescent trauma exposure on adults' brain function. In the present study, we used functional magnetic resonance imaging (fMRI) to investigate the brain activation evoked by masked trauma-related stimuli (earthquake versus neutral images) in 14 adults who lived near the epicenter of the great Sichuan earthquake when they were adolescents (trauma-exposed group) and 14 adults who lived farther from the epicenter of the earthquake when they were adolescents (control group). Compared with the control group, the trauma-exposed group showed significant elevation of activation in the right anterior cingulate cortex (ACC) and the medial prefrontal cortex (MPFC) in response to masked earthquake-related images. In the trauma-exposed group, the right ACC activation was negatively correlated with the frequency of symptoms of post-traumatic stress disorder (PTSD). These findings differ markedly from the long-term effects of trauma exposure in adults. This suggests that trauma exposure during adolescence may have a unique long-term impact on ACC/MPFC function, top-down modulation of trauma-related information, and subsequent symptoms of PTSD.

  16. Arctigenin blocks the unfolded protein response and shows therapeutic antitumor activity.

    PubMed

    Kim, Ju-Young; Hwang, Ji-Hwan; Cha, Mi-Ran; Yoon, Mi-Young; Son, Eun-Soon; Tomida, Akihiro; Ko, Bosung; Song, Si-Whan; Shin-ya, Kazuo; Hwang, Yong-il; Park, Hae-Ryong

    2010-07-01

    Cancer cells in poorly vascularized solid tumors are constantly or intermittently exposed to stressful microenvironments, including glucose deprivation, hypoxia, and other forms of nutrient starvation. These tumor-specific conditions, especially glucose deprivation, activate a signaling pathway called the unfolded protein response (UPR), which enhances cell survival by induction of the stress proteins. We have established a screening method to discover anticancer agents that could preferentially inhibit tumor cell viability under glucose-deprived conditions. Here we identify arctigenin (ARC-G) as an active compound that shows selective cytotoxicity and inhibits the UPR during glucose deprivation. Indeed, ARC-G blocked expression of UPR target genes such as phosphorylated-PERK, ATF4, CHOP, and GRP78, which was accompanied by enhanced phosphorylation of eIF2 alpha during glucose deprivation. The UPR inhibition led to apoptosis involving a mitochondrial pathway by activation of caspase-9 and -3. Furthermore, ARC-G suppressed tumor growth of colon cancer HT-29 xenografts. Our results demonstrate that ARC-G can be served as a novel type of antitumor agent targeting the UPR in glucose-deprived solid tumors.

  17. Apratoxin A Shows Novel Pancreas-Targeting Activity through the Binding of Sec 61.

    PubMed

    Huang, Kuan-Chun; Chen, Zhihong; Jiang, Yimin; Akare, Sandeep; Kolber-Simonds, Donna; Condon, Krista; Agoulnik, Sergei; Tendyke, Karen; Shen, Yongchun; Wu, Kuo-Ming; Mathieu, Steven; Choi, Hyeong-Wook; Zhu, Xiaojie; Shimizu, Hajime; Kotake, Yoshihiko; Gerwick, William H; Uenaka, Toshimitsu; Woodall-Jappe, Mary; Nomoto, Kenichi

    2016-06-01

    Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a (3)H-labeled apratoxin A probe and specific Sec 61α/β antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208-16. ©2016 AACR.

  18. Developing in vitro reporter gene assays to assess the hormone receptor activities of chemicals frequently detected in drinking water.

    PubMed

    Sun, Hong; Si, Chaozong; Bian, Qian; Chen, Xiaodong; Chen, Liansheng; Wang, Xinru

    2012-08-01

    The present study intended to develop receptor-mediated luciferase reporter gene assays to evaluate and compare the estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) activities of target chemicals. Di-2-ethylhexyl-phthalate (DEHP), chlorpyrifos (CPF), 2,4-dichlorophenoxyacetic acid (2,4-D) and bisphenol A (BPA) are some of the most common contaminants in drinking water and are frequently detected in China and worldwide. The chemicals were tested at concentrations of 0.1, 1, 10 and 100 times their maximum contaminant level in drinking water. The results showed that BPA possessed various activities on ER, AR and TR. DEHP and CPF could suppress 17β-estradiol or testosterone activity with different potencies, and DEHP possessed weaker anti-thyroid hormone activity. 2,4-D showed no agonist or antagonist activity against these hormone receptors, but it significantly enhanced the activity of testosterone through AR. Furthermore, the mixture of DEHP and CPF exhibited stronger ER and AR antagonist activities than each single component alone, but their combined effects were less than the expected effects based on the additive model. These results implied that the transcription activation mediated by hormone receptors was the potential endocrine-disrupting mechanism of the test chemicals. Our study also provided useful tools for evaluation of their endocrine disrupting activity.

  19. 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.

  20. An easy-to-perform photometric assay for methyltransferase activity measurements.

    PubMed

    Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

    2013-01-01

    Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay.

  1. A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.

    PubMed

    Slattery, Scott D; Hahn, Klaus M

    2014-12-01

    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. We describe here a method 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 (e.g., 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 effects of upstream molecules.

  2. 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.

  3. 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

  4. 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

  5. The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1.

    PubMed

    Smith, Robert A; Raugi, Dana N; Wu, Vincent H; Leong, Sally S; Parker, Kate M; Oakes, Mariah K; Sow, Papa Salif; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S

    2015-12-01

    Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4'-ethynyl stavudine, or 4'-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture. PMID:26392486

  6. The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1

    PubMed Central

    Raugi, Dana N.; Wu, Vincent H.; Leong, Sally S.; Parker, Kate M.; Oakes, Mariah K.; Sow, Papa Salif; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S.

    2015-01-01

    Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4′-ethynyl stavudine, or 4′-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture. PMID:26392486

  7. The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1.

    PubMed

    Smith, Robert A; Raugi, Dana N; Wu, Vincent H; Leong, Sally S; Parker, Kate M; Oakes, Mariah K; Sow, Papa Salif; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S

    2015-12-01

    Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4'-ethynyl stavudine, or 4'-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture.

  8. Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

    PubMed

    Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

    2011-08-15

    Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

  9. 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.

  10. 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

  11. Polyethylenimine-cyclodextrin-tegafur conjugate shows anti-cancer activity and a potential for gene delivery*

    PubMed Central

    Hu, Qi-da; Fan, Hui; Lou, Wei-jian; Wang, Qing-qing; Tang, Gu-ping

    2011-01-01

    Polyethylenimine-cyclodextrin-tegafur (PEI-CyD-tegafur) conjugate was synthesized as a novel multifunctional prodrug of tegafur for co-delivery of chemotherapeutic agent tegafur and enhanced green fluorescent protein (EGFP) reporter plasmid DNA. Conjugation of tegafur to PEI-CyD via chemical linkage was characterized by 1H NMR spectrometry and ultraviolet (UV) spectrometry. PEI-CyD-tegafur was able to condense plasmid DNA into complexes of around 150 nm with positive charge at the N/P ratio of 25, in accordance with electron microscopy observation of compact and monodisperse nanoparticles. The results of in vitro experiments showed enhanced cytotoxicity and considerable transfection efficiency in B16F10 cell line. Therefore, PEI-CyD-tegafur may have great potential as a co-delivery system with anti-cancer activity and potential for gene delivery. PMID:21887847

  12. Acidophilic actinobacteria synthesised silver nanoparticles showed remarkable activity against fungi-causing superficial mycoses in humans.

    PubMed

    Anasane, N; Golińska, P; Wypij, M; Rathod, D; Dahm, H; Rai, M

    2016-03-01

    Superficial mycoses are limited to the most external part of the skin and hair and caused by Malassezia sp., Trichophyton sp. and Candida sp. We report extracellular biosynthesis of silver nanoparticles (AgNPs) by acidophilic actinobacteria (SF23, C9) and its in vitro antifungal activity against fungi-causing superficial mycoses. The phylogenetic analysis based on the 16S rRNA gene sequence of strains SF23 and C9 showed that they are most closely related to Pilimelia columellifera subsp. pallida GU269552(T). The detection of AgNPs was confirmed by visual observation of colour changes from colourless to brown, and UV-vis spectrophotometer analysis, which showed peaks at 432 and 427 nm, respectively. These AgNPs were further characterised by nanoparticle tracking analysis (NTA), Zeta potential, Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The FTIR analysis exhibited the presence of proteins as capping agents. The TEM analysis revealed the formation of spherical and polydispersed nanoparticles in the size range of 4-36 nm and 8-60 nm, respectively. The biosynthesised AgNPs were screened against fungi-causing superficial mycoses viz., Malassezia furfur, Trichophyton rubrum, Candida albicans and C. tropicalis. The highest antifungal activity of AgNPs from SF23 and C9 against T. rubrum and the least against M. furfur and C. albicans was observed as compared to other tested fungi. The biosynthesised AgNPs were found to be potential anti-antifungal agent against fungi-causing superficial mycoses.

  13. The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

    PubMed Central

    Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

    2010-01-01

    Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

  14. Sphaeropsidin A shows promising activity against drug-resistant cancer cells by targeting regulatory volume increase.

    PubMed

    Mathieu, Véronique; Chantôme, Aurélie; Lefranc, Florence; Cimmino, Alessio; Miklos, Walter; Paulitschke, Verena; Mohr, Thomas; Maddau, Lucia; Kornienko, Alexander; Berger, Walter; Vandier, Christophe; Evidente, Antonio; Delpire, Eric; Kiss, Robert

    2015-10-01

    Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 µM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl(-) and the decreased HCO3 (-) concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na-K-2Cl electroneutral cotransporter or Cl(-)/HCO3 (-) anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells. PMID:25868554

  15. Physically active men show better semen parameters and hormone values than sedentary men.

    PubMed

    Vaamonde, Diana; Da Silva-Grigoletto, Marzo Edir; García-Manso, Juan Manuel; Barrera, Natalibeth; Vaamonde-Lemos, Ricardo

    2012-09-01

    Physical exercise promotes many health benefits. The present study was undertaken to assess possible semen and hormone differences among physically active (PA) subjects and sedentary subjects (SE). The analyzed qualitative sperm parameters were: volume, sperm count, motility, and morphology; where needed, additional testing was performed. The measured hormones were: follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), cortisol (C), and the ratio between T and C (T/C). Maximum oxygen consumption was also assessed to check for differences in fitness level. Statistically significant differences were found for several semen parameters such as total progressive motility (PA: 60.94 ± 5.03; SE: 56.07 ± 4.55) and morphology (PA: 15.54 ± 1.38, SE: 14.40 ± 1.15). The seminological values observed were supported by differences in hormones, with FSH, LH, and T being higher in PA than in SE (5.68 ± 2.51 vs. 3.14 ± 1.84; 5.95 ± 1.11 vs. 5.08 ± 0.98; 7.68 ± 0.77 vs. 6.49 ± 0.80, respectively). Likewise, the T/C ratio, index of anabolic versus catabolic status, was also higher in PA (0.46 ± 0.11 vs. 0.32 ± 0.07), which further supports the possibility of an improved hormonal environment. The present study shows that there are differences in semen and hormone values of physically active subjects and sedentary subjects. Physically active subjects seem to have a more anabolic hormonal environment and a healthier semen production.

  16. Sphaeropsidin A shows promising activity against drug-resistant cancer cells by targeting regulatory volume increase

    PubMed Central

    Mathieu, Véronique; Chantôme, Aurélie; Lefranc, Florence; Cimmino, Alessio; Miklos, Walter; Paulitschke, Verena; Mohr, Thomas; Maddau, Lucia; Kornienko, Alexander; Berger, Walter; Vandier, Christophe; Evidente, Antonio; Delpire, Eric; Kiss, Robert

    2016-01-01

    Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 μM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl− and the decreased HCO3− concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na–K–2Cl electroneutral cotransporter or Cl−/HCO3− anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells. PMID:25868554

  17. Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species.

    PubMed Central

    Harding, S A; Oh, S H; Roberts, D M

    1997-01-01

    A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed. We have used a dominant-acting calmodulin mutant (VU-3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co-enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures. VU-3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post-translationally, and as a result it hyperactivates calmodulin-dependent NAD kinase. Cells expressing VU-3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli. Increases in NADPH level were also greater in VU-3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst. These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis. PMID:9135130

  18. 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

  19. 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

  20. 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

  1. Mutagenic activity of sweepings and pigments from a household-wax factory assayed with Salmonella typhimurium.

    PubMed

    Varella, S D; Pozetti, G L; Vilegas, W; Varanda, E A

    2004-12-01

    The mutagenic activity of garbage originating from a household wax industry was determined by the Salmonella/microsome assay, using the bacterial strains TA100, TA98 and YG1024. The garbage was obtained by sweeping the floor of the factory at the end of the work shift. Organic compounds were extracted by ultrasound for 30 min in dichloromethane or 70% ethanol. After evaporation of solvent, these extracts (HFS: household-wax factory sweepings) were dissolved in DMSO, and were tested for the mutagenic activity at varying concentrations (HFS-ET: 0.08-0.68 mg/plate, HFS-DCM: 0.60-7.31 mg/plate). The colouring agents (pigments) used in the production of the wax were also dissolved in DMSO and tested with the assay. The concentrations tested for each pigment were: Amaranth: 0.46-3.65 mg/plate, Auramine: 0.15-1.2 mg/plate and Rhodamine B: 0.22-1.82 mg/plate. Both ET and DCM organic extracts had mutagenic activity, especially in the YG1024 strain. The pigments behaved in a similar way, demonstrating that YG1024 was the most sensitive strain for the detection of mutagenicity, and that metabolization increased the activity. Human exposure (occupational and non-occupational) to industrial residues generated during the household-wax manufacturing and packaging process should be monitored, since this type of garbage is normally deposited in the environment without any control.

  2. Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

    PubMed

    van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

    2014-01-15

    Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

  3. 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

  4. Screening of Dengue Virus Antiviral Activity of Marine Seaweeds by an In Situ Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Koishi, Andrea Cristine; Zanello, Paula Rodrigues; Bianco, Éverson Miguel; Bordignon, Juliano; Nunes Duarte dos Santos, Claudia

    2012-01-01

    Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization. PMID:23227238

  5. 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

  6. HATS-2b: A transiting extrasolar planet orbiting a K-type star showing starspot activity

    NASA Astrophysics Data System (ADS)

    Mohler-Fischer, M.; Mancini, L.; Hartman, J. D.; Bakos, G. Á.; Penev, K.; Bayliss, D.; Jordán, A.; Csubry, Z.; Zhou, G.; Rabus, M.; Nikolov, N.; Brahm, R.; Espinoza, N.; Buchhave, L. A.; Béky, B.; Suc, V.; Csák, B.; Henning, T.; Wright, D. J.; Tinney, C. G.; Addison, B. C.; Schmidt, B.; Noyes, R. W.; Papp, I.; Lázár, J.; Sári, P.; Conroy, P.

    2013-10-01

    We report the discovery of HATS-2b, the second transiting extrasolar planet detected by the HATSouth survey. HATS-2b is moving on a circular orbit around a V = 13.6 mag, K-type dwarf star (GSC 6665-00236), at a separation of 0.0230 ± 0.0003 AU and with a period of 1.3541 days. The planetary parameters have been robustly determined using a simultaneous fit of the HATSouth, MPG/ESO 2.2 m/GROND, Faulkes Telescope South/Spectral transit photometry, and MPG/ESO 2.2 m/FEROS, Euler 1.2 m/CORALIE, AAT 3.9 m/CYCLOPS radial-velocity measurements. HATS-2b has a mass of 1.37 ± 0.16 MJ, a radius of 1.14 ± 0.03 RJ, and an equilibrium temperature of 1567 ± 30 K. The host star has a mass of 0.88 ± 0.04 M⊙ and a radius of 0.89 ± 0.02 R⊙, and it shows starspot activity. We characterized the stellar activity by analyzing two photometric follow-up transit light curves taken with the GROND instrument, both obtained simultaneously in four optical bands (covering the wavelength range of 3860-9520 Å). The two light curves contain anomalies compatible with starspots on the photosphere of the host star along the same transit chord. Tables of the individual photometric measurements are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/558/A55

  7. A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

    PubMed

    Ibatullin, Farid M; Banasiak, Alicja; Baumann, Martin J; Greffe, Lionel; Takahashi, Junko; Mellerowicz, Ewa J; Brumer, Harry

    2009-12-01

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  8. The antioxidant activity of sulphurous thermal water protects against oxidative DNA damage: a comet assay investigation.

    PubMed

    Braga, P C; Ceci, C; Marabini, L; Nappi, G

    2013-04-01

    Various studies have recently shown that sulphurous waters acts against the oxidants released during respiratory bursts of human neutrophils, and free radicals such as HO•, O2¯•, Tempol and Fremy's salt. However, there is still a lack of data concerning their direct protection of DNA. The aim of this study was to investigate the antigenotoxicity effects of sulphurous water, which has never been previously investigated for this purpose, using the alkaline single cell gel electrophoresis (SCGE) approach (comet assay). The comet assay is a sensitive method for assessing DNA fragmentation in individual cells in genotoxicity studies but can also be used to investigate the activity of agents that protect against DNA damage. The extent of migration was measured by means of SCGE, and DNA damage was expressed as tail moment. All of these assays were made using natural sulphurous water, degassed sulphurous water (no detectable HS), and reconstituted sulphurous water (degassed plus NaHS). DNA damages was significantly inhibited by natural water with HS concentrations of 5.0 and 2.5 μg/mL. The use of degassed water did not lead to any significant differences from baseline values, whereas the reconstituted water led to significant results overlapping those obtained using natural water. These findings confirm the importance of the presence of an HS group (reductive activity) and indicate that, in addition to their known mucolytic activity and trophic effects on respiratory mucosa, HS groups in sulphurous water also protect against oxidative DNA damage and contribute to the water's therapeutic effects on upper and lower airway inflammatory diseases.

  9. 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

  10. 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

  11. 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

  12. Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity.

    PubMed

    Salinas-Jazmín, Nohemí; Estrada-Parra, Sergio; Becerril-García, Miguel Angel; Limón-Flores, Alberto Yairh; Vázquez-Leyva, Said; Medina-Rivero, Emilio; 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.

  13. Improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate

    SciTech Connect

    Junker, L.H.; Story, J.A.

    1985-10-01

    A persistent problem in measurement of cholesterol 7 alpha-hydroxylase (7 alpha-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7 alpha-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 microM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7 alpha-hydroxycholesterol formed. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.

  14. A plaque assay for malignant catarrhal fever virus and virus neutralizing activity.

    PubMed

    Hazlett, D T

    1980-05-01

    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.

  15. 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

  16. 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

  17. Sequential treatment with betulinic acid followed by 5-fluorouracil shows synergistic cytotoxic activity in ovarian cancer cells.

    PubMed

    Wang, Ying-Jian; Liu, Jun-Bao; Dou, Yu-Chang

    2015-01-01

    Betulinic acid selectively inhibits the growth of ovarian carcinoma cell lines without affecting the normal cells. In the present study, the effect of 5-fluorouracil (5-FU) and betulinic acid (BA) combination on ovarian carcinoma cells was studied. The results demonstrated that ovarian carcinoma cells on concurrent or 5-FU followed by BA treatment show increased Sub-G1 cell population, increased rate of cell apoptosis and morphological changes in mitochondrial membrane. In OVCAR 432 cells treatment with sequential combination of 5-FU and BA increased the Sub-G1 cell population to 51.3% and growth inhibition rate of > 72%. However, exposure to BA before 5-FU treatment caused a decrease in rate of inhibition to < 35%. Treatment with combination of 5 μM of 5-FU and 1 μM of BA for 48 h, led to an induction of apoptosis in 79.7% and induced morphological changes in OVCAR 432 cells. The Western blot results showed high concentration of cytochrome c in the cell cytosol after 24 h of 5-FU and BA combination treatment. Treatment of BA-responsive RMS-13 cells with 5-FU and BA combination resulted in inhibition of GLI1, GLI2, PTCH1, and IGF2 genes. In addition, we found a significant reduction in hedgehog activity of RMS-13 cells after 5-FU and BA combination treatment by means of a hedgehog-responsive reporter assay. Therefore, 5-FU and BA combination can be a promising regimen for the treatment of ovarian carcinoma. PMID:25755712

  18. A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression.

    PubMed

    Quiñones-Mateu, M E; Ball, S C; Marozsan, A J; Torre, V S; Albright, J L; Vanham, G; van Der Groen, G; Colebunders, R L; Arts, E J

    2000-10-01

    This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

  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. Hydroxyeicosapentaenoic acids from the Pacific krill show high ligand activities for PPARs[S

    PubMed Central

    Yamada, Hidetoshi; Oshiro, Eriko; Kikuchi, Sayaka; Hakozaki, Mayuka; Takahashi, Hideyuki; Kimura, Ken-ichi

    2014-01-01

    PPARs regulate the expression of genes for energy metabolism in a ligand-dependent manner. PPARs can influence fatty acid oxidation, the level of circulating triglycerides, glucose uptake and insulin sensitivity. Here, we demonstrate that 5-hydroxyeicosapentaenoic acid (HEPE), 8-HEPE, 9-HEPE, 12-HEPE and 18-HEPE (hydroxylation products of EPA) obtained from methanol extracts of Pacific krill (Euphausia pacifica) can act as PPAR ligands. Two of these products, 8-HEPE and 9-HEPE, enhanced the transcription levels of GAL4-PPARs to a significantly greater extent than 5-HEPE, 12-HEPE, 18-HEPE, EPA, and EPA ethyl-ester. 8-HEPE also activated significantly higher transcription of GAL4-PPARα, GAL4-PPARγ, and GAL4-PPARδ than EPA at concentrations greater than 4, 64, and 64 μM, respectively. We also demonstrated that 8-HEPE increased the expression levels of genes regulated by PPARs in FaO, 3T3-F442A, and C2C12 cells. Furthermore, 8-HEPE enhanced adipogenesis and glucose uptake. By contrast, at the same concentrations, EPA showed weak or little effect, indicating that 8-HEPE was the more potent inducer of physiological effects. PMID:24668940

  2. Using analogy role-play activity in an undergraduate biology classroom to show central dogma revision.

    PubMed

    Takemura, Masaharu; Kurabayashi, Mario

    2014-01-01

    For the study of biology in an undergraduate classroom, a classroom exercise was developed: an analogy role-play to learn mechanisms of gene transcription and protein translation (central dogma). To develop the central dogma role-play exercise, we made DNA and mRNA using paper sheets, tRNA using a wire dress hanger, and amino acids using Lego® blocks (Lego System A/S, Denmark). Students were studying in the course of mathematics, physics, or chemistry, so biology was not among their usual studies. In this exercise, students perform the central dogma role-play and respectively act out nuclear matrix proteins, a transcription factor, an RNA polymerase II, an mRNA transport protein, nuclear pore proteins, a large ribosomal subunit, a small ribosomal subunit, and several amino-acyl tRNA synthetases. Questionnaire results obtained after the activity show that this central dogma role-play analogy holds student interest in the practical molecular biological processes of transcription and translation.

  3. 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.

  4. 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

  5. Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays.

    PubMed

    Li, Xiao-Quan; Chen, Jing; Huang, Yan-Fen; Ding, Xi-Xia; Liu, Li-Dong; Qiu, Li-Wen; Pan, Yu-Xian; Deng, Yong-Qiang; Hu, Dong-Mei; Di, Biao; Qin, Cheng-Feng; Che, Xiao-Yan

    2013-07-01

    The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

  6. A sensitive hydroosmotic toad bladder assay. Affinity and intrinsic activity of neurohypophyseal peptides.

    PubMed

    Eggena, P; Schwartz, I L; Walter, R

    1968-09-01

    A sensitive and precise method for assaying the water permeability response evoked by neurohypophyseal hormones and their synthetic analogues on the isolated urinary bladder of the toad (Bufo marinus L.) is described. The method permits detection of 8-arginine-vasotocin at concentrations as low as 10(-12)M. This sensitivity, not achieved heretofore with this tissue, results largely from minimizing interference of inhibitory substances by means of an "in vitro circulation assembly." The precision of the method derives from a direct comparison between the cumulative dose-response curve of an agonist of unknown potency acting on one hemibladder and that of a reference compound acting on the contralateral hemibladder. Crystalline deamino-oxytocin is used as the reference standard in this assay. The intrinsic activity of 2-(O-methyltyrosine)-oxytocin, as defined by the maximal response, is 12% lower than that of deamino-oxytocin. All other hormonal peptides investigated have the same intrinsic activity as deamino-oxytocin, even 5-valine-oxytocin, in spite of its extremely low affinity. A comparison of the potencies of 8-arginine-vasotocin vs. 8-arginine-vasopressin, 8-ornithine-vasotocin vs. 8-ornithine-vasopressin, 8-alanine-oxytocin vs. 8-alanine-oxypressin, and deamino-8-alanine-oxytocin vs. deamino-8-alanine-oxypressin suggests that an isoleucine residue in position 3 imparts a higher specificity for binding of the hormonal peptide molecule to the bladder receptor than a phenylalanine residue in this locus.

  7. 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...

  8. 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 ...

  9. Active tectonics west of New Zealand's Alpine Fault: South Westland Fault Zone activity shows Australian Plate instability

    NASA Astrophysics Data System (ADS)

    De Pascale, Gregory P.; Chandler-Yates, Nicholas; Dela Pena, Federico; Wilson, Pam; May, Elijah; Twiss, Amber; Cheng, Che

    2016-04-01

    The 300 km long South Westland Fault Zone (SWFZ) is within the footwall of the Central Alpine Fault (<20 km away) and has 3500 m of dip-slip displacement, but it has been unknown if the fault is active. Here the first evidence for SWFZ thrust faulting in the "stable" Australian Plate is shown with cumulative dip-slip displacements up to 5.9 m (with 3 m throw) on Pleistocene and Holocene sediments and gentle hanging wall anticlinal folding. Cone penetration test (CPT) stratigraphy shows repeated sequences within the fault scarp (consistent with thrusting). Optically stimulated luminescence (OSL) dating constrains the most recent rupture post-12.1 ± 1.7 ka with evidence for three to four events during earthquakes of at least Mw 6.8. This study shows significant deformation is accommodated on poorly characterized Australian Plate structures northwest of the Alpine Fault and demonstrates that major active and seismogenic structures remain uncharacterized in densely forested regions on Earth.

  10. A New Assay for Determining Ganglioside Sialyltransferase Activities Lactosylceramide-2,3-Sialyltransferase (SAT I) and Monosialylganglioside-2,3-Sialyltransferase (SAT IV)

    PubMed Central

    Sun, Cynthia Q.; Hubl, Ulrike; Hoefakker, Petra; Vasudevamurthy, Madhusudan K.; Johnson, Keryn D.

    2014-01-01

    A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC 2.4.99.9) and monosialoganglioside sialyltransferase (SAT IV, EC 2.4.99.2) is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation. The analysis was performed in a quick and automated fashion. The assay showed good linearity for both BODIPY sphingolipids with a quantitative detection limit of 0.05 pmol. The high sensitivity enabled the detection of SAT I and SAT IV activities as low as 0.001 μU, at least 200 fold lower than that of most radiometric assays. This new assay was applied to the screening of SAT I and SAT IV activities in ovine and bovine organs (liver, heart, kidney, and spleen). The results provided evidence that young animals, such as calves, start to produce ganglioside sialyltransferases as early as 7 days after parturition and that levels change during maturation. Among the organs tested from a bovine source, spleen had the highest specific ganglioside sialyltransferase activity. Due to the organ size, the greatest total ganglioside sialyltransferase activities (SAT I and SAT IV) were detected in the liver of both bovine and ovine origin. PMID:24718572

  11. A new assay for determining ganglioside sialyltransferase activities lactosylceramide-2,3-sialyltransferase (SAT I) and monosialylganglioside-2,3-sialyltransferase (SAT IV).

    PubMed

    Sun, Cynthia Q; Hubl, Ulrike; Hoefakker, Petra; Vasudevamurthy, Madhusudan K; Johnson, Keryn D

    2014-01-01

    A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC 2.4.99.9) and monosialoganglioside sialyltransferase (SAT IV, EC 2.4.99.2) is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation. The analysis was performed in a quick and automated fashion. The assay showed good linearity for both BODIPY sphingolipids with a quantitative detection limit of 0.05 pmol. The high sensitivity enabled the detection of SAT I and SAT IV activities as low as 0.001 μU, at least 200 fold lower than that of most radiometric assays. This new assay was applied to the screening of SAT I and SAT IV activities in ovine and bovine organs (liver, heart, kidney, and spleen). The results provided evidence that young animals, such as calves, start to produce ganglioside sialyltransferases as early as 7 days after parturition and that levels change during maturation. Among the organs tested from a bovine source, spleen had the highest specific ganglioside sialyltransferase activity. Due to the organ size, the greatest total ganglioside sialyltransferase activities (SAT I and SAT IV) were detected in the liver of both bovine and ovine origin. PMID:24718572

  12. Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe.

    PubMed

    Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin

    2015-05-01

    An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

  13. 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.

  14. Rapid reverse phase-HPLC assay of HMG-CoA reductase activity

    PubMed Central

    Mozzicafreddo, Matteo; Cuccioloni, Massimiliano; Eleuteri, Anna Maria; Angeletti, Mauro

    2010-01-01

    Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP+) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP+, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively. PMID:20418539

  15. Chlorosulfonation of polystyrene substrates for bioanalytical assays: distribution of activated groups at the surface.

    PubMed

    del Prado, Anselmo; Briz, Nerea; Navarro, Rodrigo; Pérez, Mónica; Gallardo, Alberto; Reinecke, Helmut

    2012-12-01

    In this work the activation of transparent PS substrates by chlorosulfonation is described and their distribution in the subsurface region is analyzed. For this purpose XPS, FTIR-ATR and colorimetry have been used. It is shown that the electrophilic aromatic substitution of polystyrene in pure chlorosulfonic acid is extremely quick with complete surface coverage by chlorosulfonic groups achieved after only a 10 minute reaction time at -10 °C. It is further demonstrated that the reaction is very surface selective and that even after reaction times as long as 3 hours, the modification is limited to a layer with a thickness of less than one micron. The activated PS substrates can be further functionalized in a second step with carboxylic groups. Due to the excellent optical transparency that the samples maintain upon modification, the modified systems were successfully probed for use in ELISA assays.

  16. A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity.

    PubMed

    Liao, Ya-Fan; Hsieh, Hui-Chieh; Liu, Guang-Yaw; Hung, Hui-Chih

    2005-12-15

    A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

  17. A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon.

    PubMed

    Yang, Xue; Tong, Chunyi; Long, Ying; Liu, Bin

    2011-01-01

    A new method for assay of hSMUG1 in real-time using molecular beacon is reported. hSMUG1 could be detected linearly in the range from 0.67 nM to 10.05 nM and the detection limit is 0.168 nM. In addition, this method was applied to detect the activity of hSMUG1 in tumor cells and study kinetics. The probe with low background signal has been shown to be suitable for the real-time monitoring of hSMUG1 activity, making this a promising method of high-throughput clinical sample analysis.

  18. 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

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

    PubMed

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    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/μm(2))/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.

  20. 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

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

    PubMed

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    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/μm(2))/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

  2. Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli.

    PubMed

    Braga Goncalves, Ines; Heistermann, Michael; Santema, Peter; Dantzer, Ben; Mausbach, Jelena; Ganswindt, Andre; Manser, Marta B

    2016-01-01

    In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach. PMID:27077741

  3. Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli.

    PubMed

    Braga Goncalves, Ines; Heistermann, Michael; Santema, Peter; Dantzer, Ben; Mausbach, Jelena; Ganswindt, Andre; Manser, Marta B

    2016-01-01

    In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach.

  4. Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli

    PubMed Central

    Heistermann, Michael; Santema, Peter; Dantzer, Ben; Mausbach, Jelena; Ganswindt, Andre; Manser, Marta B.

    2016-01-01

    In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach. PMID:27077741

  5. Activity of nitro-polynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation.

    PubMed

    Nachtman, J P; Wolff, S

    1982-01-01

    Nitro-polynuclear aromatic hydrocarbons are found in diesel particulates. These compounds are potent mutagens in the Ames test. To determine whether nitro-polynuclear aromatic hydrocarbons are active in a mammalian cell assay, 1-nitropyrene, 1,8-dinitropyrene, 2-nitrofluorene, and 4-nitrobiphenyl were incubated with cultures of Chinese hamster ovary cells. The frequency of sister chromatid exchange (SCE) was measured in the presence and absence of rat liver S-9 mix. The addition of S-9 mix resulted in a large increase in the SCEs induced by all four compounds. PMID:7067667

  6. Activity of nitro-polynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation. [Hamsters

    SciTech Connect

    Nachtman, J.P.; Wolff, S.

    1982-01-01

    Nitro-polynuclear aromatic hydrocarbons are found in diesel particulates.These compounds are potent mutagens in the Ames test. To determine whether nitro-polynuclear aromatic hydrocarbons are active in a mammalian cell assay, 1-nitropyrene, 1,8-dinitropyrene, 2-nitrofluorene, and 4-nitrobiphenyl were incubated with cultures of Chinese hamster ovary cells. The frequency of sister chromatic exchange (SCE) was measured in the presence and absence of rat liver S-9 mix. The addition of S-9 mix resulted in a large increase in the SCEs induced by all four compounds.

  7. 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.

  8. 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

  9. Metabolic activation by hamster and rat hepatocytes in the Salmonella mutagenicity assay.

    PubMed

    Poiley, J A; Raineri, R; Andrews, A W; Cavanaugh, D M; Pienta, R J

    1980-12-01

    Intact and homogenized hepatocytes from untreated or Aroclor 1254-treated male and female noninbred Sprague-Dawley rats and noninbred Syrian golden hamsters were compared for their ability to metabolize chemicals in the Salmonella-mammalian microsome mutagenesis assay. The following chemicals were used: two aromatic amines, 2-amino-anthracene and N-2-fluorenylacetamide; two polycyclic aromatic hydrocarbons, 3-methylcholanthrene and benzo[a]pyrene (BP); and one nitrosamine, diethylnitrosamine (DENA). With one exception, hepatocytes from hamsters were more active than were hepatocytes from rats in the activation of these mutagens. The homogenized preparations from Aroclor 1254-treated rats were slightly more active with BP than was the equivalent hamster preparation. Intact hepatocytes from Aroclor 1254-treated hamsters were more efficient at metabolizing the aromatic amines and DENA, whereas homogenates were more effective with the hydrocarbons. Results were similar with the rat preparations, except that only large quantities of Aroclor 1254-treated intact male rat hepatocytes appeared to activate DENA. These results suggest that, in the choice of an activation system, the kind of chemical being evaluated should be considered.

  10. Can HIV reverse transcriptase activity assay be a low-cost alternative for viral load monitoring in resource-limited settings?

    PubMed Central

    Gupta, Soham; Palchaudhuri, Riya; Neogi, Ujjwal; Srinivasa, Hiresave; Ashorn, Per; De Costa, Ayesha; Källander, Clas; Shet, Anita

    2016-01-01

    Objective To evaluate the performance and cost of an HIV reverse transcriptase-enzyme activity (HIV-RT) assay in comparison to an HIV-1 RNA assay for routine viral load monitoring in resource limited settings. Design A cohort-based longitudinal study. Setting Two antiretroviral therapy (ART) centres in Karnataka state, South India, providing treatment under the Indian AIDS control programme. Participants A cohort of 327 HIV-1-infected Indian adult patients initiating first-line ART. Outcome measures Performance and cost of an HIV-RT assay (ExaVir Load V3) in comparison to a gold standard HIV-1 RNA assay (Abbott m2000rt) in a cohort of 327 Indian patients before (WK00) and 4 weeks (WK04) after initiation of first-line therapy. Results Plasma viral load was determined by an HIV-1 RNA assay and an HIV-RT assay in 629 samples (302 paired samples and 25 single time point samples at WK00) obtained from 327 patients. Overall, a strong correlation of r=0.96 was observed, with good correlation at WK00 (r=0.84) and at WK04 (r=0.77). Bland-Altman analysis of all samples showed a good level of agreement with a mean difference (bias) of 0.22 log10copies/mL. The performance of ExaVir Load V3 was not negatively affected by a nevirapine/efavirenz based antiretroviral regimen. The per test cost of measuring plasma viral load by the Abbott m2000rt and ExaVir Load V3 assays in a basic lab setting was $36.4 and $16.8, respectively. Conclusions The strong correlation between the HIV-RT and HIV-1 RNA assays suggests that the HIV-RT assay can be an affordable alternative option for monitoring patients on antiretroviral therapy in resource-limited settings. Trial registration number ISRCTN79261738. PMID:26817634

  11. Regional brain activation during meditation shows time and practice effects: an exploratory FMRI study.

    PubMed

    Baron Short, E; Kose, Samet; Mu, Qiwen; Borckardt, Jeffery; Newberg, Andrew; George, Mark S; Kozel, F Andrew

    2010-03-01

    Meditation involves attentional regulation and may lead to increased activity in brain regions associated with attention such as dorsal lateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). Using functional magnetic resonance imaging, we examined whether DLPFC and ACC were activated during meditation. Subjects who meditate were recruited and scanned on a 3.0 Tesla scanner. Subjects meditated for four sessions of 12 min and performed four sessions of a 6 min control task. Individual and group t-maps were generated of overall meditation response versus control response and late meditation response versus early meditation response for each subject and time courses were plotted. For the overall group (n = 13), and using an overall brain analysis, there were no statistically significant regional activations of interest using conservative thresholds. A region of interest analysis of the entire group time courses of DLPFC and ACC were statistically more active throughout meditation in comparison to the control task. Moreover, dividing the cohort into short (n = 8) and long-term (n = 5) practitioners (>10 years) revealed that the time courses of long-term practitioners had significantly more consistent and sustained activation in the DLPFC and the ACC during meditation versus control in comparison to short-term practitioners. The regional brain activations in the more practised subjects may correlate with better sustained attention and attentional error monitoring. In summary, brain regions associated with attention vary over the time of a meditation session and may differ between long- and short-term meditation practitioners.

  12. Male-to-female transsexuals show sex-atypical hypothalamus activation when smelling odorous steroids.

    PubMed

    Berglund, H; Lindström, P; Dhejne-Helmy, C; Savic, I

    2008-08-01

    One working hypothesis behind transsexuality is that the normal sex differentiation of certain hypothalamic networks is altered. We tested this hypothesis by investigating the pattern of cerebral activation in 12 nonhomosexual male-to-female transsexuals (MFTRs) when smelling 4,16-androstadien-3-one (AND) and estra-1,3,5(10),16-tetraen-3-ol (EST). These steroids are reported to activate the hypothalamic networks in a sex-differentiated way. Like in female controls the hypothalamus in MFTRs activated with AND, whereas smelling of EST engaged the amygdala and piriform cortex. Male controls, on the other hand, activated the hypothalamus with EST. However, when restricting the volume of interest to the hypothalamus activation was detected in MFTR also with EST, and explorative conjunctional analysis revealed that MFTR shared a hypothalamic cluster with women when smelling AND, and with men when smelling EST. Because the EST effect was limited, MFTR differed significantly only from male controls, and only for EST-AIR and EST-AND. These data suggest a pattern of activation away from the biological sex, occupying an intermediate position with predominantly female-like features. Because our MFTRs were nonhomosexual, the results are unlikely to be an effect of sexual practice. Instead, the data implicate that transsexuality may be associated with sex-atypical physiological responses in specific hypothalamic circuits, possibly as a consequence of a variant neuronal differentiation.

  13. Some Attenuated Variants of Vesicular Stomatitis Virus Show Enhanced Oncolytic Activity against Human Glioblastoma Cells relative to Normal Brain Cells▿

    PubMed Central

    Wollmann, Guido; Rogulin, Vitaliy; Simon, Ian; Rose, John K.; van den Pol, Anthony N.

    2010-01-01

    Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation. PMID:19906910

  14. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    PubMed

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne

    2014-03-15

    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  15. Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

    PubMed

    Watson, Amanda L; Chiu, Norman H L

    2016-09-01

    Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

  16. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays.

    PubMed

    Hayat, Akhtar; Cunningham, Jessica; Bulbul, Gonca; Andreescu, Silvana

    2015-07-23

    Nanomaterial-based enzyme mimics have attracted considerable interest in chemical analysis as alternative catalysts to natural enzymes. However, the conditions in which such particles can replace biological catalysts and their selectivity and reactivity profiles are not well defined. This work explored the oxidase like properties of nanoceria particles in the development of colorimetric assays for the detection of dopamine and catechol. Selectivity of the system with respect to several phenolic compounds, the effect of interferences and real sample analysis are discussed. The conditions of use such as buffer composition, selectivity, pH, reaction time and particle type are defined. Detection limits of 1.5 and 0.2μM were obtained with nanoceria for dopamine and catechol. The same assay could be used as a general sensing platform for the detection of other phenolics. However, the sensitivity of the method varies significantly with the particle type, buffer composition, pH and with the structure of the phenolic compound. The results demonstrate that nanoceria particles can be used for the development of cost effective and sensitive methods for the detection of these compounds. However, the selection of the particle system and experimental conditions is critical for achieving high sensitivity. Recommendations are provided on the selection of the particle system and reaction conditions to maximize the oxidase like activity of nanoceria. PMID:26231899

  17. A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

    PubMed

    Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

    2001-02-01

    Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.

  18. Isolation and analysis of polysaccharide showing high hyaluronidase inhibitory activity in Nostochopsis lobatus MAC0804NAN.

    PubMed

    Yamaguchi, Yuji; Koketsu, Mamoru

    2016-03-01

    An active substance with high hyaluronidase inhibitory effect was isolated from the edible cyanobacterium Nostochopsis lobatus MAC0804NAN strain and characterized. The active component in the hot water extract was purified by anion exchange and gel filtration chromatography and was found to be a polysaccharide. The IC(50) against hyaluronidase of the purified polysaccharide was 7.18 μg/ml whose inhibitory activity is 14.5 times stronger than that of disodium cromoglycate (DSCG), an anti-allergy medication. The carbohydrate composition which was analyzed by GC-MS and NMR was found to be composed mainly of glucose, glucuronic acid, fucose, 2-O-methylfucose, mannose, galactose and xylose.

  19. 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...

  20. Development of colorimetric HTS assay of cytochrome p450 for ortho-specific hydroxylation, and engineering of CYP102D1 with enhanced catalytic activity and regioselectivity.

    PubMed

    Choi, Kwon-Young; Jung, Eun-Ok; Yun, Hyungdon; Yang, Yung-Hun; Kazlauskas, Romas J; Kim, Byung-Gee

    2013-07-01

    A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examined for O-dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A-ring inactivated in order to find enhanced 3'-regioselectiviy). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L(-1) production titer and greatly increased 3'-regioselectiviy (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.

  1. A zebrafish scale assay to monitor dioxin-like activity in surface water samples.

    PubMed

    Pelayo, Sergi; López-Roldán, Ramón; González, Susana; Casado, Marta; Raldúa, Demetrio; Cortina, Jose Luis; Piña, Benjamin

    2011-10-01

    New regulations on water quality require a close control of the possible biological activities known or unexpected pollutants may bring about. We present here a protocol based on the direct exposure of zebrafish to river water and the analysis of expression of specific genes in their scales to determine the presence of compounds with dioxin-like biological activity. The method does not require the killing of animals and allows detection of the biological activity after a single day of exposure. When tested, the method with real samples from the Llobregat River, clear temporal and spatial variations were observed, demonstrating its suitability for monitoring natural variations in water quality linked to specific discharges. High biological activities were unrelated to the currently checked water quality parameters (macropollutants, turbidity, TOC, etc.), but they did correlate with the presence of micropollutants (estrogens, detergents, etc.) related to domestic and/or industrial runoffs. The scale assay therefore provides a new tool to evaluate water quality changes that cannot be easily derived from the existing standard analytical procedures. It ranks among the very few described protocols able to detect biological effects from natural water samples, without a pre-concentration step, and after only 24 h of exposure. PMID:21822775

  2. Systematic Review Shows Only Few Reliable Studies of Physical Activity Intervention in Adolescents

    PubMed Central

    Soares, Nara Michelle Moura; Leão, Arley Santos; Santos, Josivan Rosa; Monteiro, Glauber Rocha; dos Santos, Jorge Rollemberg; Thomazzi, Sara Maria; Silva, Roberto Jerônimo dos Santos

    2014-01-01

    Introduction. Several studies have pointed to the high prevalence of low levels of physical activity in adolescents, suggesting the need for more effective interventions for this group. The aim of this study was to present evidence of intervention programs for efficacy of physical activity for adolescents. Methods. Surveys in PubMed, SportDiscus, LiLacs, and SciELO databases were conducted using keywords to identify population, intervention, and outcome, as well as DeCS and MeSH terms in English, Portuguese, and Spanish, whenever appropriate. The review included observational studies with minimal intervention of six months, minimum sample size of 100 adolescents, written in any language, and those who have reached STROBE score greater than 70%. Results. Only seven studies met all inclusion criteria. Of these, five were pre- and postintervention and two had n > 2000 participants. Interventions were of several types, durations, and strategies for physical activity implementation. Behavior change was assessed in 43% of studies and three reported success in some way. Conclusion. Due to heterogeneity in their contents and methodologies, as well as the lack of jobs that accompany adolescents after the intervention period, one cannot draw conclusions about the actual effects of the intervention programs of physical activity on the behavior of young people. PMID:25152903

  3. Water Works: A Great Show on Earth. Classroom Activities for Third and Fourth Grades.

    ERIC Educational Resources Information Center

    McClure, Judy; Clark, Neil

    This curriculum guide is divided into five lessons, each containing several activities that reflect the natural path of inquiry that third or fourth grade students might take in considering the water that arrives in their bathroom sinks each morning. Starting from the familiar faucet, the students are encouraged to reflect on their own habits and…

  4. Cerato-platanin shows expansin-like activity on cellulosic materials.

    PubMed

    Baccelli, Ivan; Luti, Simone; Bernardi, Rodolfo; Scala, Aniello; Pazzagli, Luigia

    2014-01-01

    Cerato-platanin (CP) is a non-catalytic protein with a double ψβ-barrel fold located in the cell wall of the phytopathogenic fungus Ceratocystis platani. CP is released during growth and induces defence-related responses in plants. CP is also the first member of the "cerato-platanin family" (CPF) (Pfam PF07249). In the CPF, the molecular mechanism of action on plants and above all the biological role in fungal life are little-known aspects. However, an expansin-like function has recently been suggested concerning CP. Expansin-like proteins have the ability to act non-hydrolytically on cellulose. In the present work, the expansin-like activity of CP and Pop1, a CP family member, was investigated. Like expansins, CP and Pop1 were able to weaken filter paper in a concentration-dependent manner and without the production of reducing sugars. A metal-dependent polysaccharide monooxygenase-like activity was excluded. The optimum of activity was pH5.0, 38 °C. CP was also able to cause fragmentation of the crystalline cellulose Avicel and the breakage and defibration of cotton fibres. However, the interaction did not involve a stable bond with the substrates and CP did not significantly enhance the hydrolytic activity of cellulase. On the other hand, CP and Pop1 bound quickly to chitin. We consider CP as a novel one-domain expansin-like protein. We propose a structural role for CP in the fungal cell wall due to the ability to bind chitin, and hypothesize a functional role in the interaction of the fungus with the plant for the weakening activity shown on cellulose.

  5. Intervention to increase physical activity in irritable bowel syndrome shows long-term positive effects

    PubMed Central

    Johannesson, Elisabet; Ringström, Gisela; Abrahamsson, Hasse; Sadik, Riadh

    2015-01-01

    AIM: To assess the long-term effects of physical activity on irritable bowel syndrome (IBS) symptoms and on quality of life, fatigue, depression and anxiety. METHODS: Seventy-six patients from a previous randomized controlled interventional study on increased physical activity in IBS were asked to participate in this long-term follow-up study. The included patients attended one visit in which they filled out questionnaires and they underwent a submaximal cycle ergometer test. The primary end point was the change in the IBS Severity Scoring System (IBS-SSS) at baseline, i.e., before the intervention and at follow-up. The secondary endpoints were changes in quality of life, fatigue, depression and anxiety. RESULTS: A total of 39 [32 women, median age 45 (28-61) years] patients were included in this follow-up. Median follow-up time was 5.2 (range: 3.8-6.2) years. The IBS symptoms were improved compared with baseline [IBS-SSS: 276 (169-360) vs 218 (82-328), P = 0.001]. This was also true for the majority of the dimensions of psychological symptoms such as disease specific quality of life, fatigue, depression and anxiety. The reported time of physical activity during the week before the visit had increased from 3.2 (0.0-10.0) h at baseline to 5.2 (0.0-15.0) h at follow-up, P = 0.019. The most common activities reported were walking, aerobics and cycling. There was no significant difference in the oxygen uptake 31.8 (19.7-45.8) mL per min per kg at baseline vs 34.6 (19.0-54.6) mL/min per kg at follow-up. CONCLUSION: An intervention to increase physical activity has positive long-term effects on IBS symptoms and psychological symptoms. PMID:25593485

  6. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    PubMed

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms.

  7. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    PubMed

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. PMID:21674619

  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. Development of an activity assay for discovery of inhibitors of lipopolysaccharide transport.

    PubMed

    Gronenberg, Luisa S; Kahne, Daniel

    2010-03-01

    The outer membrane of gram-negative bacteria contains an outer leaflet composed of lipopolysaccharide (LPS) that is transported to this location by a pathway that is essential for viability. It has been suggested that inhibitors of this pathway could be useful antibiotics. Herein we reconstitute the activity of the ATPase component (LptB) of the ABC transporter that initiates LPS transport and assembly. We developed a high-throughput assay and screened a library of kinase inhibitors against LptB. We identified two classes of ATP-competitive inhibitors. These are the first inhibitors of the ATPase component of any bacterial ABC transporter. The small-molecule inhibitors will be very useful tools for further biochemical studies of the proteins involved in LPS transport and assembly.

  11. 5-epi-Torrubiellutin C shows antiproliferative activity on DU145 prostate cancer cells through inactivation of the AKT/mTOR pathway.

    PubMed

    Singh, Ashita; Mahipal, Bodugam; Chandrasekhar, Srivari; Ummanni, Ramesh

    2014-04-01

    Cell-based assays for evaluation of the anticancer potential of a focused small molecule library have identified a few potential hit molecules. Among the hits identified, Torrubiellutins (3a) showed good anticancer potential across the cells used in screening assays. Torrubiellutins are isolated from fungal insects Torrubiella luteorostrata and diverse pharmacological effects for these have been reported. However, it is not known as to how Torrubiellutins act through signaling pathways inhibiting the growth of eukaryotic cells. The current study aimed to determine the anticancer potential of Torrubiellutins by defining the molecular mechanism of cytotoxicity using DU145 cells. The results showed that the inhibition of prostate cancer cell growth by 3a was associated with inhibition of anchorage-independent growth, cell migration, and, to a small extent, apoptosis-mediated cell death by caspase activation. The growth-inhibitory effects of 3a are supported by inactivation of prosurvival pathways. Immunoblot analysis showed that the treatment of DU145 cells with 3a resulted in specific downregulation of AKT/mammalian target of rapamycin (mTOR) and its downstream effector proteins p70S6K, GSK3β, and STAT3. On the basis of these findings, we propose that the changes observed in the AKT/mTOR signaling axis are new targets of 3a that are involved in its inhibitory activity on the proliferation of prostate cancer cells, suggesting its potential for further investigation as a promising anticancer agent. PMID:24445589

  12. 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

  13. Conjugated polyelectrolyte based fluorescence turn-on assay for real-time monitoring of protease activity.

    PubMed

    Wang, Yanyan; Zhang, Yong; Liu, Bin

    2010-10-15

    A fluorescence "turn-on" assay for monitoring protease activity is developed on the basis of a water-soluble carboxylated polyfluorene derivative, PFP-CO₂Na, and its different fluorescence response toward cytochrome c (cyt c) and its fragments. PFP-CO₂Na is synthesized via Suzuki coupling polymerization between 2,7-dibromo-9,9-bis(3'-tert-butyl propanoate)fluorene and 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzene, followed by treatment with trifluoroacetic acid and Na₂CO₃. The fluorescence of PFP-CO₂Na can be significantly quenched by cyt c due to complexation-mediated electron transfer between the polymer and protein. Using the complex of PFP-CO₂Na/cyt c as a substrate, a real-time fluorescence turn-on assay for trypsin activity study has been developed. Addition of trypsin to the substrate solution induces gradual recovery of the fluorescence intensity for PFP-CO₂Na due to trypsin-catalyzed hydrolysis of cyt c, which dissociates the heme moiety from the polymer vicinity. The time-dependent fluorescence intensity increase of PFP-CO₂Na in the presence of trypsin allows us to derive the initial reaction rates and k(cat)/K(m) (5350 M⁻¹ s⁻¹) for trypsin-catalyzed hydrolysis. Addition of trypsin inhibitor efficiently inhibits trypsin-catalyzed hydrolysis reaction of cyt c, which leads to a decreased fluorescence turn-on response of PFP-CO₂Na.

  14. Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells.

    PubMed

    Thougaard, Annemette V; Christiansen, Joan; Mow, Tomas; Hornberg, Jorrit J

    2014-12-01

    Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96-well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell-based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell-based genotoxicity assays. The resulting dataset was used to set the relevant cut-off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well-suited for use in early drug discovery projects. Environ.

  15. 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...

  16. 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...

  17. Suramin inhibits helicase activity of NS3 protein of dengue virus in a fluorescence-based high throughput assay format.

    PubMed

    Basavannacharya, Chandrakala; Vasudevan, Subhash G

    2014-10-24

    Dengue fever is a major health concern worldwide. The virus encoded non-structural protein 3 (NS3) is a multifunctional protein endowed with protease, helicase, nucleoside triphosphatase (NTPase) and RNA 5' triphosphatase (RTPase) activities. Helicase activity of NS3 catalyzes the unwinding of double stranded polynucleotides by utilizing the energy released from ATP hydrolysis. As this activity is essential for replication, NS3 helicase represents an attractive drug target for developing a dengue antiviral drug. Here, we report fluorescence based molecular beacon helicase assay using a duplex RNA substrate that contains a fluorophore on the 5' end and a quencher on the 3' end of one of the strands. The assay was optimized with respect to several parameters and adapted to 384-well high-throughput screening format, with an average Z' factor of 0.65. Assay validation with a small diverse set library of 1600 compounds identified, suramin as a significant inhibitor of the helicase activity of NS3. Helicase activity deficient NS3 K199A was used in a counter-screen to identify compounds interfering with the assay. Suramin inhibited DENV (dengue virus) NS3 helicase activity with a Ki of 0.75±0.03μM as a non-competitive inhibitor. The molecular beacon helicase assay together with the counter screen and suramin as a tool compound can be used to identify novel inhibitors of DENV helicase.

  18. X-ray microtomography shows pore structure and tortuosity in alkali-activated binders

    SciTech Connect

    Provis, John L.; Myers, Rupert J.; White, Claire E.; Rose, Volker; Deventer, Jannie S.J. van

    2012-06-15

    Durability of alkali-activated binders is of vital importance in their commercial application, and depends strongly on microstructure and pore network characteristics. X-ray microtomography ({mu}CT) offers, for the first time, direct insight into microstructural and pore structure characteristics in three dimensions. Here, {mu}CT is performed on a set of sodium metasilicate-activated fly ash/slag blends, using a synchrotron beamline instrument. Segmentation of the samples into pore and solid regions is then conducted, and pore tortuosity is calculated by a random walker method. Segmented porosity and diffusion tortuosity are correlated, and vary as a function of slag content (slag addition reduces porosity and increases tortuosity), and sample age (extended curing gives lower porosity and higher tortuosity). This is particularly notable for samples with {>=} 50% slag content, where a space-filling calcium (alumino)silicate hydrate gel provides porosity reductions which are not observed for the sodium aluminosilicate ('geopolymer') gels which do not chemically bind water of hydration.

  19. Bacillus cereus strain S2 shows high nematicidal activity against Meloidogyne incognita by producing sphingosine

    PubMed Central

    Gao, Huijuan; Qi, Gaofu; Yin, Rong; Zhang, Hongchun; Li, Chenggang; Zhao, Xiuyun

    2016-01-01

    Plant-parasitic nematodes cause serious crop losses worldwidely. This study intended to discover the antagonistic mechanism of Bacillus cereus strain S2 against Meloidogyne incognita. Treatment with B. cereus strain S2 resulted in a mortality of 77.89% to Caenorhabditis elegans (a model organism) and 90.96% to M. incognita. In pot experiment, control efficiency of B. cereus S2 culture or supernatants were 81.36% and 67.42% towards M. incognita, respectively. In field experiment, control efficiency was 58.97% towards M. incognita. Nematicidal substances were isolated from culture supernatant of B. cereus S2 by polarity gradient extraction, silica gel column chromatography and HPLC. Two nematicidal compounds were identified as C16 sphingosine and phytosphingosine by LC-MS. The median lethal concentration of sphingosine was determined as 0.64 μg/ml. Sphingosine could obviously inhibit reproduction of C. elegans, with an inhibition rate of 42.72% for 24 h. After treatment with sphingosine, ROS was induced in intestinal tract, and genital area disappeared in nematode. Furthermore, B. cereus S2 could induce systemic resistance in tomato, and enhance activity of defense-related enzymes for biocontrol of M. incognita. This study demonstrates the nematicidal activity of B. cereus and its product sphingosine, as well provides a possibility for biocontrol of M. incognita. PMID:27338781

  20. In Vitro Analysis of Albendazole Sulfoxide Enantiomers Shows that (+)-(R)-Albendazole Sulfoxide Is the Active Enantiomer against Taenia solium

    PubMed Central

    Paredes, Adriana; de Campos Lourenço, Tiago; Marzal, Miguel; Rivera, Andrea; Dorny, Pierre; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H.; Cass, Quezia B.

    2013-01-01

    Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (−)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (−)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (−)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole. PMID:23229490

  1. In vitro analysis of albendazole sulfoxide enantiomers shows that (+)-(R)-albendazole sulfoxide is the active enantiomer against Taenia solium.

    PubMed

    Paredes, Adriana; de Campos Lourenço, Tiago; Marzal, Miguel; Rivera, Andrea; Dorny, Pierre; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E; Cass, Quezia B

    2013-02-01

    Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (-)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (-)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (-)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.

  2. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.

    PubMed

    Hageman, Jurre; van Waarde, Maria A W H; Zylicz, Alicja; Walerych, Dawid; Kampinga, Harm H

    2011-04-01

    Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.

  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. A sensitive fluorescent assay for thiamine based on metal-organic frameworks with intrinsic peroxidase-like activity.

    PubMed

    Tan, Hongliang; Li, Qian; Zhou, Zhengchen; Ma, Chanjiao; Song, Yonghai; Xu, Fugang; Wang, Li

    2015-01-26

    Metal-organic frameworks (MOFs) with tunable structures and properties have recently been emerged as very interesting functional materials. However, the catalytic properties of MOFs as enzymatic mimics remain to be further investigated. In this work, we for the first time demonstrated the peroxidase-like activity of copper-based MOFs (HKUST-1) by employing thiamine (TH) as a peroxidase substrate. In the presence of H2O2, HKUST-1 can catalyze efficiently the conversion of non-fluorescent TH to strong fluorescent thiochrome. The catalytic activity of HKUST-1 is highly dependent on the temperature, pH and H2O2 concentrations. As a peroxidase mimic, HKUST-1 not only has the features of low cost, high stability and easy preparation, but also follows Michaelis-Menten behaviors and shows stronger affinity to TH than horseradish peroxidase (HRP). Based on the peroxidase-like activity of HKUST-1, a simple and sensitive fluorescent method for TH detection has been developed. As low as 1 μM TH can be detected with a linear range from 4 to 700 μM. The detection limit for TH is about 50 fold lower than that of HRP-based fluorescent assay. The proposed method was successfully applied to detect TH in tablets and urine samples and showed a satisfactory result. We believed that the present work could improve the understanding of catalytic behaviors of MOFs as enzymatic mimics and find out a wider application in bioanalysis.

  5. Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds.

    PubMed

    Bracke, Marc E; Roman, Bart I; Stevens, Christian V; Mus, Liselot M; Parmar, Virinder S; De Wever, Olivier; Mareel, Marc M

    2015-01-01

    The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account

  6. Montmorillonite enhanced ciprofloxacin transport in saturated porous media with sorbed ciprofloxacin showing antibiotic activity

    NASA Astrophysics Data System (ADS)

    Chen, Hao; Gao, Bin; Yang, Liu-Yan; Ma, Lena Q.

    2015-02-01

    Antibiotic ciprofloxacin (CIP) is immobile in the subsurface but it has been frequently detected in the aquatic system. Therefore it is important to investigate the factors impacting CIP's mobilization in aquifer. Laboratory columns packed with sand were used to test colloid-facilitated CIP transport by 1) using kaolinite or montmorillonite to mobilize presorbed-CIP in a column or 2) co-transporting with CIP by pre-mixing them before transport. The Langmuir model showed that CIP sorption by montmorillonite (23 g kg- 1) was 100 times more effective than sand or kaolinite. Even with strong CIP complexation ability to Fe/Al coating on sand surface, montmorillonite promoted CIP transport, but not kaolinite. All presorbed-CIP by sand was mobilized by montmorillonite after 3 pore volumes through co-transporting of CIP with montmorillonite. The majority of CIP was fixed onto the montmorillonite interlayer but still showed inhibition of bacteria growth. Our results suggested that montmorillonite with high CIP sorption ability can act as a carrier to enhance CIP's mobility in aquifer.

  7. Montmorillonite enhanced ciprofloxacin transport in saturated porous media with sorbed ciprofloxacin showing antibiotic activity.

    PubMed

    Chen, Hao; Gao, Bin; Yang, Liu-Yan; Ma, Lena Q

    2015-02-01

    Antibiotic ciprofloxacin (CIP) is immobile in the subsurface but it has been frequently detected in the aquatic system. Therefore it is important to investigate the factors impacting CIP's mobilization in aquifer. Laboratory columns packed with sand were used to test colloid-facilitated CIP transport by 1) using kaolinite or montmorillonite to mobilize presorbed-CIP in a column or 2) co-transporting with CIP by pre-mixing them before transport. The Langmuir model showed that CIP sorption by montmorillonite (23gkg(-1)) was 100 times more effective than sand or kaolinite. Even with strong CIP complexation ability to Fe/Al coating on sand surface, montmorillonite promoted CIP transport, but not kaolinite. All presorbed-CIP by sand was mobilized by montmorillonite after 3 pore volumes through co-transporting of CIP with montmorillonite. The majority of CIP was fixed onto the montmorillonite interlayer but still showed inhibition of bacteria growth. Our results suggested that montmorillonite with high CIP sorption ability can act as a carrier to enhance CIP's mobility in aquifer. PMID:25528132

  8. Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants.

    PubMed

    Broothaerts, W; McPherson, J; Li, B; Randall, E; Lane, W D; Wiersma, P A

    2000-12-01

    A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed. PMID:11141262

  9. Assay of Chilling Injury in Wild and Domestic Tomatoes Based on Photosystem Activity of the Chilled Leaves

    PubMed Central

    Smillie, Robert M.; Nott, Robyn

    1979-01-01

    Tomato leaves were detached and stored at 0 C for various periods of time. Chloroplasts were isolated from the leaves and their photoreductive activities were determined. Comparisons were made between two altitudinal forms of the wild tomato Lycopersicon hirsutum Humb. and Bonpl. (a tropical lowlands form and a highlands form adapted to growth at 3,100 meters), and two cultivars of the domestic tomato L. esculentum Mill. In each case the capacity of the isolated chloroplasts to photoreduce ferricyanide declined linearly with time of storage of the leaves at 0 C, but not at 10 C. This injury developed more slowly in the high altitudinal form of the wild tomato compared with the low altitudinal form and the two domestic cultivars indicating an enhanced resistance toward chilling injury in the tomato from 3,100 meters. Chloroplast activity declined in green tomato fruit held at 0 C, at about the same rate as in the chilled leaves. Measurements of photochemical activities in the isolated chloroplasts and in vivo measurements of cytochrome-554 photooxidation in chilled leaves showed that the site of action of the chilling effect was water donation to photosystem II. The chilling-induced impairment of photoreductive activity in chloroplasts provides a useful assay for detecting and measuring differences in the susceptibility of plants to chilling injury. PMID:16660815

  10. 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

  11. Downscaling CMIP5 climate models shows increased tropical cyclone activity over the 21st century

    PubMed Central

    Emanuel, Kerry A.

    2013-01-01

    A recently developed technique for simulating large [O(104)] numbers of tropical cyclones in climate states described by global gridded data is applied to simulations of historical and future climate states simulated by six Coupled Model Intercomparison Project 5 (CMIP5) global climate models. Tropical cyclones downscaled from the climate of the period 1950–2005 are compared with those of the 21st century in simulations that stipulate that the radiative forcing from greenhouse gases increases by over preindustrial values. In contrast to storms that appear explicitly in most global models, the frequency of downscaled tropical cyclones increases during the 21st century in most locations. The intensity of such storms, as measured by their maximum wind speeds, also increases, in agreement with previous results. Increases in tropical cyclone activity are most prominent in the western North Pacific, but are evident in other regions except for the southwestern Pacific. The increased frequency of events is consistent with increases in a genesis potential index based on monthly mean global model output. These results are compared and contrasted with other inferences concerning the effect of global warming on tropical cyclones. PMID:23836646

  12. Downscaling CMIP5 climate models shows increased tropical cyclone activity over the 21st century.

    PubMed

    Emanuel, Kerry A

    2013-07-23

    A recently developed technique for simulating large [O(10(4))] numbers of tropical cyclones in climate states described by global gridded data is applied to simulations of historical and future climate states simulated by six Coupled Model Intercomparison Project 5 (CMIP5) global climate models. Tropical cyclones downscaled from the climate of the period 1950-2005 are compared with those of the 21st century in simulations that stipulate that the radiative forcing from greenhouse gases increases by over preindustrial values. In contrast to storms that appear explicitly in most global models, the frequency of downscaled tropical cyclones increases during the 21st century in most locations. The intensity of such storms, as measured by their maximum wind speeds, also increases, in agreement with previous results. Increases in tropical cyclone activity are most prominent in the western North Pacific, but are evident in other regions except for the southwestern Pacific. The increased frequency of events is consistent with increases in a genesis potential index based on monthly mean global model output. These results are compared and contrasted with other inferences concerning the effect of global warming on tropical cyclones.

  13. Hyperthermostable Thermotoga maritima xylanase XYN10B shows high activity at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids.

    PubMed

    Yu, Tianyi; Anbarasan, Sasikala; Wang, Yawei; Telli, Kübra; Aslan, Aşkın Sevinç; Su, Zhengding; Zhou, Yin; Zhang, Li; Iivonen, Piia; Havukainen, Sami; Mentunen, Tero; Hummel, Michael; Sixta, Herbert; Binay, Baris; Turunen, Ossi; Xiong, Hairong

    2016-07-01

    The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15-35 % hydrophilic ILs, even at 75-90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15-25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower V max. However, only minor changes in kinetic parameter K m showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential. PMID:27240671

  14. Yeast DEL assay detects clastogens.

    PubMed

    Kirpnick, Zhanna; Homiski, Michael; Rubitski, Elizabeth; Repnevskaya, Marina; Howlett, Niall; Aubrecht, Jiri; Schiestl, Robert H

    2005-04-01

    Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in

  15. 2,2'-Azobis(isobutyronitrile)-derived alkylperoxyl radical scavenging activity assay of hydrophilic antioxidants by employing EPR spin trap method

    PubMed Central

    Kohri, Shunji; Fujii, Hirotada

    2013-01-01

    As interest in the study of antioxidant intake from foods and other agricultural products increases, methods for performing radical scavenging activity assays based on the electron paramagnetic resonance spectroscopic method, in which there is no interference from the sample color and turbidity, are required. In this study, we have developed a rapid and simple electron paramagnetic resonance based assay to evaluate the alkylperoxyl radical scavenging activity of several antioxidants. The alkylperoxyl radical species was generated by the photolysis of azo-radical initiator 2,2'-azobis(isobutyronitrile), in which the radical generation rate and period were controlled by the illumination light. The relative alkylperoxyl radical scavenging activity was obtained by a simple formula of competing reaction of antioxidant and spin trap toward the oxygen radical. The scavenging activities toward alkylperoxyl radical and alkoxy radical species were evaluated in six antioxidants. Although quercetin showed the highest activity toward both radicals, the order of the relative activities in the other antioxidants was different mutually between the alkylperoxyl radical and the alkoxyl radical. This alkylperoxyl radical scavenging activity assay based on electron paramagnetic resonance spectroscopy is useful for evaluation of colored and turbid food samples. PMID:24249966

  16. 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.

  17. 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

  18. 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

  19. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

    PubMed Central

    Cederbaum, Arthur I.

    2014-01-01

    The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed. PMID:25454746

  20. Detection of sodium channel activators by a rapid fluorimetric microplate assay.

    PubMed

    Louzao, M C; Vieytes, M R; Yasumoto, T; Botana, L M

    2004-04-01

    Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, time-consuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i). the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potential-dependent, and (ii). dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.

  1. 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

  2. Exonuclease I manipulating primer-modified gold nanoparticles for colorimetric telomerase activity assay.

    PubMed

    Zhang, Lei; Zhang, Sijin; Pan, Wei; Liang, Qingcheng; Song, Xingyu

    2016-03-15

    Telomerase is a widely accepted cancer biomarker. The conventional method for telomerase activity assay, the telomeric repeat amplification protocol (TRAP), is time-consuming and susceptible to contaminants. Therefore, development of simple and sensitive strategies for telomerase detection is still a challenging subject. Here we develop a highly sensitive method for telomerase detection based on primer-modified gold nanoparticles (GNPs) manipulated by exonuclease I (Exo I). In the absence of telomerase, Exo I digests the substrate nucleic acid on the surface of GNPs, inducing the GNPs' aggregation. In the presence of telomerase, the telomerase elongation products which fold into G-quadruplex are resistant to the digestion of Exo I, and protect the GNPs from aggregation. By using this method, we can detect telomerase activity in 100 HL-60 cancer cells mL(-1) by naked eyes, and the detection limit is 29 HL-60 cells mL(-1). This method is very simple and reliable, without any separation and amplification procedure. We also demonstrate the feasibility of this protocol for screening of telomerase inhibitors as anticancer agents. This method is promising to be applied in early clinical diagnosis and drug discovery. PMID:26402592

  3. Nondestructive assay of fission products in spent-fuel assemblies using gamma and photoneutron activation

    NASA Astrophysics Data System (ADS)

    Lakosi, L.; Veres, Á.

    1990-12-01

    Hard γ-radiation (above 1.078 MeV) from spent reactor fuel was detected by means of excitation of 115In to its 4.5 h half-life metastable state induced by the (γ, γ') reaction and subsequent counting of the 336 keV isomeric transition. Resonance-energy quanta were produced by Compton scattering in the source, i.e. the spent fuel itself. The sensitivity of the activation method above 1.67 MeV γ-energy was enhanced by introducing a Be photoneutron converter in order to produce neutrons for exploiting their much larger activation cross sections. For short cooling times (10-40 d) the hard-γ signature of the fuel was due to the fission product 140Ba 140La, detection of which facilitated monitoring of the reactor power which existed in the core just before reactor shutdown. A linear relationship was found between the γ-signal and the fissile content in the fuel. For 100-1000 d cooled fuel the 144Ce 144Pr content could be detected, which was only sensitive to the cooling time. Spent-fuel assemblies of both a research and a power reactor were assayed by these novel methods for reactor operational and nuclear-material safeguard purposes.

  4. Alkaline phosphatase activity: new assay for the Reflotron system. Results of the evaluation in eight clinical laboratories.

    PubMed

    Schumann, G; Dominick, H C; Hellmann, D; Klauke, R; Möckesch, M; Stekel, H; von Schenck, H; Kraft, M; Nagel, R; Hänseler, E

    2001-01-01

    A new reagent carrier, Reflotron ALP, has been developed for the Reflotron system, allowing easy and rapid measurement (in less than 3 minutes) of alkaline phosphatase (ALP) activity in capillary blood, venous blood, heparinized plasma or serum. The evaluation of the analytical performance of the assay was carried out at eight clinical laboratories. The study of the imprecision using the measurements in human samples resulted in coefficients of variation ranging from 1.3% to 4.6% (within-run) and from 3.2% to 4.0% (day-to-day). The analytical specificity of the Reflotron ALP assay agrees well with ALP methods using a N-methyl-D-glucamine buffer solution. The calibration of the Reflotron ALP assay, however, is related to the reference intervals for ALP methods using a diethanolamine buffer solution. Method comparisons were performed with the ALP method on Hitachi instruments using diethanolamine buffer. Reflotron ALP measurements in blood and plasma in 157 randomly selected split samples showed excellent agreement (slope: 0.99; intercept: 0.7 U/l; median bias: 2.3%; median difference from the comparison method: -0.3%). Specimens from pregnant women and adolescents were excluded from this study. Differing values were obtained in a method comparison using 48 samples containing predominantly the ALP bone isoform (slope: 0.81; intercept: 31.5 U/l; median bias: 5.7%; median difference from the comparison method: -12.2%). Regression analysis of the results from 21 sera with prevailing placental ALP gave a slope of 1.51, and an intercept of -41.1 U/l (median bias: 8.6%; median difference from the comparison method: 35.6%). Reflotron ALP was compared with three different wet chemistry procedures using different buffer compounds: N-methyl-D-glucamine or diethanolamine or 2-amino-2-methyl-1-propanol. In samples containing predominantly ALP isoforms not of liver origin, the measurements with N-methyl-D-glucamine buffer gave the best fit with respect to Reflotron. In an

  5. 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

    -positive individuals with low CD4 counts who are seriously ill, LF-LAM may help with the diagnosis of TB. PLAIN LANGUAGE SUMMARY The lateral flow urine lipoarabinomannan (LF-LAM) test for diagnosis of tuberculosis in people living with human immunodeficiency virus (HIV) Background Tuberculosis (TB) is a common cause of death in people with human immunodeficiency virus (HIV) infection, but diagnosis is difficult, and depends on testing for TB in the sputum and other sites, which may take weeks to give results. A rapid and accurate point-of-care test could reduce delays in diagnosis, allow treatment to start promptly, and improve linkage between diagnosis and treatment. Test evaluated by this review The lateral flow urine lipoarabinomannan assay (LF-LAM, Alere Determine™ TB LAM Ag, Alere Inc, Waltham, MA, USA) is a commercially available point-of-care test for active TB (pulmonary and extrapulmonary TB). The test detects lipoarabinomannan (LAM), a component of the bacterial cell walls, which is present in some people with active TB. The test is performed by placing urine on one end of a test strip, with results appearing as a line (that is, a band) on the strip if TB is present. The test is simple, requires no special equipment, and shows results in 25 minutes. During the period we conducted the review, the manufacturer issued new recommendations for defining a positive test. We collected data based on both the original and the new recommendations Objectives We aimed to see how accurately LF-LAM diagnosed TB in people living with HIV with TB symptoms, and how accurately LF-LAM diagnosed TB in people living with HIV being screened for TB whether or not they had TB symptoms. Main results We examined evidence up to 5 February 2015 and included 12 studies: six studies evaluated LF-LAM for TB diagnosis and six studies evaluated the test for TB screening. All studies were conducted in low- or middle-income countries. Quality of the evidence We assessed quality by describing how participants

  6. Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads.

    PubMed

    Wu, Ding; Nair-Gill, Evan; Sher, Dorie A; Parker, Laurie L; Campbell, Jennifer M; Siddiqui, Mariah; Stock, Wendy; Kron, Stephen J

    2005-12-01

    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.

  7. 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

  8. 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

  9. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    PubMed

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  10. Isolation and Identification of Twelve Metabolites of Isocorynoxeine in Rat Urine and their Neuroprotective Activities in HT22 Cell Assay

    PubMed Central

    Qi, Wen; Chen, Fangfang; Sun, Jiahong; Simpkins, James W.; Yuan, Dan

    2015-01-01

    Isocorynoxeine, one of the major alkaloids from Uncaria Hook, shows the effects of lowering blood pressure, vasodilatation, and protection against ischemia-induced neuronal damage. In this paper, the metabolism of isocorynoxeine was investigated in rats. Twelve metabolites and the parent drug were isolated by using solvent extraction and repeated chromatographic methods, and determined by spectroscopic methods including UV, MS, NMR, and CD experiments. Seven new compounds were identified as 11-hydroxyisocorynoxeine, 5-oxoisocorynoxeinic acid-22-O-β-D-glucuronide, 10-hydroxyisocorynoxeine, 17-O-demethyl-16,17-dihydro-5-oxoisocorynoxeine, 5-oxoisocorynoxeinic acid, 21-hydroxy-5-oxoisocorynoxeine, and oxireno[18,19]-5-oxoisocorynoxeine, together with six known compounds identified as isocorynoxeine, 18,19-dehydrocorynoxinic acid, 18,19-dehydrocorynoxinic acid B, corynoxeine, isocorynoxeine-N-oxide, and corynoxeine-N-oxide. Possible metabolic pathways of isocorynoxeine are proposed. Furthermore, the activity assay for the parent drug and some of its metabolites showed that isocorynoxeine exhibited a significant neuroprotective effect against glutamate-induced HT22 cell death at the maximum concentration. However, little or weak neuroprotective activities were observed for M-3, M-6, M-7, and M-10. Our present study is important to further understand their metabolic fate and disposition in humans. PMID:25519834

  11. Urolithins, intestinal microbial metabolites of Pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay.

    PubMed

    Bialonska, Dobroslawa; Kasimsetty, Sashi G; Khan, Shabana I; Ferreira, Daneel

    2009-11-11

    Many health benefits of pomegranate products have been attributed to the potent antioxidant action of their tannin components, mainly punicalagins and ellagic acid. While moving through the intestines, ellagitannins are metabolized by gut bacteria into urolithins that readily enter systemic circulation. In this study, the antioxidant properties of seven urolithin derivatives were evaluated in a cell-based assay. This method is biologically more relevant because it reflects bioavailability of the test compound to the cells, and the antioxidant action is determined in the cellular environment. Our results showed that the antioxidant activity of urolithins was correlated with the number of hydroxy groups as well as the lipophilicity of the molecule. The most potent antioxidants are urolithins C and D with IC(50) values of 0.16 and 0.33 microM, respectively, when compared to IC(50) values of 1.1 and 1.4 microM of the parent ellagic acid and punicalagins, respectively. The dihydroxylated urolithin A showed weaker antioxidant activity, with an IC(50) value 13.6 microM, however, the potency was within the range of urolithin A plasma concentrations. Therefore, products of the intestinal microbial transformation of pomegranate ellagitannins may account for systemic antioxidant effects.

  12. Three-stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant.

    PubMed

    Mielicki, W P; Gordon, S G

    1993-06-01

    The cysteine proteinase, cancer procoagulant (CP; EC 3.4.22.26) was isolated from human amnion-chorion and purified by precipitation with polyethylene glycol and either ion exchange or immunoaffinity chromatography. A new, sensitive, three-stage chromogenic assay was developed for determination of CP factor X-activating activity. Using this assay some properties including dose-response, effect of calcium, phospholipid and pH on the activation of factor X by CP was determined. There was an excellent linear correlation (r2 = 0.99) between concentration and the enzymatic activity of CP. The activation of factor X by purified CP was calcium dependent with an optimum calcium concentration of 7 mM. CP was not phospholipid dependent. There was a rather broad pH optimum between pH 6.9 and 7.25 for the activation of factor X by CP.

  13. Toxicity effect of dichlorvos on loach (Misgurnus anguillicaudatus) assessed by micronucleus test, hepatase activity analysis and comet assay.

    PubMed

    Nan, Ping; Yan, Shuaiguo; Li, Li; Chen, Jianjun; Du, Qiyan; Chang, Zhongjie

    2015-06-01

    Pesticides and other chemicals at environmental concentrations often have detrimental effects. Many aquatic species are particularly threatened because of their susceptibility and also because water environment are often polluted. This study preliminarily evaluated the toxicity effect of dichlorvos (DDVP) on loach (Misgurnus anguillicaudatus) using the methods of micronucleus (MN) test, hepatase activity and comet assay. The tested results showed that indeed very little DDVP had strong toxicity effect on loach and its 50% lethal concentration (LC50) at 24 h, 48 h and 96 h was 8.38 μg l(-1), 7.168 μg l(-1) and 6.411 μg l(-1), respectively; The glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activity of loach liver decreased; meanwhile, the GPT and GOT activity of loach serum, the MN rate (‰) and three comet parameters of tested fish increased with the increase in the treatment concentration and treatment time of DDVP, and there was significant difference between control group and each treatment group (p < 0.05). These results suggested that DDVP residues might become toxic chemical contaminant in environment and would threaten aquatic and other organisms.

  14. 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

  15. 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

  16. 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.

  17. Development of a new catalase activity assay for biological samples using optical CUPRAC sensor.

    PubMed

    Bekdeşer, Burcu; Özyürek, Mustafa; Güçlü, Kubilay; Alkan, Fulya Üstün; Apak, Reşat

    2014-11-11

    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.

  18. Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays.

    PubMed

    Kintzios, Spiridon; Papageorgiou, Katerina; Yiakoumettis, Iakovos; Baricevic, Dea; Kusar, Anita

    2010-11-01

    We investigated the antioxidant activity of methanolic and water extracts of Slovene accessions of four medicinal plant species (Salvia officinalis, Achillea millefolium, Origanum vulgare subsp. vulgare and Gentiana lutea). Their free radical-scavenging activity against the DPPH. free radical was studied with a spectrophotometric assay, while their biological activity with the help of a laboratory-made biosensor based on immobilized fibroblast cells (assay duration: 3 min). The observed antioxidant activity of the extracts from the four investigated medicinal plant species was dependent on both the solvent used for extraction and the assay method (conventional or biosensor-based). Independently from the assay method and the solvent used for extraction, the lowest scavenging activity was observed in root extracts of G. lutea. Treatment of the immobilized cells with the plant extracts resulted in an increase of the cell membrane potential (membrane hyperpolarization), possibly due to the reduction of membrane damage due to oxidation. The novel cell biosensor could be utilized as a rapid, high throughput tool for screening the antioxidant properties of plant-derived compounds.

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

    PubMed

    Pohanka, Miroslav

    2015-06-11

    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.

  20. 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

  1. A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

    PubMed Central

    2014-01-01

    Background Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative. Results Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency. Conclusions Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research. PMID:24694320

  2. Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

    PubMed

    Gilmore, Marcella A; Williams, Dudley; Okawa, Yumiko; Holguin, Bret; James, Nicholas G; Ross, Justin A; Roger Aoki, K; Jameson, David M; Steward, Lance E

    2011-06-01

    The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

  3. Candida tropicalis from veterinary and human sources shows similar in vitro hemolytic activity, antifungal biofilm susceptibility and pathogenesis against Caenorhabditis elegans.

    PubMed

    Brilhante, Raimunda Sâmia Nogueira; Oliveira, Jonathas Sales de; Evangelista, Antônio José de Jesus; Serpa, Rosana; Silva, Aline Lobão da; Aguiar, Felipe Rodrigues Magalhães de; Pereira, Vandbergue Santos; Castelo-Branco, Débora de Souza Collares Maia; Pereira-Neto, Waldemiro Aquino; Cordeiro, Rossana de Aguiar; Sidrim, José Júlio Costa; Rocha, Marcos Fábio Gadelha

    2016-08-30

    The aim of this study was to evaluate the in vitro hemolytic activity and biofilm antifungal susceptibility of veterinary and human Candida tropicalis strains, as well as their pathogenesis against Caenorhabditis elegans. Twenty veterinary isolates and 20 human clinical isolates of C. tropicalis were used. The strains were evaluated for their hemolytic activity and biofilm production. Biofilm susceptibility to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin was assessed using broth microdilution assay. The in vivo evaluation of strain pathogenicity was investigated using the nematode C. elegans. Hemolytic factor was observed in 95% of the strains and 97.5% of the isolates showed ability to form biofilm. Caspofungin and amphotericin B showed better results than azole antifungals against mature biofilms. Paradoxical effect on mature biofilm metabolic activity was observed at elevated concentrations of caspofungin (8-64μg/mL). Azole antifungals were not able to inhibit mature C. tropicalis biofilms, even at the higher tested concentrations. High mortality rates of C. elegans were observed when the worms were exposed to with C. tropicalis strains, reaching up to 96%, 96h after exposure of the worms to C. tropicalis strains. These results reinforce the high pathogenicity of C. tropicalis from veterinary and human sources and show the effectiveness of caspofungin and amphotericin B against mature biofilms of this species. PMID:27527785

  4. Dual-readout fluorescent assay of protein kinase activity by use of TiO2-coated magnetic microspheres.

    PubMed

    Bai, Jie; Zhao, Yunjie; Wang, Zhibin; Liu, Chenghui; Wang, Yucong; Li, Zhengping

    2013-05-01

    A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO2 layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science. PMID:23581884

  5. 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...

  6. Lack of DNA-damaging activity of five non-nutritive sweeteners in the rat hepatocyte/DNA repair assay.

    PubMed

    Jeffrey, A M; Williams, G M

    2000-04-01

    The non-nutritive sweeteners acesulfame-K, aspartame, cyclamate, saccharin and sucralose were tested for DNA damaging activity in the rat hepatocyte/DNA repair assay. Using hepatocytes from F344 and Sprague-Dawley male rats, all were inactive despite strong responses for the positive control, 2-aminofluorene.

  7. 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

  8. 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

  9. 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...

  10. Endocrine activity of persistent organic pollutants accumulated in human silicone implants--Dosing in vitro assays by partitioning from silicone.

    PubMed

    Gilbert, Dorothea; Mayer, Philipp; Pedersen, Mikael; Vinggaard, Anne Marie

    2015-11-01

    Persistent organic pollutants (POPs) accumulated in human tissues may pose a risk for human health by interfering with the endocrine system. This study establishes a new link between actual human internal POP levels and the endocrine active dose in vitro, applying partitioning-controlled dosing from silicone to the H295R steroidogenesis assay: (1) Measured concentrations of POPs in silicone breast implants were taken from a recent study and silicone disks were loaded according to these measurements. (2) Silicone disks were transferred into H295R cell culture plates in order to control exposure of the adrenal cells by equilibrium partitioning. (3) Hormone production of the adrenal cells was measured as toxicity endpoint. 4-Nonylphenol was used for method development, and the new dosing method was compared to conventional solvent-dosing. The two dosing modes yielded similar dose-dependent hormonal responses of H295R cells. However, with the partitioning-controlled freely dissolved concentrations (Cfree) as dose metrics, dose-response curves were left-shifted by two orders of magnitude relative to spiked concentrations. Partitioning-controlled dosing of POPs resulted in up to 2-fold increases in progestagen and corticosteroid levels at Cfree of individual POPs in or below the femtomolar range. Silicone acted not only as source of the POPs but also as a sorption sink for lipophilic hormones, stimulating the cellular hormone production. Methodologically, the study showed that silicone can be used as reference partitioning phase to transfer in vivo exposure in humans (silicone implants) to in vitro assays (partition-controlled dosing). The main finding was that POPs at the levels at which they are found in humans can interfere with steroidogenesis in a human adrenocortical cell line. PMID:26264162

  11. Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay.

    PubMed

    Liman, Recep; Ciğerci, İbrahim Hakkı; Öztürk, Nur Serap

    2015-02-01

    Imazethapyr (IM) is an imidazolinone herbicide that is currently used for broad-spectrum weed control in soybean and other legume crops. In this study, cytotoxic and genotoxic effects of IM were investigated by using mitotic index (MI), mitotic phases, chromosomal abnormalities (CAs) and DNA damage on the root meristem cells of Allium cepa. In Allium root growth inhibition test, EC50 value was determined as 20 ppm, and 0.5xEC50, EC50 and 2xEC50 concentrations of IM herbicide were introduced to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 mg/L) were used as a negative and positive control, respectively. As A. cepa cell cycle is 24 hours, so, application process was carried out for 24, 48, 72 and 96 hours. All the applied doses decreased MIs compared to control group and these declines were found to be statistically meaningful. Analysis of the chromosomes showed that 10 ppm IM except for 48 h induced CAs but 40 ppm IM except for 72 h decreased CAs. DNA damage was found significantly higher in 20 and 40 ppm of IM compared to the control in comet assay. These results indicated that IM herbicide exhibits cytotoxic activity but not genotoxic activity (except 10 ppm) and induced DNA damage in a dose dependent manner in A. cepa root meristematic cells. PMID:25752428

  12. The polysaccharide isolated from Pleurotus nebrodensis (PN-S) shows immune-stimulating activity in RAW264.7 macrophages.

    PubMed

    Cui, Hai-Yan; Wang, Chang-Lu; Wang, Yu-Rong; Li, Zhen-Jing; Zhang, Ya-Nan

    2015-05-01

    A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.

  13. Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

    PubMed

    Jensen, T H; Neville, M; Rain, J C; McCarthy, T; Legrain, P; Rosbash, M

    2000-11-01

    Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

  14. Application of active and passive neutron non destructive assay methods to concrete radioactive waste drums

    NASA Astrophysics Data System (ADS)

    Jallu, F.; Passard, C.; Brackx, E.

    2011-09-01

    This paper deals with the application of non-destructive neutron measurement methods to control and characterize 200 l radioactive waste drums filled with a concrete matrix. Due to its composition, and particularly to hydrogen, concrete penalizes the use of such methods to quantify uranium (U) and plutonium (Pu) components, which are mainly responsible of the α-activity of the waste. The determination of the alpha activity is the main objective of neutron measurements, in view to verify acceptance criteria in surface storage. Calibration experiments of the Active Neutron Interrogation (ANI) method lead to Detection Limit Masses (DLM) of about 1 mg of 239Pu eff in the total counting mode, and of about 10 mg of 239Pu eff in the coincidence counting mode, in case of a homogeneous Pu source and measurement times between one and two hours. Monte Carlo calculation results show a very satisfactory agreement between experimental values and calculated ones. Results of the application of passive and active neutron methods to control two real drums are presented in the last part of the paper. They show a good agreement between measured data and values declared by the waste producers. The main difficulties that had to be overcome are the low neutron signal in passive and active coincidence counting modes due to concrete, the analysis of the passive neutron signal in presence of 244Cm in the drum, which is a strong spontaneous fission neutron emitter, the variation of the active background with the concrete composition, and the analysis of the active prompt neutron signal due to the simultaneous presence of U and Pu in the drums.

  15. Comparison of tetrazolium salt assays for evaluation of drug activity against Leishmania spp.

    PubMed

    Ginouves, Marine; Carme, Bernard; Couppie, Pierre; Prevot, Ghislaine

    2014-06-01

    In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)-for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

  16. Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

    PubMed Central

    Ginouves, Marine; Carme, Bernard; Couppie, Pierre

    2014-01-01

    In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

  17. Antioxidant activity measured in different solvent fractions obtained from Mentha spicata Linn.: an analysis by ABTS*+ decolorization assay.

    PubMed

    Arumugam, Ponnan; Ramamurthy, Perumal; Santhiya, Sathiyavedu Thyagarajan; Ramesh, Arabandi

    2006-01-01

    Antioxidant compounds are abundantly available in plants and play an important role in scavenging free radicals, thus providing protection to humans against oxidative DNA damage. Mentha spicata Linn., commonly called spearmint, belongs to the family lamiaceae. It was selected in the present study because Mentha extracts have antioxidant properties due to the presence of eugenol, caffeic acid, rosmarinic acid and alpha-tocopherol. Four solvent fractions (hexane, chloroform, ethyl acetate and water) of ethanolic extract of dried leaves powder of M. spicata were analyzed for total antioxidant activity (TAA) and relative antioxidant activity (RAA) and compared with standard antioxidants such as Quercetin, beta-carotene, L-ascorbic acid and glutathione using ABTS*+ decolorization assay (ABTS/Potassium persulphate). The antioxidant activity was assumed to be from the total phenolic content of the ethanolic extract. Total phenolics are found to be highest in ethyl acetate fraction (54 mg/g) and least in hexane fraction (13 mg/g) and more or less similar in water and chloroform fractions (30-32 mg/g). TAA is found to be less in hexane and chloroform fractions (<53% at 50 microg/ml) and highest in ethyl acetate (95% at 20 microg/ml) and water (84% at 30 microg/ml) fractions. The RAA of ethyl acetate fraction is 1.1 compared to quercetin (at 5 microM/ml), but greater when compared to beta-carotene (15 microM/ml), L-ascorbic acid (15 microM/ml) and glutathione (15 microM/ml). The RAAs with these antioxidants are in the range of 1.31 -1.6. The values of RAAs for water fraction also show similar trend and are in the range of 1.0-1.4. The antioxidant activities of the solvent factions are closely related to the content of total phenolics present in them.

  18. Sesquiterpene lactone containing extracts from two cultivars of forage chicory (Cichorium intybus) show distinctive chemical profiles and in vitro activity against Ostertagia ostertagi

    PubMed Central

    Peña-Espinoza, Miguel; Boas, Ulrik; Williams, Andrew R.; Thamsborg, Stig M.; Simonsen, Henrik T.; Enemark, Heidi L.

    2015-01-01

    The study investigated direct anthelmintic effects of sesquiterpene lactones (SL)-containing extracts from forage chicory against free-living and parasitic stages of Ostertagia ostertagi. Freeze-dried leaves from chicory cultivars ‘Spadona’ and ‘Puna II’ were extracted using methanol/water. Total SL were further fractionated by solid-phase extraction and resulting extracts were characterised by high-performance liquid chromatography (HPLC). O. ostertagi eggs from faeces of mono-infected calves were hatched and L1 were used in a larval feeding inhibition assay (LFIA), while cultured L3 were used in a larval exsheathment inhibition assay (LEIA). Adult worms were immediately recovered after slaughter and used for motility inhibition assays (AMIA). Electron microscopy (EM) was performed on adult O. ostertagi exposed to 1000 μg extract mL−1 of both chicory cultivars. In all assays, decreasing concentrations of SL-containing extracts in PBS (1% DMSO) were tested in replicates with 1% DMSO in PBS as negative controls. HPLC demonstrated similar concentrations of most SL in both extracts. However, Spadona-extract contained significantly higher concentrations of 11, 13-dihydro-8-deoxylactucin (P = 0.01), while Puna II-extract had increased levels of 11, 13-dihydrolactucin (P < 0.0001). In the LFIA, both extracts reduced larval feeding at increasing concentrations, but Spadona-extract showed higher potency confirmed by significantly lower EC50 (P < 0.0001). In the LEIA, neither of the two extracts interfered with the exsheathment of L3 (P > 0.05). In the AMIA, both SL-containing extracts induced a dose-dependent effect but Spadona-extract showed greater activity and exerted faster worm paralysis than Puna II-extract with significantly lower EC50 (P < 0.0001). No cuticular damage was observed by EM in worms exposed to any of the extracts. We have demonstrated that SL-containing extracts from forage chicory can inhibit feeding of free-living larvae and

  19. Sesquiterpene lactone containing extracts from two cultivars of forage chicory (Cichorium intybus) show distinctive chemical profiles and in vitro activity against Ostertagia ostertagi.

    PubMed

    Peña-Espinoza, Miguel; Boas, Ulrik; Williams, Andrew R; Thamsborg, Stig M; Simonsen, Henrik T; Enemark, Heidi L

    2015-12-01

    The study investigated direct anthelmintic effects of sesquiterpene lactones (SL)-containing extracts from forage chicory against free-living and parasitic stages of Ostertagia ostertagi. Freeze-dried leaves from chicory cultivars 'Spadona' and 'Puna II' were extracted using methanol/water. Total SL were further fractionated by solid-phase extraction and resulting extracts were characterised by high-performance liquid chromatography (HPLC). O. ostertagi eggs from faeces of mono-infected calves were hatched and L1 were used in a larval feeding inhibition assay (LFIA), while cultured L3 were used in a larval exsheathment inhibition assay (LEIA). Adult worms were immediately recovered after slaughter and used for motility inhibition assays (AMIA). Electron microscopy (EM) was performed on adult O. ostertagi exposed to 1000 μg extract mL(-1) of both chicory cultivars. In all assays, decreasing concentrations of SL-containing extracts in PBS (1% DMSO) were tested in replicates with 1% DMSO in PBS as negative controls. HPLC demonstrated similar concentrations of most SL in both extracts. However, Spadona-extract contained significantly higher concentrations of 11, 13-dihydro-8-deoxylactucin (P = 0.01), while Puna II-extract had increased levels of 11, 13-dihydrolactucin (P < 0.0001). In the LFIA, both extracts reduced larval feeding at increasing concentrations, but Spadona-extract showed higher potency confirmed by significantly lower EC50 (P < 0.0001). In the LEIA, neither of the two extracts interfered with the exsheathment of L3 (P > 0.05). In the AMIA, both SL-containing extracts induced a dose-dependent effect but Spadona-extract showed greater activity and exerted faster worm paralysis than Puna II-extract with significantly lower EC50 (P < 0.0001). No cuticular damage was observed by EM in worms exposed to any of the extracts. We have demonstrated that SL-containing extracts from forage chicory can inhibit feeding of free-living larvae and exert

  20. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

    PubMed Central

    Weininger, Ulrich; Jakob, Roman P; Kovermann, Michael; Balbach, Jochen; Schmid, Franz X

    2010-01-01

    PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition. PMID:19866485

  1. Novel antiviral characteristics of nanosized copper(I) iodide particles showing inactivation activity against 2009 pandemic H1N1 influenza virus.

    PubMed

    Fujimori, Yoshie; Sato, Tetsuya; Hayata, Taishi; Nagao, Tomokazu; Nakayama, Mikio; Nakayama, Tsuruo; Sugamata, Ryuichi; Suzuki, Kazuo

    2012-02-01

    We investigated the antiviral activity of nanosized copper(I) iodide (CuI) particles having an average size of 160 nm. CuI particles showed aqueous stability and generated hydroxyl radicals, which were probably derived from monovalent copper (Cu(+)). We confirmed that CuI particles showed antiviral activity against an influenza A virus of swine origin (pandemic [H1N1] 2009) by plaque titration assay. The virus titer decreased in a dose-dependent manner upon incubation with CuI particles, with the 50% effective concentration being approximately 17 μg/ml after exposure for 60 min. SDS-PAGE analysis confirmed the inactivation of the virus due to the degradation of viral proteins such as hemagglutinin and neuraminidase by CuI. Electron spin resonance (ESR) spectroscopy revealed that CuI generates hydroxyl radicals in aqueous solution, and radical production was found to be blocked by the radical scavenger N-acetylcysteine. Taken together, these findings indicate that CuI particles exert antiviral activity by generating hydroxyl radicals. Thus, CuI may be a useful material for protecting against viral attacks and may be suitable for applications such as filters, face masks, protective clothing, and kitchen cloths.

  2. Novel Antiviral Characteristics of Nanosized Copper(I) Iodide Particles Showing Inactivation Activity against 2009 Pandemic H1N1 Influenza Virus

    PubMed Central

    Sato, Tetsuya; Hayata, Taishi; Nagao, Tomokazu; Nakayama, Mikio; Nakayama, Tsuruo; Sugamata, Ryuichi; Suzuki, Kazuo

    2012-01-01

    We investigated the antiviral activity of nanosized copper(I) iodide (CuI) particles having an average size of 160 nm. CuI particles showed aqueous stability and generated hydroxyl radicals, which were probably derived from monovalent copper (Cu+). We confirmed that CuI particles showed antiviral activity against an influenza A virus of swine origin (pandemic [H1N1] 2009) by plaque titration assay. The virus titer decreased in a dose-dependent manner upon incubation with CuI particles, with the 50% effective concentration being approximately 17 μg/ml after exposure for 60 min. SDS-PAGE analysis confirmed the inactivation of the virus due to the degradation of viral proteins such as hemagglutinin and neuraminidase by CuI. Electron spin resonance (ESR) spectroscopy revealed that CuI generates hydroxyl radicals in aqueous solution, and radical production was found to be blocked by the radical scavenger N-acetylcysteine. Taken together, these findings indicate that CuI particles exert antiviral activity by generating hydroxyl radicals. Thus, CuI may be a useful material for protecting against viral attacks and may be suitable for applications such as filters, face masks, protective clothing, and kitchen cloths. PMID:22156433

  3. Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model.

    PubMed

    Lam, Kai Heng; Alex, Deepa; Lam, In Kei; Tsui, Stephen Kwok Wing; Yang, Zi Feng; Lee, Simon Ming Yuen

    2011-11-01

    Traditional Chinese medicinal herbs are a rich source of compounds with reported anti-inflammatory and anti-carcinogenic effects. Growing evidence shows the codependence of chronic inflammation and angiogenesis, and the potential benefits of targeting angiogenesis in the treatment of chronic inflammation and targeting inflammation in the treatment of diseases with impaired angiogenesis. We hypothesized that the anti-inflammatory activity of the natural compounds may owe at least some of its efficacy to their anti-angiogenic activity and hence we investigated the anti-angiogenic activity of these compounds in vivo in zebrafish embryos and in vitro in human umbilical vein endothelial cells (HUVECs). Nobiletin, a polymethoxylated flavonoid from citrus fruits, showed anti-angiogenic activity in both assays. Nobiletin inhibited the formation of intersegmental vessels (ISVs) in live transgenic zebrafish embryos expressing green fluorescent protein (GFP) in the vasculature. Cell cycle analysis of dissociated zebrafish embryo cells showed that nobiletin induced G0/G1 phase accumulation in a dose-dependent manner in GFP-positive endothelial cells. Nobiletin also dose-dependently induced VEGF-A mRNA expression. In HUVECs, nobiletin inhibited endothelial cell proliferation and, to a greater extent, tube formation in a dose-dependent manner. As in the in vivo study, nobiletin induced G0/G1 cell cycle arrest in HUVECs. However, this arrest was not accompanied by an increase in apoptosis, indicating a cytostatic effect of nobiletin. This study, for the first time, identifies nobiletin as having potent anti-angiogenic activity and suggests that nobiletin has a great potential for future research and development as a cytostatic anti-proliferative agent.

  4. Comparison of plaque- and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein.

    PubMed

    Liu, Lidong; Wen, Kun; Li, Jie; Hu, Dongmei; Huang, Yanfen; Qiu, Liwen; Cai, Jianpiao; Che, Xiaoyan

    2012-01-01

    The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC(50)) of these MAbs. Using PRNT as the reference and treating IC(50) values higher than 50 μg/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R(2) = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV.

  5. Comparison of Plaque- and Enzyme-Linked Immunospot-Based Assays To Measure the Neutralizing Activities of Monoclonal Antibodies Specific to Domain III of Dengue Virus Envelope Protein

    PubMed Central

    Liu, Lidong; Wen, Kun; Li, Jie; Hu, Dongmei; Huang, Yanfen; Qiu, Liwen; Cai, Jianpiao

    2012-01-01

    The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC50) of these MAbs. Using PRNT as the reference and treating IC50 values higher than 50 μg/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R2 = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV. PMID:22116689

  6. Nonlinear analysis of motor activity shows differences between schizophrenia and depression: a study using Fourier analysis and sample entropy.

    PubMed

    Hauge, Erik R; Berle, Jan Øystein; Oedegaard, Ketil J; Holsten, Fred; Fasmer, Ole Bernt

    2011-01-28

    The purpose of this study has been to describe motor activity data obtained by using wrist-worn actigraphs in patients with schizophrenia and major depression by the use of linear and non-linear methods of analysis. Different time frames were investigated, i.e., activity counts measured every minute for up to five hours and activity counts made hourly for up to two weeks. The results show that motor activity was lower in the schizophrenic patients and in patients with major depression, compared to controls. Using one minute intervals the depressed patients had a higher standard deviation (SD) compared to both the schizophrenic patients and the controls. The ratio between the root mean square successive differences (RMSSD) and SD was higher in the schizophrenic patients compared to controls. The Fourier analysis of the activity counts measured every minute showed that the relation between variance in the low and the high frequency range was lower in the schizophrenic patients compared to the controls. The sample entropy was higher in the schizophrenic patients compared to controls in the time series from the activity counts made every minute. The main conclusions of the study are that schizophrenic and depressive patients have distinctly different profiles of motor activity and that the results differ according to period length analysed.

  7. Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay.

    PubMed

    Haugan, I R; Nilsen, B M; Worland, S; Olsen, L; Helland, D E

    1995-12-26

    Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evaluate the binding of DNA to HIV-1 integrase. In this assay the DNA-binding was found to be independent of Mn2+ concentration, inhibited by concentrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibited by monoclonal antibodies reacting with either the N-terminal or C-terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is concluded that both the N-terminal and the C-terminal regions of integrase are involved in DNA-binding and that the reported double-layered dot blot radio assay is well suited for further characterization of the integrase.

  8. Free radical scavenging activity and characterization of sesquiterpenoids in four species of Curcuma using a TLC bioautography assay and GC-MS analysis.

    PubMed

    Zhao, Jing; Zhang, Jiang-Sheng; Yang, Bin; Lv, Guang-Ping; Li, Shao-Ping

    2010-10-27

    The sesquiterpenoids are one of major groups of antioxidants in Curcuma besides curcuminoids. However, the real substances contributing to the antioxidant activity are still unknown. In this paper, the antioxidant activity of sesquiterpenoids in four species and two essential oils from Curcuma genus was determined and compared based on TLC separation and DPPH bioautography assay. Their antioxidant capacities were quantitatively evaluated using densitometry with detection at 530 nm (λ(reference )= 800 nm) using vitamin C as reference. The results showed that Curcuma longa rhizomes had the highest antioxidant capacity while C. phaeocaulis presented the lowest one among the four species of Curcuma. Moreover, essential oil of C. wenyujin showed higher antioxidant potential than that of C. longa. The main TLC bands with antioxidant activity of the four species of Curcuma were collected and characterized using GC-MS, and thus curzerene, furanodiene, α-turmerone, β-turmerone and β-sesquiphellandrene were determined as major sesquiterpenoids with antioxidant activity in Curcuma.

  9. Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays

    PubMed Central

    2014-01-01

    Background In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Methods Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. Results 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. Conclusion A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy. PMID:24592852

  10. 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.

  11. Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

    PubMed

    Kwon, Young Chul; Kim, Sinil; Lee, Yong Seok; Lee, Je Chul; Cho, Myung-Je; Lee, Woo-Kon; Kang, Hyung-Lyun; Song, Jae-Young; Baik, Seung Chul; Ro, Hyeon Su

    2016-05-01

    HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells. PMID:27095458

  12. Cytotoxicity and Antiproliferative Activity Assay of Clove Mistletoe (Dendrophthoe pentandra (L.) Miq.) Leaves Extracts.

    PubMed

    Elsyana, Vida; Bintang, Maria; Priosoeryanto, Bambang Pontjo

    2016-01-01

    Clove mistletoe (Dendrophthoe pentandra (L.) Miq.) is a semiparasitic plant that belongs to Loranthaceae family. Clove mistletoe was traditionally used for cancer treatment in Indonesia. In the present study, we examined cytotoxicity of clove mistletoe leaves extracts against brine shrimps and conducted their antiproliferative activity on K562 (human chronic myelogenous leukemia) and MCM-B2 (canine benign mixed mammary) cancer cell lines in vitro. The tested samples were water extract, ethanol extract, ethanol fraction, ethyl acetate fraction, and n-hexane fraction. Cytotoxicity was screened using Brine Shrimp Lethality Test (BSLT). Antiproliferative activity was conducted using Trypan Blue Dye Method and cells were counted using haemocytometer. The results showed that n-hexane fraction exhibited significant cytotoxicity with LC50 value of 55.31 μg/mL. The n-hexane fraction was then considered for further examination. The n-hexane fraction of clove mistletoe could inhibit growth of K562 and MCM-B2 cancer cell lines in vitro. The inhibition activity of clove mistletoe n-hexane fraction at concentration of 125 μg/mL on K562 cancer cell lines was 38.69%, while on MCM-B2 it was 41.5%. Therefore, it was suggested that clove mistletoe had potential natural anticancer activity. PMID:27099614

  13. Assessment of dioxin-like activity in ambient air particulate matter using recombinant yeast assays

    NASA Astrophysics Data System (ADS)

    Olivares, Alba; van Drooge, Barend L.; Pérez Ballesta, Pascual; Grimalt, Joan O.; Piña, Benjamin

    2011-01-01

    Ectopic activation of the aryl hydrocarbon receptor (AhR), also known as dioxin-like activity, is a major component of the toxicity associated with polycyclic aromatic hydrocarbons (PAH). Filtration of ambient air particulate matter through PM 10 filters followed by chemical determination of PAH concentrations and a yeast-based bioassay (RYA) were combined to evaluate and characterize dioxin-like activity in ambient air. Samples