Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

PubMed

Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

2017-01-05

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

PubMed

Price, Joseph A

2015-09-01

Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity.

PubMed

Sommer, Jurg M; Buyue, Yang; Bardan, Sara; Peters, Robert T; Jiang, Haiyan; Kamphaus, George D; Gray, Elaine; Pierce, Glenn F

2014-11-01

Due to variability in the one-stage clotting assay, the performance of new factor IX (FIX) products should be assessed in this assay. The objective of this field study was to evaluate the accuracy of measuring recombinant FIX Fc fusion protein (rFIXFc) activity in clinical haemostasis laboratories using the one-stage clotting assay. Human haemophilic donor plasma was spiked with rFIXFc or BeneFIX® at 0.80, 0.20, or 0.05 IU/ml based on label potency. Laboratories tested blinded samples using their routine one-stage assay and in-house FIX plasma standard. The mean spike recoveries for BeneFIX (n=30 laboratories) were 121 %, 144 %, and 168 % of expected at nominal 0.80, 0.20, and 0.05 IU/ml concentrations, respectively. Corresponding rFIXFc spike recoveries were 88 %, 107 %, and 132 % of expected, respectively. All BeneFIX concentrations were consistently overestimated by most laboratories. rFIXFc activity was reagent-dependent; ellagic acid and silica gave higher values than kaolin, which underestimated rFIXFc. BeneFIX demonstrated significantly reduced chromogenic assay activity relative to one-stage assay results and nominal activity, while rFIXFc activity was close to nominal activity at three concentrations with better dilution linearity than the typical one-stage assay. In conclusion, laboratory- and reagent-specific assay variabilities were revealed, with progressively higher variability at lower FIX concentrations. Non-parallelism against the FIX plasma standard was observed in all one-stage assays with rFIXFc and BeneFIX, leading to significant overestimation of FIX activity at lower levels and generally high inter-laboratory variability. Compared to the accuracy currently achieved in clinical laboratories when measuring other rFIX products, most laboratories measured rFIXFc activity with acceptable accuracy and reliability using routine one-stage assay methods and commercially available plasma standards.

Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay.

PubMed

Teh, Chien Huey; Nazni, Wasi Ahmad; Nurulhusna, Ab Hamid; Norazah, Ahmad; Lee, Han Lim

2017-02-16

Antimicrobial resistance is currently a major global issue. As the rate of emergence of antimicrobial resistance has superseded the rate of discovery and introduction of new effective drugs, the medical arsenal now is experiencing shortage of effective drugs to combat diseases, particularly against diseases caused by the dreadful multidrug-resistant strains, such as the methicillin-resistant Staphylococcus aureus (MRSA). The ability of fly larvae to thrive in septic habitats has prompted us to determine the antibacterial activity and minimum inhibitory concentrations (MICs) of larval extract of flies, namely Lucilia cuprina, Sarcophaga peregrina and Musca domestica against 4 pathogenic bacteria [Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli] via a simple and sensitive antibacterial assay, resazurin-based turbidometric (TB) assay as well as to demonstrate the preliminary chemical profile of larval extracts using gas chromatography-mass spectrophotometry (GC-MS). The resazurin-based TB assay demonstrated that the L. cuprina larval extract was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only inhibitory against the MRSA, with a MIC of 100 mg ml -1 . Subsequent sub-culture of aliquots revealed that the larval extract of L. cuprina was bactericidal against MRSA whilst the larval extracts of S. peregrina and M. domestica were bacteriostatic against MRSA. The GC-MS analysis had quantitatively identified 20 organic compounds (fatty acids or their derivatives, aromatic acid esters, glycosides and phenol) from the larval extract of L. cuprina; and 5 fatty acid derivatives with known antimicrobial activities from S. peregrina and M. domestica. The resazurin-based turbidometric assay is a simple, reliable and feasible screening assay which evidently demonstrated the antibacterial activity of all fly larval extracts, primarily against the MRSA. The larval

In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

PubMed Central

Rasooly, L; Rose, N R; Shah, D B; Rasooly, A

1997-01-01

Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food. PMID:9172356

A microplate assay to measure classical and alternative complement activity.

PubMed

Puissant-Lubrano, Bénédicte; Fortenfant, Françoise; Winterton, Peter; Blancher, Antoine

2017-05-01

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay.

PubMed

Merrigan, Stephen D; Owen, William E; Straseski, Joely A

2015-01-01

Vitamin B12 (cobalamin) is a necessary cofactor in methionine and succinyl-CoA metabolism. Studies estimate the deficiency prevalence as high as 30% in the elderly population. Ten to thirty percent of circulating cobalamin is bound to transcobalamin (holotranscobalamin, holoTC) which can readily enter cells and is therefore considered the bioactive form. The objective of our study was to evaluate the analytical performance of a high-throughput, automated holoTC assay (ARCHITECT i2000(SR) Active-B12 (Holotranscobalamin)) and compare it to other available methods. Manufacturer-specified limits of blank (LoB), detection (LoD), and quantitation (LoQ), imprecision, interference, and linearity were evaluated for the ARCHITECT HoloTC assay. Residual de-identified serum samples were used to compare the ARCHITECT HoloTC assay with the automated AxSYM Active-B12 (Holotranscobalamin) assay (Abbott Diagnostics) and the manual Active-B12 (Holotranscobalamin) Enzyme Immunoassay (EIA) (Axis-Shield Diagnostics, Dundee, Scotland, UK). Manufacturer's claims of LoB, LoD, LoQ, imprecision, interference, and linearity to the highest point tested (113.4 pmol/L) were verified for the ARCHITECT HoloTC assay. Method comparison of the ARCHITECT HoloTC to the AxSYM HoloTC produced the following Deming regression statistics: (ARCHITECT(HoloTc)) = 0.941 (AxSYM(HoloTC)) + 1.2 pmol/L, S(y/x) = 6.4, r = 0.947 (n = 98). Comparison to the Active-B12 EIA produced: (ARCHITECT(HoloTC)) = 1.105 (EIA(Active-B12)) - 6.8 pmol/L, S(y/x) = 11.0, r = 0.950 (n = 221). This assay performed acceptably for LoB, LoD, LoQ, imprecision, interference, linearity and method comparison to the predicate device (AxSYM). An additional comparison to a manual Active-B12 EIA method performed similarly, with minor exceptions. This study determined that the ARCHITECT HoloTC assay is suitable for routine clinical use, which provides a high-throughput alternative for automated testing of this emerging marker of cobalamin

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

PubMed

Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

2018-01-01

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

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.

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

PubMed

Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

A protein chip membrane-capture assay for botulinum neurotoxin activity

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

Marconi, Severine; Universite de la Mediterranee-Aix Marseille 2, Faculte de medecine secteur nord, Bd P. Dramard, Marseille F-13916; Ferracci, Geraldine

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 capturemore » 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.« less

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.

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

PubMed

Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian

2004-12-30

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

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

PubMed

Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier

PubMed Central

Breslow, David K.; Koslover, Elena F.; Seydel, Federica; Spakowitz, Andrew J.

2013-01-01

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia. PMID:24100294

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

PubMed

Bainbridge, Travis W; Dunshee, Diana Ronai; Kljavin, Noelyn M; Skelton, Nicholas J; Sonoda, Junichiro; Ernst, James A

2017-10-02

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

CroFabtrade mark total anti-venom activity measured by SE-HPLC, and anti-PLA(2) activity assayed in vitro at physiological pH.

PubMed

Price, Joseph A; Sanny, Charles G

2007-05-01

One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 degrees C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A(2) (PLA(2)) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA(2) and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab anti-venin) inactivation of C. atrox PLA(2) activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.

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

PubMed

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

2012-12-01

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.

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.

PubMed

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

2010-05-01

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression. 2010 Elsevier Inc. All rights reserved.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2015-01-01

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

A High-Throughput Screening Assay to Detect ...

EPA Pesticide Factsheets

In support of the Endocrine Disruption Screening Program (EDSP21), the US EPA ToxCast program is developing assays to enable screening for chemicals that may disrupt thyroid hormone synthesis. Thyroperoxidase (TPO) is critical for TH synthesis and is a known target of thyroid-disrupting chemicals that adversely impact neurodevelopment. The AUR-TPO assay was recently developed to screen >1,900 ToxCast chemicals for potential TPO inhibition activity. Parallel assays were used to determine which AUR-TPO actives were more selective for TPO inhibition. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO assay and an orthogonal peroxidase oxidation assay using guaiacol as substrate to confirm putative TPO inhibition profiles. Bioactivity results from the AUR-TPO assay were used to identify chemical substructures associated with in vitro TPO inhibition. Substructure profiles were generated for each chemical in the ToxCast test set using the publicly-available ToxPrint 2.0 chemotypes. Chemotypes enriched among the putative TPO inhibitors were identified using a cumulative hypergeometric probability (p < 0.01). Of the total 729 chemotypes evaluated, 44 were overrepresented among TPO inhibitors. Another 24 chemotypes were found to be significantly underrepresented among AUR-TPO actives. Examination of these chemotypes revealed four basic pharmacophores that accounted for 70% of the ToxCast chemicals active in the AUR-TPO assay:

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

PubMed Central

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

2014-01-01

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

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

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

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

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

PubMed

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

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

Evaluation of differential representative values between Chinese hamster cells and human lymphocytes in mitomycin C-induced cytogenetic assays and caspase-3 activity.

PubMed

Liao, Pei-Hu; Lin, Ruey-Hseng; Yang, Ming-Ling; Li, Yi-Ching; Kuan, Yu-Hsiang

2012-03-01

Chinese hamster ovary (CHO) cells, its lung fibroblasts (V79), and human lymphocytes are routinely used in in vitro cytogenetic assays, which include micronuclei (MN), sister chromatid exchange (SCE), and chromosome aberration (CA) assays. Mitomycin C (MMC), a DNA cross-link alkylating agent, is both an anticancer medicine and a carcinogen. To study the differential representative values of cell types in MMC-treated cytogenetic assays and its upstream factor, cysteine aspartic acid-specific protease (caspase)-3. Among the chosen cell types, lymphocytes expressed the highest sensitivity in all three MMC-induced assays, whereas CHO and V79 showed varied sensitivity in different assays. In MN assay, the sensitivity of CHO is higher than or equal to V79; in SCE assay, the sensitivity of CHO is the same as V79; and in CA assay, the sensitivity of CHO is higher than V79. In-depth analysis of CA revealed that in chromatid breaks and dicentrics formation, lymphocyte was the most sensitive of all and CHO was more sensitive than V79; and in acentrics and interchanges formation, lymphocyte was much more sensitive than the others. Furthermore, we found caspase-3 activity plays an important role in MMC-induced cytogenetic assays, with MMC-induced caspase-3 activity resulting in more sensitivity in lymphocytes than in CHO and V79. Based on these findings, lymphocyte will make a suitable predictive or representative control reference in cytogenetic assays and caspase-3 activity with its high specificity, positive predictive value, and sensitivity.

Effect of hawthorn (Crataegus oxycantha) crude extract and chromatographic fractions on multiple activities in a cultured cardiomyocyte assay.

PubMed

Long, S R; Carey, R A; Crofoot, K M; Proteau, P J; Filtz, T M

2006-11-01

Extracts of hawthorn (Crataegus oxycantha) have become popular herbal supplements for their well-recognized cardiotonic effects. Many commercial preparations have been used successfully in the treatment of congestive heart failure, although the active principles within these extracts have yet to be conclusively identified. Several hawthorn preparations were studied and found to have negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay using unpaced cells. As compared to conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extract has a unique activity profile. Hawthorn extract appears to be anti-arrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extract does not cause beta-adrenergic receptor blockade at concentrations which cause negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries have similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple cardiac activities. Assays with chromatographic fractions reveal that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging.

A sensitive assay reveals structural requirements for α-synuclein fibril growth

PubMed Central

Tsai, Christina; Bagchi, Devika P.; Engel, Laura A.; Sarezky, Jonathan; Kotzbauer, Paul T.

2017-01-01

The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble α-syn monomers associate to form seeds, followed by fibril growth during which monomeric α-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant α-syn protein containing an N-terminal bicysteine tag (C2-α-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-α-syn monomer, we observed that some familial PD-associated α-syn mutations (i.e. H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that α-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of α-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD. PMID:28373279

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

PubMed

Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

2018-05-17

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds.

PubMed

Zhao, Ziyan; Henowitz, Liza; Zweifach, Adam

2018-05-01

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

A combination of new screening assays for prioritization of transmission-blocking antimalarials reveals distinct dynamics of marketed and experimental drugs.

PubMed

Bolscher, J M; Koolen, K M J; van Gemert, G J; van de Vegte-Bolmer, M G; Bousema, T; Leroy, D; Sauerwein, R W; Dechering, K J

2015-05-01

The development of drugs to reduce malaria transmission is an important part of malaria eradication plans. We set out to develop and validate a combination of new screening assays for prioritization of transmission-blocking molecules. We developed high-throughput assays for screening compounds against gametocytes, the parasite stages responsible for onward transmission to mosquitoes. An existing gametocyte parasitic lactate dehydrogenase (pLDH) assay was adapted for use in 384-well plates, and a novel homogeneous immunoassay to monitor the functional transition of female gametocytes into gametes was developed. A collection of 48 marketed and experimental antimalarials was screened and subsequently tested for impact on sporogony in Anopheles mosquitoes, to directly quantify the transmission-blocking properties of antimalarials in relation to their effects on gametocyte pLDH activity or gametogenesis. The novel screening assays revealed distinct stage-specific kinetics and dynamics of drug effects. Peroxides showed the most potent transmission-blocking effects, with an intermediate speed of action and IC50 values that were 20-40-fold higher than the IC50s against the asexual stages causing clinical malaria. Finally, the novel synthetic peroxide OZ439 appeared to be a promising drug candidate as it exerted gametocytocidal and transmission-blocking effects at clinically relevant concentrations. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

PubMed Central

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

2015-01-01

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

Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

PubMed Central

Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

1999-01-01

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

New generation QuIC assays for prion seeding activity.

PubMed

Orrù, Christina D; Wilham, Jason M; Vascellari, Sarah; Hughson, Andrew G; Caughey, Byron

2012-01-01

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP(Sen) has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.

PubMed

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

2016-01-01

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

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

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

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

A new effective assay to detect antimicrobial activity of filamentous fungi.

PubMed

Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

2013-01-15

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

PubMed

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

2012-12-01

In recent years the global rise in antibiotic resistance and environmental consciousness lead to a renewed fervour to find and develop novel antibiotics, including antifungals. However, the influence of the environment on antifungal activity is often disregarded and many in vitro assays may cause the activity of certain antifungals to be overestimated or underestimated. The general antifungal test assays that are economically accessible to the majority of scientists primarily rely on visual examination or on spectrophotometric analysis. The effect of certain morphogenic antifungals, which may lead to hyperbranching of filamentous fungi, unfortunately renders these methods unreliable. To minimise the difficulties experienced as a result of hyperbranching, we developed a straightforward, economical 96-well gel-based method, independent of spectrophotometric analysis, for highly repeatable determination of antifungal activity. For the calculation of inhibition parameters, this method relies on the visualisation of assay results by digitisation. The antifungal activity results from our novel micro-gel dilution assay are comparable to that of the micro-broth dilution assay used as standard reference test of The Clinical and Laboratory Standard Institute. Furthermore, our economical assay is multifunctional as it permits microscopic analysis of the preserved assay results, as well as rendering highly reliable data. Copyright © 2012 Elsevier B.V. All rights reserved.

Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

PubMed

Wolfe, Kelly L; Liu, Rui Hai

2007-10-31

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

NASA Astrophysics Data System (ADS)

Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

2014-11-01

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

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. Copyright © 2011 John Wiley & Sons, Ltd.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

PubMed

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

PubMed Central

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S

2011-01-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein. PMID:21918373

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

PubMed

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

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

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

DOE PAGES

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; ...

2018-05-14

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Cell based assays for anti-Plasmodium activity evaluation.

PubMed

Mokgethi-Morule, Thabang; N'Da, David D

2016-03-10

Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

PubMed Central

Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

2015-01-01

NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

[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.

Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

PubMed

Valdez-Flores, Marco A; Vargas-Poussou, Rosa; Verkaart, Sjoerd; Tutakhel, Omar A Z; Valdez-Ortiz, Angel; Blanchard, Anne; Treard, Cyrielle; Hoenderop, Joost G J; Bindels, René J M; Jeleń, Sabina

2016-12-01

Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations. Copyright © 2016 the American Physiological Society.

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.

PubMed

Bandhuvula, Padmavathi; Fyrst, Henrik; Saba, Julie D

2007-12-01

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.

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.

Advanced analysis techniques for uranium assay

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

Geist, W. H.; Ensslin, Norbert; Carrillo, L. A.

2001-01-01

Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples countmore » rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.« less

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

PubMed Central

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

Optimized UDP-glucuronosyltransferase (UGT) activity assay for trout liver S9 fractions

EPA Pesticide Factsheets

This publication provides an optimized UGT assay for trout liver S9 fractions which can be used to perform in vitro-in vivo extrapolations of measured UGT activityThis dataset is associated with the following publication:Ladd, M., P. Fitzsimmons , and J. Nichols. Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, USA, 46(12): 1066-1075, (2016).

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

PubMed

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

2014-08-01

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

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.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

PubMed

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

Install active/passive neutron examination and assay (APNEA)

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

Not Available

1996-04-01

This document describes activities pertinent to the installation of the prototype Active/Passive Neutron Examination and Assay (APNEA) system built in Area 336 into its specially designed trailer. It also documents the basic theory of operation, design and protective features, basic personnel training, and the proposed characterization site location at Lockheed Martin Specialty Components, Inc., (Specialty Components) with the estimated 10 mrem/year boundary. Additionally, the document includes the Preventive Change Analysis (PCA) form, and a checklist of items for verification prior to unrestricted system use.

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

PubMed Central

Newberry, Kim; Wang, Shuya; Hoque, Nina; Kiss, Laszlo; Ahlijanian, Michael K.; Herrington, James

2016-01-01

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 Ca2+ 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

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

PubMed Central

Barkal, Layla J.; Walsh, Naomi M.; Botts, Michael R.; Beebe, David J.; Hull, Christina M.

2016-01-01

Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination. PMID:27026574

Automated chromatographic laccase-mediator-system activity assay.

PubMed

Anders, Nico; Schelden, Maximilian; Roth, Simon; Spiess, Antje C

2017-08-01

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

Enzymatic assay for calmodulins based on plant NAD kinase activity

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

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

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 formore » 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.« less

Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

PubMed Central

Lek, Sovanarak; Vargas-Medrano, Javier; Villanueva, Ernesto; Marcus, Brian; Godfrey, Wesley; Perez, Ruth G.

2017-01-01

α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo. PMID:28829427

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

PubMed

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

2009-11-15

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

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

PubMed

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

Comparison of three anti-dsDNA assays: performance and correlation with systemic lupus erythematosus disease activity.

PubMed

Venner, Allison A; Ibañez, Dominique; Gladman, Dafna D; Urowitz, Murray B; MacKinnon, Anne; Blasutig, Ivan M; Yip, Paul M

2013-03-01

To investigate the BioPlex 2200 multiplex immunoassay and Farrzyme ELISA assays as alternatives to the established Farr radioimmunoassay for the correlation of anti-dsDNA antibodies in the assessment of disease activity in systemic lupus erythematosus (SLE). Standard protocols were used to verify analytical performance claims. Anti-dsDNA antibody levels in SLE patient specimens (N=105) were measured and assessed for clinical performance using manufacturer cut-off limits along with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score. Assay precision, measurable range and normal reference interval met the manufacturers' stated claims. Agreement between Farr and BioPlex assays was moderate (positive agreement=62%; negative agreement=85%; kappa=0.48), as was agreement between Farr and Farrzyme assays (positive agreement=56%; negative agreement=91%; kappa=0.51). Mean SLEDAI-2K scores differed significantly between the anti-dsDNA positive and negative groups for BioPlex (p=0.0006), but not Farr (p=0.11) or Farrzyme (p=0.34). ROC curve analysis showed a similar area under the curve (AUC) for all three assays (0.76, 0.74, and 0.73 for Farr, BioPlex, and Farrzyme, respectively) in the discrimination of clinically active disease. Furthermore, increased anti-dsDNA levels from BioPlex showed significant correlation with active renal disease. However, results suggested a lower cut-off for the Farrzyme assay for assessment of global disease activity. BioPlex and Farrzyme assays had similar overall agreement with the Farr assay, with BioPlex best reflecting disease activity in SLE patients. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology.

PubMed

Abderrahim, Mohamed; Arribas, Silvia M; Condezo-Hoyos, Luis

2017-05-01

Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H 2 O 2 ) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL -1 ), HRP (1UmL -1 ) and H 2 O 2 (250µmolL -1 ) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL -1 ) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H 2 O 2 as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL -1 (R 2 =0.993) and very sensitive with limits of detection (LOD) of 0.005UmL -1 and quantitation (LOQ) of 0.01UmL -1 . PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

An improved 96-well turbidity assay for T4 lysozyme activity

DTIC Science & Technology

2015-05-13

enzyme present in the reaction, resulting in a measure of activity in Unitsmg1. 10. To improve the reliability of the activity values, perform the...quantification of lysozyme activity with significantly lower enzyme concentrations, and the signal intensity can be enhanced by using greater amounts of... enzyme at the expense of a shorter linear reaction time. Several parameters of the assay are critical for obtaining reproducible activity

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue

Flow-injection assay of catalase activity.

PubMed

Ukeda, Hiroyuki; Adachi, Yukiko; Sawamura, Masayoshi

2004-03-01

A novel flow-injection assay (FIA) system with a double line for catalase activity was constructed in which an oxidase is immobilized and the substrate is continuously pumped to reduce the dissolved oxygen and to generate a given level of hydrogen peroxide. The catalase in a sample decomposed the hydrogen peroxide, and thus the increase in dissolved oxygen dependent on the activity was amperometrically monitored using a Clark-type oxygen electrode. Among the examined several oxidases, uricase was most suitable for the continuous formation of hydrogen peroxide from a consideration of the stability and the conversion efficiency. Under the optimum conditions, a linear calibration curve was obtained in the range from 21 to 210 units/mg and the reproducibility (CV) was better than 2% by 35 successive determinations of 210 units/ml catalase preparation. The sampling frequency was about 15 samples/h. The present FIA system was applicable to monitor the inactivation of catalase by glycation.

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.

An improved 96-well turbidity assay for T4 lysozyme activity

PubMed Central

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. PMID:26150996

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Anti-Candida activity and brine shrimp toxicity assay of Ganoderma boninense.

PubMed

Daruliza, K M A; Fernandez, L; Jegathambigai, R; Sasidharan, S

2012-01-01

Ganoderma (G.) boninense is a white rot fungus, which can be found in the palm oil tree. Several studies have shown that G. boninense has antimicrobial and antagonistic properties. However, there is limited information reported on antifungal properties especially on Candida (C) albicans. Hence, this study was conducted to determine the anti-Candida activity of G. boninense against C albicans. Crude methanolic extracts of G. boninense was obtained by maceration method with 70% methanol. Anti-Candida test was carried out using disc diffusion assay, broth dilution method, time killing profile and brine shrimp toxicity assay. Anti-Candida activity indicated that the mean zone of inhibition was 12.5 +/- 0.6 mm. The MIC value for C. albicans found to be 3.125 mg/ml. The result from time-killing profile showed that the growth of C albicans was inhibited hence decreases its exponential phase. For brine shrimp toxicity assay, the LC50 value was 3.59 mg/ml which proved that the extract of G. boninense is not toxic.

Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods.

PubMed

Yokotani, Kaori; Umegaki, Keizo

2017-02-01

The contribution of (-)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500 mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where <3 μmol/L EGCg in the presence of protein exhibited minimal antioxidant activity, as measured by both FRAP and ORAC. As the concentration of plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.

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.

Heme polymerization inhibition activity (HPIA) assay of synthesized xanthone derivative as antimalarial compound

NASA Astrophysics Data System (ADS)

Fitriastuti, Dhina; Jumina, Priatmoko

2017-03-01

Xanthone is a phenolic secondary metabolite of Garcinia and Calophyllum herbs which has been clinically proven to display anti malaria activity. In the present paper, 2,3,4-trihydroxy-5-methyl xanthone which has been synthesized from gallic acid and o-cresol in Eaton's reagent was tested for its activity as antimalarial. Thus, HPIA assay of the synthesized xanthones was successfully conducted. The HPIA assay was carried out towards the xanthone, chloroquine diphosphate as positive control and distilled water as negative control in various concentration. The samples were reacted with hematin (ferriprotoporphyrin IX hydroxide) and the absorbance of the precipitate was observed by using Elisa reader. The results of HPIA assay showed that 2,3,4-trihydroxy-5-methyl xanthone and chloroquine have IC50 values of 0.755 and 1.462 mg/mL or 2.92 and 4.57 mM, respectively. 2,3,4-Trihydroxy-5-methyl xanthone displayed better antimalarial activity than chloroquine.

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…

Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.

PubMed

Paskin, Taylor R; Jellies, John; Bacher, Jessica; Beane, Wendy S

2014-01-01

Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli), planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green), as well as ultraviolet (UV) and infrared (IR) which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV) causing the most intense photophobic responses while longer wavelengths produce no effect (red) or an apparent attraction (IR). In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength) and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment.

From the Cover: Development and Application of a Dual Rat and Human AHR Activation Assay.

PubMed

Brown, Martin R; Garside, Helen; Thompson, Emma; Atwal, Saseela; Bean, Chloe; Goodall, Tony; Sullivan, Michael; Graham, Mark J

2017-12-01

Significant prolonged aryl hydrocarbon receptor (AHR) activation, classically exhibited following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, can cause a variety of undesirable toxicological effects. Novel pharmaceutical chemistries also have the potential to cause activation of AHR and consequent toxicities in pre-clinical species and man. Previous methods either employed relatively expensive and low-throughput primary hepatocyte dosing with PCR endpoint, or low resolution overexpressing reporter gene assays. We have developed, validated and applied an in vitro microtitre plate imaging-based medium throughput screening assay for the assessment of endogenous species-specific AHR activation potential via detection of induction of the surrogate transcriptional target Cytochrome P450 CYP1A1. Routine testing of pharmaceutical drug development candidate chemistries using this assay can influence the chemical design process and highlight AHR liabilities. This assay should be introduced such that human AHR activation liability is flagged early for confirmatory testing. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

PubMed

Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

2016-01-01

The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.

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

PubMed

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

2014-10-01

The present study investigated and compared the estrogenic and androgenic activities of the three different classes of environmental pollutants and their metabolites using the XenoScreen XL YES/YAS assay, which has advantages compared with the original YES/YAS protocol. Contrary to the parent brominated flame retardants TBB and TBPH, which demonstrated no or very weak (anti)estrogenic or (anti)androgenic activities, their metabolites, TBBA and TBMEPH, exhibited anti-estrogenic (IC50 for TBBA=31.75 μM and IC50 for TBMEPH=0.265 μM) and anti-androgenic (IC50 for TBBA=73.95 μM and IC50 for TBMEPH=2.92 μM) activities. These results reveal that metabolism can enhance the anti-estrogenic and anti-androgenic effects of these two novel brominated flame retardants. Based on the activities of BPAF, BPF, BPA and MBP, we can conclude that the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti)androgenic assays that are reported in the literature. For BPA, it was confirmed previously that the metabolite formed after an ipso-reaction (hydroxycumyl alcohol) exhibited higher estrogenic activity compared with the parent BPA, but this was not confirmed for BPAF and BPF ipso-metabolites, which were not active in the XenoScreen YES/YAS assay. Among the substituted BPA analogues, bis-GMA exhibited weak anti-estrogenic activity, BADGE demonstrated weak anti-estrogenic and anti-androgenic activities (IC50=13.73 μM), and the hydrolysed product BADGE·2H2O demonstrated no (anti)estrogenic or (anti)androgenic activities. Copyright © 2014. Published by Elsevier Ltd.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

PubMed Central

Deschuyteneer, Aude; Boeckstaens, Mélanie; De Mees, Christelle; Van Vooren, Pascale; Wintjens, René; Marini, Anna Maria

2013-01-01

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants. PMID:23967154

IFN-γ Release Assay Result Is Associated with Disease Site and Death in Active Tuberculosis.

PubMed

Auld, Sara C; Lee, Scott H; Click, Eleanor S; Miramontes, Roque; Day, Cheryl L; Gandhi, Neel R; Heilig, Charles M

2016-12-01

The IFN-γ release assays and tuberculin skin tests are used to support the diagnosis of both latent and active tuberculosis. However, we previously demonstrated that a negative tuberculin test in active tuberculosis is associated with disseminated disease and death. It is unknown whether the same associations exist for IFN-γ release assays. To determine the association between these tests and site of tuberculosis and death among persons with active tuberculosis. We analyzed IFN-γ release assays and tuberculin test results for all persons with culture-confirmed tuberculosis reported to the U.S. National Tuberculosis Surveillance System from 2010 to 2014. We used logistic regression to calculate the association between these tests and site of disease and death. A total of 24,803 persons with culture-confirmed tuberculosis had either of these test results available for analysis. Persons with a positive tuberculin test had lower odds of disseminated disease (i.e., miliary or combined pulmonary and extrapulmonary disease), but there was no difference in the odds of disseminated disease with a positive IFN-γ release assay. However, persons who were positive to either of these tests had lower odds of death. An indeterminate IFN-γ release assay result was associated with greater odds of both disseminated disease and death. Despite perceived equivalence in clinical practice, IFN-γ release assays and tuberculin test results have different associations with tuberculosis site, yet similar associations with the risk of death. Furthermore, an indeterminate IFN-γ release assay result in a person with active tuberculosis is not unimportant, and rather carries greater odds of disseminated disease and death. Prospective study may improve our understanding of the underlying mechanisms by which these tests are associated with disease localization and death.

Blood-brain barrier specific permeability assay reveals N-methylated tyramine derivatives in standardised leaf extracts and herbal products of Ginkgo biloba.

PubMed

Könczöl, Árpád; Rendes, Kata; Dékány, Miklós; Müller, Judit; Riethmüller, Eszter; Balogh, György Tibor

2016-11-30

The linkage between the central nervous system availability and neuropharmacological activity of the constituents of Ginkgo biloba L. extracts (GBE) is still incomplete. In this study, the in vitro blood-brain barrier (BBB) permeability profile of the standardised GBE was investigated by the parallel artificial membrane permeability assay (PAMPA). Biomarkers, such as terpene trilactones, flavonoid aglycones and ginkgotoxin exerted moderate or good BBB-permeability potential (BBB+), while glycosides and biflavones were predicted as unable to pass the BBB. N-methyltyramine (NMT) and N,N-dimethyltyramine or hordenine (Hor) were identified among BBB+ compounds, while subsequent direct HRMS analysis revealed tyramine (Tyr) and N,N,N-trimethyltyramine or candicine (Can) in GBE as trace constituents. Distribution of Tyr, NMT, Hor and Can was determined by a validated ion-exchange mechanism-based liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method in G. biloba samples, such as herbal drugs and dietary supplements. The total content of the four tyramine derivatives in various GBEs ranged from 7.3 up to 6357μg/g dry extract with NMT and Hor as most abundant ones. Considering the pharmacological activities and the revealed fluctuation in the concentration of the analysed adrenergic protoalkaloids, the presented rapid LC-ESI-MS method is proposed for monitoring of the levels of Tyr, NMT, Hor and Can in G. biloba products. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay.

PubMed

Maruki-Uchida, Hiroko; Sai, Masahiko; Sekimizu, Kazuhisa

2017-11-22

We evaluated the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay. Sake cake, a raw material used to make amazake, had high innate immunity-stimulating activity, whereas rice malt, another raw material used to make amazake, did not, even after fermentation. These results suggest that the silkworm muscle contraction assay is a useful tool to screen foods with high innate immune-stimulating activity and that amazake made from sake cake has immunomodulatory potential.

Differential sensitivity of von Willebrand factor (VWF) 'activity' assays to large and small VWF molecular weight forms: a cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays.

PubMed

Favaloro, E J; Bonar, R; Chapman, K; Meiring, M; Funk Adcock, D

2012-06-01

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura. © 2012 International Society on Thrombosis and Haemostasis.

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

PubMed

John, Victoria H; Dale, Tim J; Hollands, Emma C; Chen, Mao Xiang; Partington, Leanne; Downie, David L; Meadows, Helen J; Trezise, Derek J

2007-02-01

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

PubMed

Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

2018-05-25

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay.

PubMed

Adcock, Robert S; Chu, Yong-Kyu; Golden, Jennifer E; Chung, Dong-Hoon

2017-02-01

virus). From these assays, we discovered that brequinar has potent anti-ZIKV activity. Our results show that a broad anti-ZIKV screen of compound libraries with our CPE-based HTS assay will reveal multiple chemotypes that could be pursued as lead compounds for therapies to treat ZIKV-associated diseases or as molecular probes to study the biology of the ZIKV replication mechanism. Copyright © 2016 Elsevier B.V. All rights reserved.

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

PubMed Central

Burchett, Gina G.; Folsom, Charles G.; Lane, Kimberly T.

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins. PMID:26121040

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase.

PubMed

Burchett, Gina G; Folsom, Charles G; Lane, Kimberly T

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

PubMed

Mahboubi, Arash; Sadjady, Seyyed Kazem; Mirzaei Saleh Abadi, Mohammad; Azadi, Saeed; Solaimanian, Roya

2012-01-01

DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

PubMed

Sajayan, Arya; Seghal Kiran, G; Priyadharshini, S; Poulose, Navya; Selvin, Joseph

2017-09-01

A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

Roelofs, Maarke J.E., E-mail: m.j.e.roelofs@uu.nl; Center for Health Protection, National Institute for Public Health and the Environment; Piersma, Aldert H.

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 2.2more » 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

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

PubMed Central

Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

2017-01-01

Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

Development of a homogeneous, fluorescence resonance energy transfer-based in vitro recruitment assay for peroxisome proliferator-activated receptor delta via selection of active LXXLL coactivator peptides.

PubMed

Drake, Katherine A; Zhang, Ji-Hu; Harrison, Richard K; McGeehan, Gerald M

2002-05-01

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by fatty acids and their metabolites. The PPARdelta subtype is believed to be involved in lipoprotein regulation and may have a role in reverse cholesterol transport. While the range of biological roles of PPARdelta still remains unclear, it is of therapeutic interest in cardiovascular diseases. Here we report a homogeneous in vitro assay for studying ligand activation of PPARdelta. We surveyed a panel of peptides containing the LXXLL motifs derived from coactivator protein sequences. Peptides with the best response were used to develop a sensitive and homogeneous recruitment assay for PPARdelta. The optimized assay has a signal-to-background ratio of about 8:1 and an assay quality parameter Z'-factor value of 0.8. The assay signal generated is stable for hours to even overnight. This simple recruitment assay can provide agonist and/or antagonist information that cannot be assessed by receptor-binding assay, and can be used for characterization and screening of ligands that modulate the activation of PPARdelta. (c)2002 Elsevier Science (USA).

Optimized angiotensin-converting enzyme activity assay for the accurate diagnosis of sarcoidosis.

PubMed

Csongrádi, Alexandra; Enyedi, Attila; Takács, István; Végh, Tamás; Mányiné, Ivetta S; Pólik, Zsófia; Altorjay, István Tibor; Balla, József; Balla, György; Édes, István; Kappelmayer, János; Tóth, Attila; Papp, Zoltán; Fagyas, Miklós

2018-06-27

Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study. Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined. Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 μM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency. An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.

Honey promotes angiogeneic activity in the rat aortic ring assay.

PubMed

Rossiter, K; Cooper, A J; Voegeli, D; Lwaleed, B A

2010-10-01

To investigate possible effects of honey on angiogenesis, using in vitro analogues of angiogenesis and an endothelial proliferation assay. Using an in vitro rat aortic ring assay we compared pseudotubule formation by medicinal honey (Activon), supermarket honey (Rowse) and a honey-based ointment (Mesitran), with that of artificial honey (70% w/w sugar glucose/fructose). Pseudotubules were analysed using TCS Cellworks AngioSys software. The Angiokit sytem was used to validate the results. Using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium. Bromide] assay, toxicity was also assessed on human umbilical vein endothelial cells (HUVEC) directly adherent to plastic. All honey preparations stimulated pseudotubule formation, maximal at around 0.2% honey. Medicinal honeys were more active than Rowse. The effect was not attributable to the sugar content. Among the honeys tested, the Manuka-based Activon preparation reduced residual viable biomass compared with a sugar control at > 0.32% v/v concentration. Rowse had a similar effect only at 2.5%, the highest dose tested. The influence of honey constituents on angiogenesis in a wound dressing context is likely to be positive, but would depend on the effective dilution of the honey and the penetration of the active constituents against an osmotic gradient. The extent to which this occurs has yet to be established. This work was conceived, designed and executed by the authors. Medical honey preparations were supplied unconditionally but free of charge by the distributors.

RAS - Screens & Assays - Drug Discovery

Cancer.gov

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD12.

PubMed

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

2016-01-01

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

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.

Active B12: a rapid, automated assay for holotranscobalamin on the Abbott AxSYM analyzer.

PubMed

Brady, Jeff; Wilson, Lesley; McGregor, Lynda; Valente, Edward; Orning, Lars

2008-03-01

Conventional tests for vitamin B(12) deficiency measure total serum vitamin B12, whereas only that portion of vitamin B12 carried by transcobalamin (holotranscobalamin) is metabolically active. Measurement of holotranscobalamin (holoTC) may be more diagnostically accurate for detecting B(12) deficiency that requires therapy. We developed an automated assay for holoTC that can be used on the Abbott AxSYM immunoassay analyzer. AxSYM Active B12 is a 2-step sandwich microparticle enzyme immunoassay. In step 1, a holoTC-specific antibody immobilized onto latex microparticles captures holoTC in samples of serum or plasma. In step 2, the captured holoTC is detected with a conjugate of alkaline phosphatase and antiTC antibody. Neither apoTC nor haptocorrin exhibited detectable cross-reactivity. The detection limit was < or = 0.1 pmol/L. Within-run and total imprecision (CV ranges) were 3.4%-5.1% and 6.3%-8.5%, respectively. Assay CVs were < 20% from at least 3 pmol/L to 107 pmol/L. With diluted serum samples, measured concentrations were 104%-114% of the expected values in the working range of the assay. No interference from bilirubin, hemoglobin, triglycerides, erythrocytes, rheumatoid factor, or total protein was detected at expected (abnormal) concentrations. A comparison of the AxSYM Active B12 assay with a commercial RIA for holoTC yielded the regression equation: AxSYM = 0.98RIA + 4.7 pmol/L (S(y x), 11.4 pmol/L; n = 204). Assay throughput was 45 tests/h. A 95% reference interval of 19-134 pmol/L holoTC was established with samples from 292 healthy individuals. The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.

Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

PubMed Central

van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

2015-01-01

The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

Estrogenic activity of constituents of underarm deodorants determined by E-Screen assay.

PubMed

Lange, Claudia; Kuch, Bertram; Metzger, Jörg W

2014-08-01

The purpose of this study was to ascertain whether different kinds of underarm deodorants commercially available in Germany might contain substances with estrogenic potential which after use enter the aquatic environment via wastewater. Twenty five deodorants produced by ten different manufacturers in the form of sprays, roll-ons and sticks were investigated using an in vitro-test system (E-Screen assay) for the determination of estrogenic activity based on the human breast cancer cell line MCF-7. Seven out of ten spray deodorant samples showed a quantifiable estrogenic activity. In the case of the sticks and roll-ons it was only one out of six and one out of nine, respectively. The 17β-estradiol equivalent concentrations (EEQs) of the samples ranged from 0.1 ng g(-1) to 9 ng g(-1) deodorant. Spray deodorant samples showed the highest activities in the E-Screen assay compared to the stick and roll-on deodorants. In order to identify substances possibly contributing to the observed biological activity the samples were additionally analyzed by GC/MS. The obtained results of this non-target screening led to the selection of 62 single substances present in the deodorants which for their part were analyzed by E-Screen assay. Eight of these single substances, all of them fragrances, showed estrogenic effects with estradiol equivalence factors (EEFs) similar to parabens, a group of 4-hydroxybenzoic acid esters commonly used as preservatives in personal care products, which are known to have a slight estrogenic effect. Thus, these fragrances are obviously responsible to a substantial degree for the observed estrogenic activity of the deodorants. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

2010-11-02

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. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Profiling 976 ToxCast Chemicals across 331 Enzymatic and Receptor Signaling Assays

PubMed Central

2013-01-01

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

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

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

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain

2016-09-15

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

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

PubMed

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

2012-12-01

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

Novel enzymatic method for assaying Lp-PLA2 in serum.

PubMed

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

2018-06-01

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

Lack of activity of cadmium in in vitro estrogenicity assays

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

Silva, Elisabete; Lopez-Espinosa, Maria Jose; Molina-Molina, Jose-Manuel

2006-10-01

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

Modification of the cellular antioxidant activity (CAA) assay to study phenolic antioxidants in a Caco-2 cell line.

PubMed

Kellett, Mary E; Greenspan, Phillip; Pegg, Ronald B

2018-04-01

In vitro assays are widely used to analyze the antioxidant potential of compounds, but they cannot accurately predict antioxidant behavior in living systems. Cell-based assays, like the cellular antioxidant activity (CAA) assay, are gaining importance as they provide a biological perspective. When the CAA assay was employed to study phenolic antioxidants using hepatocarcinoma (HepG2) cells, quercetin showed antioxidant activity in HepG2 cells; 25 and 250μM quercetin reduced fluorescence by 17.1±0.9% and 58.6±2.4%, respectively. (+)-Catechin, a phenolic antioxidant present in many foods, bestowed virtually no CAA in HepG2 cells. When Caco-2 cells were employed, more robust antioxidant activity was observed; 50μM (+)-catechin and quercetin reduced fluorescence by 54.1±1.4% and 63.6±0.9%, respectively. Based on these results, likely due to differences in active membrane transport between the cell types, the Caco-2-based CAA assay appears to be a more appropriate method for the study of certain dietary phenolics. Copyright © 2017 Elsevier Ltd. All rights reserved.

S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

PubMed

Lv, Zongyang; Rickman, Kimberly A; Yuan, Lingmin; Williams, Katelyn; Selvam, Shanmugam Panneer; Woosley, Alec N; Howe, Philip H; Ogretmen, Besim; Smogorzewska, Agata; Olsen, Shaun K

2017-02-16

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Assay of Deoxyhypusine Synthase Activity

PubMed Central

Wolff, Edith C.; Lee, Seung Bum; Park, Myung Hee

2011-01-01

Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H] deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter. PMID:21318875

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.

Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.

PubMed

Ranganathan, Raj; Lenti, Gena; Tassone, Nicholas M; Scannell, Brian J; Southern, Cathrine A; Karver, Caitlin E

2018-02-15

Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (K M  = 15 ± 2 μM), WEHD-MCA (K M  = 93 ± 19 μM), WEHDG-MCA (K M  = 21 ± 6 μM) and WEHDA-MCA (K M  = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

PubMed Central

Hurt, Ed; Hannus, Stefan; Schmelzl, Birgit; Lau, Denise; Tollervey, David; Simos, George

1999-01-01

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes. PMID:9971735

Resonance scattering spectra of micrococcus lysodeikticus and its application to assay of lysozyme activity.

PubMed

Jiang, Zhi-Liang; Huang, Guo-Xia

2007-02-01

Several methods, including turbidimetric and colorimetric methods, have been reported for the detection of lysozyme activity. However, there is no report about the resonance scattering spectral (RSS) assay, which is based on the catalytic effect of lysozyme on the hydrolysis of micrococcus lysodeikticus (ML) and its resonance scattering effect. ML has 5 resonance scattering peaks at 360 400, 420, 470, and 520 nm with the strongest one at 470 nm. The concentration of ML in the range of 2.0x10(6)-9.3x10(8) cells/ml is proportional to the RS intensity at 470 nm (I(470 nm)). A new catalytic RSS method has been proposed for 0.24-40.0 U/ml (or 0.012-2.0 mug/ml) lysozyme activity, with a detection limit (3sigma) of 0.014 U/ml (or 0.0007 microg/ml). Saliva samples were assayed by this method, and it is in agreement with the results of turbidimetric method. The slope, intercept and the correlation coefficient of the regression analysis of the 2 assays were 0.9665, -87.50, and 0.9973, respectively. The assay has high sensitivity and simplicity.

A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

EPA Science Inventory

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently...

Combined IFN-γ and TNF-α release assay for differentiating active tuberculosis from latent tuberculosis infection.

PubMed

Kim, Ji Yeun; Park, Joung Ha; Kim, Min-Chul; Cha, Hye Hee; Jeon, Na-Young; Park, Seong Yeon; Kim, Min-Jae; Chong, Yong Pil; Lee, Sang-Oh; Choi, Sang-Ho; Kim, Yang Soo; Woo, Jun Hee; Kim, Sung-Han

2018-05-08

The IFN-γ-release assay (IGRA) cannot differentiate active tuberculosis (TB) from latent TB infection (LTBI). We hypothesized that the TNF-α-release assay (TARA) combined with IGRA might discriminate active TB from not active TB without LTBI. Adult patients with suspected TB, and with unrelated diseases such as herpes zoster as controls, were enrolled in an intermediate TB-burden country. Patients with confirmed or probable TB were regarded as active TB, and patients with not active TB were further classified as those having not active TB with and without LTBI based on IGRA results. The IGRA and TARA by using ELISPOT assays were performed on peripheral mononuclear cells. Thirty six patients with active TB and 53 patients including 18 not active TB with LTBI and 35 not active TB without LTBI were finally included. The sensitivity and specificity of the IGRA for those patients found to have active TB were 94% (CI, 80-99) and 66% (CI 52-78), respectively. Combining the IGRA and the TARA substantially increased the specificity for active TB (93%, CI, 82-98; P = 0.001) compared with the IGRA only, without compromising sensitivity (89%, CI, 73-96; P = 0.67). Combining the IGRA and TARA appears to be useful for diagnosing active TB. Copyright © 2018. Published by Elsevier Ltd.

No clastogenic activity of a senna extract in the mouse micronucleus assay.

PubMed

Mengs, U; Grimminger, W; Krumbiegel, G; Schuler, D; Silber, W; Völkner, W

1999-08-18

In previous studies, an analytically well-defined senna extract, commonly used as a laxative, gave positive responses in vitro in the Ames test and in the CHO assay. Therefore, the objective of this study was to investigate the genotoxic activity of the same senna extract in an in vivo genotoxicity assay by means of the generally acknowledged MNT. After administration of an oral dose of 2000 mg senna extract/kg to NMRI mice of both genders, which is equivalent to 119 mg potential rhein/kg, 5.74 mg potential aloeemodin/kg and 0. 28 mg potential emodin/kg, there were no elevated levels of micronuclei in bone marrow cells. Kinetic studies were performed in parallel to demonstrate target organ availability. Highest concentrations in the plasma were reached after 1 h with 3.4 microg rhein/ml and 0.065 microg aloeemodin/ml. In all cases, emodin was below the limit of quantification. From the results, the in vitro clastogenic activity of the senna extract could not be confirmed in the mouse micronucleus assay. Together with further negative in vivo genotoxicity studies with anthranoids, the conclusion can be drawn that there is no indication so far demonstrating a genotoxic risk for patients taking senna laxatives.

Optimization of a resazurin-based microplate assay for large-scale compound screenings against Klebsiella pneumoniae.

PubMed

Kim, Hyung Jun; Jang, Soojin

2018-01-01

A new resazurin-based assay was evaluated and optimized using a microplate (384-well) format for high-throughput screening of antibacterial molecules against Klebsiella pneumoniae . Growth of the bacteria in 384-well plates was more effectively measured and had a > sixfold higher signal-to-background ratio using the resazurin-based assay compared with absorbance measurements at 600 nm. Determination of minimum inhibitory concentrations of the antibiotics revealed that the optimized assay quantitatively measured antibacterial activity of various antibiotics. An edge effect observed in the initial assay was significantly reduced using a 1-h incubation of the bacteria-containing plates at room temperature. There was an approximately 10% decrease in signal variability between the edge and the middle wells along with improvement in the assay robustness ( Z ' = 0.99). This optimized resazurin-based assay is an efficient, inexpensive, and robust assay that can quantitatively measure antibacterial activity using a high-throughput screening system to assess a large number of compounds for discovery of new antibiotics against K. pneumoniae .

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

PubMed

Greer, Michael S; Zhou, Ting; Weselake, Randall J

2014-08-01

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-(14)C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Antibacterial assay-guided isolation of active compounds from Artocarpus heterophyllus heartwoods.

PubMed

Septama, Abdi Wira; Panichayupakaranant, Pharkphoom

2015-01-01

Preparations from Artocarpus heterophyllus Lam. (Moraceae) heartwoods are used in the traditional folk medicine for the treatment of inflammation, malarial fever, and to prevent bacterial and fungal infections. The objective of this study was to isolate pure antibacterial compounds from A. heterophyllus heartwoods. The dried and powdered A. heterophyllus heartwoods were successively extracted with the following solvents: hexane, ethyl acetate, and methanol. Each of the extracts was screened for their antibacterial activities using a disc diffusion method (10 mg/disc). Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using a broth microdilution method. The extract that showed the strongest antibacterial activities was fractionated to isolate the active compounds by an antibacterial assay-guided isolation process. The ethyl acetate extract exhibited the strongest antibacterial activities against Streptococcus mutans, S. pyogenes, and Bacillus subtilis with MIC values of 78, 39, and 9.8 µg/mL, respectively. Based on an antibacterial assay-guided isolation, four antibacterial compounds: cycloartocarpin (1), artocarpin (2), artocarpanone (3), and cyanomaclurin (4) were purified. Among these isolated compounds, artocarpin exhibited the strongest antibacterial activity against Gram-positive bacteria, including S. mutans, S. pyogenes, B. subtilis, Staphylococcus aureus, and S. epidermidis with MICs of 4.4, 4.4, 17.8, 8.9, and 8.9 µM, respectively, and MBCs of 8.9, 8.9, 17.8, 8.9, and 8.9 µM, respectively, while artocarpanone showed the strongest activity against Escherichia coli, a Gram-negative bacteria with MIC and MBC values of 12.9 and 25.8 µM, respectively. Only artocarpin showed inhibitory activity against Pseudomonas aeruginosa with an MIC of 286.4 µM.

Plasma Endothelial Microparticles in Multiple Sclerosis: A Novel Metric Assay of Disease Activity and Response to Treatment

DTIC Science & Technology

2011-09-01

direct link to a source of increased tissue iron deposition which is characteristic for multiple sclerosis . The results of these data and their... Multiple Sclerosis : A Novel Metric Assay of Disease Activity and Response to Treatment PRINCIPAL INVESTIGATOR: Jonathan Steven...SUBTITLE 5a. CONTRACT NUMBER Plasma Endothelial Microparticles in Multiple Sclerosis : A Novel Metric Assay of Disease Activity and Response to

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

PubMed

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

2007-09-01

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

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity.

PubMed

Glogowski, J; Jankowski, J; Faruga, A; Ottobre, J S; Ciereszko, A

2001-09-15

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

Automated locomotor activity monitoring as a quality control assay for mass-reared tephritid flies.

PubMed

Dominiak, Bernard C; Fanson, Benjamin G; Collins, Samuel R; Taylor, Phillip W

2014-02-01

The Sterile Insect Technique (SIT) requires vast numbers of consistently high quality insects to be produced over long periods. Quality control (QC) procedures are critical to effective SIT, both providing quality assurance and warning of operational deficiencies. We here present a potential new QC assay for mass rearing of Queensland fruit flies (Bactrocera tryoni Froggatt) for SIT; locomotor activity monitoring. We investigated whether automated locomotor activity monitors (LAMs) that simply detect how often a fly passes an infrared sensor in a glass tube might provide similar insights but with much greater economy. Activity levels were generally lower for females than for males, and declined over five days in the monitor for both sexes. Female activity levels were not affected by irradiation, but males irradiated at 60 or 70 Gy had reduced activity levels compared with unirradiated controls. We also found some evidence that mild heat shock of pupae results in adults with reduced activity. LAM offers a convenient, effective and economical assay to probe such changes. © 2013 Society of Chemical Industry.

Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

PubMed

Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

2017-06-28

The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

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

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

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung

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 themore » 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.« less

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils.

PubMed

Etebu, E; Osborn, A M

2009-05-01

The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0-5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3.88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.

Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

PubMed

Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

2009-01-01

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.

Beta-endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays.

PubMed

Ho, C L; Li, C H

1985-03-01

Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.

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. Copyright © 2012 Elsevier Inc. All rights reserved.

RAS - Screens & Assays

Cancer.gov

A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays.

PubMed

Mahapatra, Debabrata; Franzosa, Jill A; Roell, Kyle; Kuenemann, Melaine Agnes; Houck, Keith A; Reif, David M; Fourches, Denis; Kullman, Seth W

2018-06-11

High throughput screening (HTS) programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals. In this study, we examined the Tox21 qHTS data set generated against VDR for reproducibility and concordance and elucidated functional insights into VDR-xenobiotic interactions. Twenty-one potential VDR agonists and 19 VDR antagonists were identified from a subset of >400 compounds with putative VDR activity and examined for VDR functionality utilizing select orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp24 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. Mammalian-2-hybrid assay (M2H) was then used to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited significant recruitment of co-regulators and co-activators whereas antagonists exhibited considerable attenuation of recruitment by VDR. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Cheminformatics modeling of VDR-ligand interactions were conducted and revealed selective ligand VDR interaction. Overall, data emphasizes the molecular complexity of ligand-mediated interactions with VDR and suggest that VDR transactivation may be a target site of action for diverse xenobiotics.

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.

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

PubMed Central

2012-01-01

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.

PubMed

Ruggiero, Alicia M; Wright, Jane; Ferguson, Shawn M; Lewis, Michelle; Emerson, Katie S; Iwamoto, Hideki; Ivy, Michael T; Holmstrand, Ericka C; Ennis, Elizabeth A; Weaver, C David; Blakely, Randy D

2012-10-17

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

PubMed

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

2013-01-01

The intermediate-conductance Ca(2+)-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 Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) 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.

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...

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.

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

PubMed

Pick, Edgar

2014-01-01

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

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

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

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

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 addedmore » 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.« less

Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF-7 Cells.

PubMed

Potratz, Sarah; Tarnow, Patrick; Jungnickel, Harald; Baumann, Sven; von Bergen, Martin; Tralau, Tewes; Luch, Andreas

2017-04-17

The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17β-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes in vitro. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd 2+ ). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd 2+ . Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound's estrogenic potency. Co-exposure to Cd 2+ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd 2+ -specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

PubMed

Murakami, Chiaki; Mizuno, Satoru; Kado, Sayaka; Sakane, Fumio

2017-06-01

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. Copyright © 2017 Elsevier Inc. All rights reserved.

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of a New IFN-γ Release Assay for Rapid Diagnosis of Active Tuberculosis in a High-Incidence Setting.

PubMed

Li, Gen; Li, Feng; Zhao, Hui-Min; Wen, Han-Li; Li, Hai-Cong; Li, Chun-Ling; Ji, Ping; Xu, Peng; Wu, Kang; Hu, Zhi-Dong; Lu, Shui-Hua; Lowrie, Douglas B; Lv, Jian-Xin; Fan, Xiao-Yong

2017-01-01

Blood-based interferon-gamma (IFN-γ) release assays (IGRAs) have been proven to be useful in the diagnosis of Mycobacterium tuberculosis ( Mtb ) infection. However, IGRAs have not been recommended for clinical practice in most low-income settings due to cost-intensive limitations and shortage of clinical data available. The established T-SPOT. TB assay containing Mtb -specific antigens ESAT-6 and CFP10 are widely used for immunodiagonsis of Mtb infection, but the high cost is one of the restricting factors against its clinical application in the developing countries. More recently, a cost-saving IGRA assay, TS-SPOT, was approved in China. This new assay contains an additional antigen Rv3615c. Rv3615c contains broadly recognized CD4 + and CD8 + epitopes, and T-cell responses to Rv3615c are as specific for Mtb infection as the responses to ESAT-6 and CFP10 in both Mtb -infected humans and M. bovis -infected cattle. Therefore, we assessed the likely effect of inclusion of Rv3615c as stimulus besides ESAT-6 and CFP10 in an IGRA assay and evaluated the performance of TS-SPOT for diagnosis of Mtb infection and active TB compared with T-SPOT. TB . We tested 155 active TB patients, 90 non-TB lung disease patients, and 55 healthy individuals. The results presented an improved positive rate for diagnosis of active TB and Mtb infection, that could be attributable to inclusion of Rv3615c in the mixture of stimulatory antigens. The diagnostic efficiency of TS-SPOT assay for active TB was as follows: sensitivity 80.00%, specificity 83.45%, positive predictive value (PPV) 83.78%, negative predictive value (NPV) 83.45%, positive likelihood ratio (LR+) 4.83, and negative likelihood ratio (LR-) 0.24. The results were similar to those of T-SPOT. TB , with an excellent agreement (κ = 0.91, 95% CI: 0.85-0.95) being observed between these two assays. The sensitivities of the TS-SPOT assay varied for patients with different forms of active TB, with the highest sensitivity for patients

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

NASA Astrophysics Data System (ADS)

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

2012-04-01

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

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

PubMed

Petryayeva, Eleonora; Algar, W Russ

2014-03-18

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

Evaluation of a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) activity enzyme-linked immunosorbent assay for measuring plasma ADAMTS13 activity in dogs.

PubMed

Maruyama, Haruhiko; Kaneko, Michiko; Otake, Taiga; Kano, Rui; Yamaya, Yoshiki; Watari, Toshihiro; Hasegawa, Atsuhiko; Kamata, Hiroshi

2014-03-01

A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0-12.4%, and interassay CV was 11.5-12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs (P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.

Screening of Neem extracts for microbial anti-chaperone activity by employing in vitro enzyme refolding assay.

PubMed

Patki, Jyoti M; Shah, Priyanka

2017-10-01

Microbial heat shock proteins (Hsps) play an important role in pathogenesis and development of resistance to existing drugs. New compounds that target microbial molecular chaperones have the potential of combating the challenge of anti-microbial resistance. The present study was aimed at assessing the employment of in vitro enzyme refolding assay to detect anti-chaperone activity of Neem ( Azadirachta indica ) extracts. Protein extracts of thermotolerant Escherichia coli cells were used as a source of Hsps or chaperones. Thermotolerance was found to be induced by pre-treating E. coli cells at 47 °C before subjecting them to a lethal temperature of 55 °C. This thermotolerance correlated with over-expression of specific proteins and reduced aggregation as evident from the SDS-PAGE profiles. Refolding assays of denatured enzymes exhibited 45% activity regain in presence of cell protein extracts containing chaperones compared to less than 5% regain in BSA negative controls. The chaperone activity was found to be ATP dependent. Addition of Neem extracts to refolding reaction mixtures distinctly reduced the activity regain (20%) in a dose dependent manner (500 and 1000 ppm). The negative influence of plant extract on refolding of the enzyme in the presence of chaperones gives evidence to its anti-chaperone activity. We propose that the employment of in vitro enzyme refolding assays will help not only to analyze the activity of known and putative chaperones but also to screen natural compounds for anti-microbial-Hsp activity.

A high-throughput, modified ALS activity assay for Cyperus difformis and Schoenoplectus mucronatus seedlings.

PubMed

Pedroso, Rafael M; Al-Khatib, Kassim; Hanson, Bradley D; Fischer, Albert J

2017-01-01

Cyperus difformis L. (CYPDI) and Schoenoplectus mucronatus (L.) Palla (SCHMU) are major weeds of California (CA) rice, where resistance to acetolactate synthase (ALS)-inhibitors was identified in several CYPDI and SCHMU populations that have also evolved resistance to photosystem II (PSII)-inhibiting herbicides. The mechanism of ALS resistance in these populations remains to be clarified but this information is crucial in a weed management program, especially in a scenario where resistance to multiple herbicides has been identified. ALS activity assays are commonly used to diagnose resistance to ALS-inhibitors, but protocols currently available are burdensome for the study of CYPDI and SCHMU, as they require large amounts of plant material from young seedlings and have low yields. Our objective was to investigate the ALS resistance mechanism in suspected ALS-resistant (R) CYPDI and SCHMU biotypes using a modified ALS activity assay that requires less plant material. ALS enzymes from suspected R biotypes were at least 10,000-fold less sensitive to bensulfuron-methyl than susceptible (S) cohorts, indicating ALS resistance that is likely due to an altered target-site. Protein concentration (mgg -1 tissue) did not differ between R and S biotypes within each species, suggesting that R biotypes do not over produce ALS enzymes. CYPDI biotypes had up to 4-fold more protein per mg of tissue than SCHMU biotypes, but up to 7-fold more acetoin per mg -1 protein was quantified in SCHMU, suggesting greater ALS catalytic ability in SCHMU biotypes, regardless of their herbicide resistance status. Our optimized protocol to measure ALS activity allowed for up to a 3-fold increase in the number of assays performed per g of leaf tissue. The modified assay may be useful for measuring ALS activity in other weed species that also produce small amount of foliage in early growth stages when protein in tissue is most abundant. Copyright © 2016 Elsevier B.V. All rights reserved.

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity.

PubMed

Zheng, Jianyong; Wei, Wei; Lan, Xing; Zhang, Yinjun; Wang, Zhao

2018-05-15

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

PubMed Central

Sadikot, Takrima; Swink, Megan; Eskew, Jeffery D.; Brown, Douglas; Zhao, Huiping; Kusuma, Bhaskar R.; Rajewski, Roger A.; Blagg, Brian S. J.; Matts, Robert L.; Holzbeierlein, Jeffrey M.

2013-01-01

Abstract The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu. PMID:24127661

A novel assay system for macrophage-activating factor activity using a human U937 cell line.

PubMed

Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2014-08-01

Macrophages play important roles in antitumor immunity, and immunotherapy with the group-specific component protein-derived macrophage-activating factor (GcMAF) has been reported to be effective in patients with various types of cancers. However, in macrophage research, it is important to properly evaluate macrophage activity. U937 macrophages were induced by 12-O-tetradecanoyl-13-phorbolacetate (TPA). The phagocytic activity of macrophages was evaluated as the internalized beads ratio. The MAF activity was assessed at 30 min after MAF addition as the activation ratio. We established a novel assay for phagocytic activities using differentiated U937 macrophages. The novel protocol was simple and rapid and was sensitive for GcMAF. This protocol should be useful not only for basic studies, such as those on molecular mechanisms underlying macrophage activation, but also for clinical studies, such as assessment of GcMAF activity prior to clinical use. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

"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.

A vapour phase assay for evaluating the antimicrobial activities of essential oils against bovine respiratory bacterial pathogens.

PubMed

Amat, S; Baines, D; Alexander, T W

2017-12-01

The objectives of this study were to develop a new assay for the evaluation of the antimicrobial activities of essential oils (EOs) in vapour phase and to demonstrate the antimicrobial activities of commercial EOs against BRPs. To achieve the first objective, a microtube cap containing 100 μl of EO was embedded in an agar plate. An agar plug (diameter 13 mm) inoculated with a bacterial suspension containing10 8  CFU per ml was then placed over the cap and incubated at 37°C for 24 h. Subsequently, bacteria were recovered from the agar plug by immersion in 5 ml of broth for 10 min, followed by vortexing for 30 s, and the broths were then plated for enumeration. To demonstrate the usefulness of the assay, nine commercial EOs derived from the following specific plants: ajowan, carrot seed, cinnamon leaf, citronella, fennel, ginger grass, lavender, rosemary and thyme were first evaluated for their vapour phase antimicrobial activities against Mannheimia haemolytica serotype 1. Selected EOs were further tested against Pasteurella multocida and Histophilus somni. The EOs of ajowan, thyme and cinnamon leaf completely or partially inhibited BRPs growth. This new assay provided reproducible results on the vapour phase antimicrobial activities of EOs against BRPs. These results support further study of EOs as a potential mitigation strategy against BRPs. In this study, we present a new vapour phase assay for evaluating the antimicrobial activities of essential oils (EO) against bovine respiratory pathogens (BRPs). Using this assay, we identified EOs, such as ajowan, thyme and cinnamon leaf, that can effectively inhibit growth of the BRPs Mannheimia haemolytica serotype 1, Pasteurella multocida and Histophilus somni. This is the first study to demonstrate the vapour phase antimicrobial activity of EOs against BRPs. © 2017 Her Majesty the Queen in Right of Canada. © 2017 The Society for Applied Microbiology Reproduced with the permission of the Minister of the

Accounting Artifacts in High-Throughput Toxicity Assays.

PubMed

Hsieh, Jui-Hua

2016-01-01

Compound activity identification is the primary goal in high-throughput screening (HTS) assays. However, assay artifacts including both systematic (e.g., compound auto-fluorescence) and nonsystematic (e.g., noise) complicate activity interpretation. In addition, other than the traditional potency parameter, half-maximal effect concentration (EC50), additional activity parameters (e.g., point-of-departure, POD) could be derived from HTS data for activity profiling. A data analysis pipeline has been developed to handle the artifacts and to provide compound activity characterization with either binary or continuous metrics. This chapter outlines the steps in the pipeline using Tox21 glucocorticoid receptor (GR) β-lactamase assays, including the formats to identify either agonists or antagonists, as well as the counter-screen assays for identifying artifacts as examples. The steps can be applied to other lower-throughput assays with concentration-response data.

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

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

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE PAGES

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; ...

2017-03-08

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

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.

A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation.

PubMed

Eisenhardt, Nathalie; Chaugule, Viduth K; Pichler, Andrea

2016-01-01

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.

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. Copyright © 2011 Elsevier Inc. All rights reserved.

A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

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

Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors thatmore » block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.« less

EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

PubMed

Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

2013-01-01

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

PubMed Central

Sykes, Melissa L.; Avery, Vicky M.

2015-01-01

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed. PMID:27120069

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes.

PubMed

Sykes, Melissa L; Avery, Vicky M

2015-12-01

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Soil-free System for Assaying Nematicidal Activity of Chemicals

PubMed Central

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

1981-01-01

A biological assay system for studying the nematicidal activity of chemicals has been devised using a model consisting of cucumber (Cucumis sativus L. cv. Long Marketer) seedlings growing in the diSPo® growth-pouch apparatus. Meloidogyne incognita was used as the test organism. The response was quantified in terms of the numbers of galls produced. Statistical procedures were applied to estimate the ED50 values of currently available nematicides. This system permits accurate quantification of galling and requires much less space and effort than the currently used methods. PMID:19300800

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

PubMed

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

1981-10-01

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

Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study.

PubMed

Bowyer, A E; Hillarp, A; Ezban, M; Persson, P; Kitchen, S

2016-07-01

Essentials Validated assays are required to precisely measure factor IX (FIX) activity in FIX products. N9-GP and two other FIX products were assessed in various coagulation assay systems at two sites. Large variations in FIX activity measurements were observed for N9-GP using some assays. One-stage and chromogenic assays accurately measuring FIX activity for N9-GP were identified. Background Measurement of factor IX activity (FIX:C) with activated partial thromboplastin time-based one-stage clotting assays is associated with a large degree of interlaboratory variation in samples containing glycoPEGylated recombinant FIX (rFIX), i.e. nonacog beta pegol (N9-GP). Validation and qualification of specific assays and conditions are necessary for the accurate assessment of FIX:C in samples containing N9-GP. Objectives To assess the accuracy of various one-stage clotting and chromogenic assays for measuring FIX:C in samples containing N9-GP as compared with samples containing rFIX or plasma-derived FIX (pdFIX) across two laboratory sites. Methods FIX:C, in severe hemophilia B plasma spiked with a range of concentrations (from very low, i.e. 0.03 IU mL(-1) , to high, i.e. 0.90 IU mL(-1) ) of N9-GP, rFIX (BeneFIX), and pdFIX (Mononine), was determined at two laboratory sites with 10 commercially available one-stage clotting assays and two chromogenic FIX:C assays. Assays were performed with a plasma calibrator and different analyzers. Results A high degree of variation in FIX:C measurement was observed for one-stage clotting assays for N9-GP as compared with rFIX or pdFIX. Acceptable N9-GP recovery was observed in the low-concentration to high-concentration samples tested with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays. Similar patterns of FIX:C measurement were observed at both laboratory sites, with minor differences probably being attributable to the use of different analyzers. Conclusions These results suggest that, of the

Comparative antioxidant activity of cultivated and wild Vaccinium species investigated by EPR, human neutrophil burst and COMET assay.

PubMed

Braga, P C; Antonacci, R; Wang, Y Y; Lattuada, N; Dal Sasso, M; Marabini, L; Fibiani, M; Lo Scalzo, R

2013-01-01

The Vaccinium (V.) spp. berries are considered a source of antioxidants, mainly belonging to polyphenols, specifically flavonoids and anthocyanins. Wild genotypes generally contain more antioxidants than cultivated counterparts. So, seven different antioxidants assays on extracts from cultivated and wild Vaccinium berries were performed, to evaluate their difference in terms of bioactivity on oxidative protection and minimum dosage to have a significant action. Four cell-free antioxidant assays (ABTS radical scavenging and electronic paramagnetic resonance using Fremy's salt, superoxide anion and hydroxyl radical), and three assays on human cells (two luminol amplified chemiluminescence, LACL, one on DNA damage, COMET) were used to measure the effects of cultivated blueberry (V. corymbosum) and wild bilberry (V. myrtillus) on the differently induced oxidative stress. Concentrations vs activity patterns were obtained by successive dilutions of extracts in order to identify both EC50 and minimum significant activity (MSA). All the assays (except for the hydroxyl radical scavenging) showed a good relationship mainly with anthocyanin and polyphenol content and the significant greater activity of wild Vaccinium extracts. In fact, LACL data gave an EC50 of 11.8 and an MSA of 5.2 g were calculated as fresh weight dosage in cultivated berries, compared with lower doses in wild berries, EC50 of 5.7 g and MSA of 3.4 g. Wild Vaccinium extracts averaged 3.04 and 2.40 fold more activity than cultivated extracts by EC50 and MSA, respectively. COMET assay confirmed the stronger action on DNA protection in wild samples.

Application of manual assessment of oxygen radical absorbent capacity (ORAC) for use in high throughput assay of "total" antioxidant activity of drugs and natural products.

PubMed

Price, Joseph A; Sanny, Charles G; Shevlin, Dennis

2006-01-01

Antioxidants are of particular interest in a spectrum of diseases, and thus are an active area of drug discovery and design. It is important to make considered choices as to which assay chemistry will best serve for particular investigations. We examined the manual oxygen radical absorbent capacity (ORAC) assay for "total" antioxidant activity, including a direct comparison to an alternative technique, the AOP-490 assay, using a panel of extracts from 12 phylogenetically unrelated algae. The AOP-490 assay was done per manufacturer's protocol. The ORAC assay was done by hand, in 96-well plates, not by machine as had been previously published. Our ORAC calculations were done using an in-experiment antioxidant standard curve. Results were reported as equivalents of the antioxidant Trolox, which was used as a standard. With the AOP-490 kit (from Oxis Research) widespread activity was found, but not in all samples. When the ORAC method was used to assay aliquots of the same extracts there was significant activity detected in all samples, and the rank order of activity by the two methods was not identical. The data showed the wide occurrence of antioxidants in algae. The standard curve with the manual ORAC assay was linear in the range tested (0-100 mM Trolox) and had excellent reproducibility. The importance of the beneficial effects of antioxidants is currently an area of active interest for drug development, and thus it is of great value to have an assay that is robust and approximates "total" antioxidant activity in a high throughput format. The ORAC (oxygen radical absorbent capacity) method was adapted to microplates and an eight-channel pipette and was more effective in detecting "total" antioxidant activity than the AOP-490 assay. These results might vary with other types of samples, and would depend on the active agents measured, but do suggest the practical value of the ORAC assay for any laboratory not ready for robotics but using manual 96-well format assays

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

PubMed

Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

2014-07-01

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. © 2014 Society for Laboratory Automation and Screening.

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

Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins ▿

PubMed Central

Carney, Paul J.; Lipatov, Aleksandr S.; Monto, Arnold S.; Donis, Ruben O.; Stevens, James

2010-01-01

During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities. PMID:20660137

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

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

Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong

2009-11-10

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenzamore » H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.« less

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

PubMed

Banasik, Michał; Sachadyn, Paweł

2016-09-01

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

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...

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...

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

PubMed

Pavelka, S

2014-01-01

We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

PubMed

Zhang, Hua; Zhao, Zhen; Lei, Zhen; Wang, Zhenxin

2016-12-06

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL -1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

PubMed

Caron, C; Meley, R; Le Cam Duchez, V; Aillaud, M F; Lavenu-Bombled, C; Dutrillaux, F; Flaujac, C; Ryman, A; Ternisien, C; Lasne, D; Galinat, H; Pouplard, C

2017-06-01

Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay ® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom ® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay ® with the Berichrom ® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom ® FXIII activity but normal antigen level and clot solubility as expected. The measurement of FXIII antigen using the K-Assay ® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available. © 2017 John Wiley & Sons Ltd.

An electro-active system of immuno-assay (EASI assay) utilising self assembled monolayer modified electrodes.

PubMed

Porter, R; van der Logt, P; Howell, S; Kyröläinen-Reay, M; Badley, A

2001-12-01

Most immunoassays currently rely on optical methods for signal generation e.g. in ELISA and rapid assay formats. It has become apparent as in the Glucose sensor market that there is a need for simple direct electrical immuno-sensors. We have investigated the novel use of organic conducting monolayers used as a direct electrochemical detection support for an immuno-reaction. It was found that antibodies raised to a carbazole dimer monolayer could increase the charge movement across that monolayer surface. Antibody fragments were taken from a specific anti-carbazole antibody fragment library and combined with an antibody fragment directed to the hormone estrone 3 glucuronide (E3G), the target antigen to form a bispecific antibody fragment. The device utilised these specific antibody fragments and incorporated them on the top plate of a capillary fill format as the immuno-assay components. The immuno-reaction utilised a competition assay. Free E3G analyte in the sample displaced the bispecific antibody fragment from the immuno-surface leaving it free to bind the carbazole monolayer surface. There the binding was detected using amperometric or coulometric methods. By combining all there element it was possible to develop a sensitive immuno-assay that could detect E3G in a reproducible calibrated fashion down to 10 ng/ml.

Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

PubMed

Lumor, Stephen E; Fredrickson, Neal R; Ronningen, Ian; Deen, Bronwyn D; Smith, Kenneth; Diez-Gonzalez, Francisco; Labuza, Theodore P

2012-06-01

This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

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. Copyright © 2014 John Wiley & Sons, Inc.

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.

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

PubMed

Barabas, Sascha; Spindler, Theresa; Kiener, Richard; Tonar, Charlotte; Lugner, Tamara; Batzilla, Julia; Bendfeldt, Hanna; Rascle, Anne; Asbach, Benedikt; Wagner, Ralf; Deml, Ludwig

2017-03-07

In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions. Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay. Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 10 4 and 2 × 10 5 PBMC per well upon stimulation with T-activated® IE-1 (R 2  = 0.97) and pp65 (R 2  = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3 + CD4 + (Th), CD3 + CD8 + (CTL), CD3 - CD56 + (NK) and CD3 + CD56 + (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive. The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay.

PubMed

Houk, V S; Claxton, L D

1986-03-01

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bjørseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.

Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

PubMed

Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

2016-03-17

L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

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

PubMed

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

2011-04-01

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

Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

PubMed

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

2016-07-15

Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

PubMed Central

de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

2016-01-01

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

PubMed Central

Fonnum, F.

1969-01-01

1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-14C]acetate or sodium [3H]acetate by incubation with CoA, ATP, Mg2+ and extract from acetone-dried pigeon liver were used. 3. [1-14C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [3H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [3H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively. PMID:4982085

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

PubMed

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

2017-02-01

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

In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrP(C) to PrP(Sc).

PubMed

Pagadala, Nataraj S; Perez-Pineiro, Rolando; Wishart, David S; Tuszynski, Jack A

2015-02-16

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 μM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 μM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between β2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while

Studies on the genotoxic properties of essential oils with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay.

PubMed

Zani, F; Massimo, G; Benvenuti, S; Bianchi, A; Albasini, A; Melegari, M; Vampa, G; Bellotti, A; Mazza, P

1991-06-01

Genotoxic properties of essential oils from Anthemis nobilis L., Artemisia dracunculus L., Salvia officinalis L., Salvia sclarea L., Satureja hortensis L., Satureja montana L., Thymus capitatus L., Thymus citriodorus Schreb., Thymus vulgaris L., Citrus bergamia Risso, were studied with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay. The essential oil of Artemisia dracunculus L. "Piemontese" turned out to be active in the rec-assay but not in the Salmonella test. DNA-damaging activity was demonstrated to be due to the estragol component of the oil. Advantages of the combined use of these two short-term microbial assays in genotoxic studies are discussed.

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.

An assay of optimal cytochrome c oxidase activity in fish gills.

PubMed

Hu, Yau-Chung; Chung, Meng-Han; Lee, Tsung-Han

2018-07-15

Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 μg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

2015-06-06

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

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

Luminogenic cytochrome P450 assays.

PubMed

Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

2006-08-01

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

PubMed Central

2012-01-01

Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes. PMID:22424588

Luciferase assay to study the activity of a cloned promoter DNA fragment.

PubMed

Solberg, Nina; Krauss, Stefan

2013-01-01

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(®) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.

An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

PubMed Central

Tanigawa, Mirai

2017-01-01

ABSTRACT Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism. PMID:28483912

Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

PubMed

Bausinger, Julia; Schütz, Petra; Piberger, Ann Liza; Speit, Günter

2016-03-01

The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the

A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

PubMed

Gagnon, David; Archambault, Jacques

2015-01-01

A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

PubMed Central

Jackson, Colin R.; Tyler, Heather L.; Millar, Justin J.

2013-01-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample. PMID:24121617

Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.

PubMed

Jackson, Colin R; Tyler, Heather L; Millar, Justin J

2013-10-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Indole-based assay to assess the effect of ethanol on Pseudomonas putida F1 dioxygenase activity.

PubMed

da Silva, Márcio Luis Busi; Alvarez, Pedro J J

2010-06-01

Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 +/- 0.0006 OD(610) min(-1). The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.

Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR

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

Tao, Jiahui; Petrova, Kseniya; Ron, David

2010-05-25

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 {angstrom} resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting withmore » misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).« less

Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.

PubMed

Fezza, Filomena; Gasperi, Valeria; Mazzei, Cinzia; Maccarrone, Mauro

2005-04-01

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

PubMed Central

Macovei, Anca; Faè, Matteo; Biggiogera, Marco; de Sousa Araújo, Susana; Carbonera, Daniela; Balestrazzi, Alma

2018-01-01

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells. PMID:29868059

Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay.

PubMed

Lee, Kwang Jin; Oh, You Chang; Cho, Won Kyung; Ma, Jin Yeul

2015-01-01

This study investigated the antioxidant activity of one hundred kinds of pure chemical compounds found within a number of natural substances and oriental medicinal herbs (OMH). Three different methods were used to evaluate the antioxidant activity of DPPH radical-scavenging activity, ABTS radical-scavenging activity, and online screening HPLC-ABTS assays. The results indicated that 17 compounds exhibited better inhibitory activity against ABTS radical than DPPH radical. The IC50 rate of a more practical substance is determined, and the ABTS assay IC50 values of gallic acid hydrate, (+)-catechin hydrate, caffeic acid, rutin hydrate, hyperoside, quercetin, and kaempferol compounds were 1.03 ± 0.25, 3.12 ± 0.51, 1.59 ± 0.06, 4.68 ± 1.24, 3.54 ± 0.39, 1.89 ± 0.33, and 3.70 ± 0.15 μg/mL, respectively. The ABTS assay is more sensitive to identifying the antioxidant activity since it has faster reaction kinetics and a heightened response to antioxidants. In addition, there was a very small margin of error between the results of the offline-ABTS assay and those of the online screening HPLC-ABTS assay. We also evaluated the effects of 17 compounds on the NO secretion in LPS-stimulated RAW 264.7 cells and also investigated the cytotoxicity of 17 compounds using a cell counting kit (CCK) in order to determine the optimal concentration that would provide an effective anti-inflammatory action with minimum toxicity. These results will be compiled into a database, and this method can be a powerful preselection tool for compounds intended to be studied for their potential bioactivity and antioxidant activity related to their radical-scavenging capacity.

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

PubMed

Kudryashova, Elena V; Sukhoverkov, Kirill V

2016-02-01

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

New and highly sensitive assay for L-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography-voltammetry.

PubMed

Rahman, M K; Nagatsu, T; Kato, T

1980-12-12

This paper describes a new, inexpensive and highly sensitive assay for aromatic L-amino acid decarboxylase (AADC) activity, using L-5-hydroxytryptophan (L-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. L-5-HTP was used as substrate and D-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from L-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using L-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.

On the Use of the Platelet Activity State Assay for the In Vitro Quantification of Platelet Activation in Blood Recirculating Devices for Extracorporeal Circulation.

PubMed

Consolo, Filippo; Valerio, Lorenzo; Brizzola, Stefano; Rota, Paolo; Marazzato, Giulia; Vincoli, Valentina; Reggiani, Stefano; Redaelli, Alberto; Fiore, Gianfranco

2016-10-01

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact. Copyright © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries.

PubMed

Morisseau, Christophe; Sahdeo, Sunil; Cortopassi, Gino; Hammock, Bruce D

2013-03-01

The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z'>0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (K(I)=550 nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor. Copyright © 2012 Elsevier Inc. All rights reserved.

Spiral Salmonella assay: validation against the standard pour-plate assay.

PubMed

Diehl, M; Fort, F

1996-01-01

The spiral Ames assay, an automated approach to bacterial mutagenicity testing which simplifies the test procedure and reduces the amount of drug required to generate mutagenic dose-response information, has been evaluated and validated for routine screening. The spiral plater delivers the Salmonella bacteria, exogenous metabolic activation system and drug to the surface of a rotating agar plate one on top of another in such a way that a uniform density of bacteria is exposed to a logarithmically decreasing volume of drug. Following an incubation of 48 hr at 37 degrees C, the plates are scanned by a laser counter, and the data are subjected to a computerized analysis. Petri plates of 15 cm diameter were used to provide a concentration range of about 250-fold per plate. The Salmonella were concentrated 20-fold to increase sensitivity. Thirty-eight compounds from a variety of chemical classes, including both pharmaceuticals and known mutagens of moderate to strong potency, were tested in both the spiral and the standard pour-plate assays. There was overall test agreement on positive or negative results for 82% of the compounds tested. When only the results from strains TA98 plus TA100 were considered, the agreement was 87%. When positive results were obtained, the fold increase over vehicle control was on average twice as great for the spiral assay compared to the pour-plate assay. It was concluded that the two assay procedures generally provided comparable results, with the spiral assay being somewhat more sensitive in terms of dose-response than the pour-plate assay.

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

PubMed Central

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

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

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

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.

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

PubMed

Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard

2017-12-01

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

PubMed

Uemura, Ryota; Ogura, Mikako; Matsumaru, Chihiro; Akiyama, Tsuyoshi; Maeda, Megumi; Kimura, Yoshinobu

2018-04-15

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Ferrocenes as potential chemotherapeutic drugs: Synthesis, cytotoxic activity, reactive oxygen species production and micronucleus assay

PubMed Central

Pérez, Wanda I.; Soto, Yarelys; Ortíz, Carmen; Matta, Jaime; Meléndez, Enrique

2014-01-01

Three new ferrocene complexes were synthesized with 4-(1H-pyrrol-1-yl)phenol group appended to one of the Cp ring. These are: 1,1′-4-(1H-pyrrol-1-yl)phenyl ferrocenedicarboxylate, (“Fc-(CO2-Ph-4-Py)2”), 1,4-(1H-pyrrol-1-yl)phenyl, 1′-carboxyl ferrocenecarboxylate (“Fc-(CO2-Ph-4-Py)CO2H”) and 4-(1H-pyrrol-1-yl)phenyl ferroceneacetylate (“Fc-CH2CO2-Ph-4-Py”). The new species were characterized by standard analytical methods. Cyclic voltammetry experiments showed that Fc-CH2CO2-Ph-4-Py has redox potential very similar to the Fc/Fc+ redox couple whereas Fc-(CO2-Ph-4-Py)2 and Fc-(CO2-Ph-4-Py)CO2H have redox potentials of over 400 mV higher than Fc/Fc+ redox couple. The in vitro studies on Fc-(CO2-Ph-4-Py)2 and Fc-(CO2-Ph-4-Py)CO2H revealed that these two compounds have moderate anti-proliferative activity on MCF-7 breast cancer cell line. In contrast Fc-CH2CO2-Ph-4-Py which displayed low anti-proliferative activity. In the HT-29 colon cancer cell line, the new species showed low anti-proliferaive activity. Cytokinesis-block micronucleus assay (CBMN) was performed on these ferrocenes and it was determined they induce micronucleus formation on binucleated cells and moderate genotoxic effects on the MCF-7 breast cancer cell line. There is a correlation between the IC50 values of the ferrocenes and the amount of micronucleus formation activity on binucleated cells and the reactive oxygen species (ROS) production on MCF-7 cell line. PMID:25555734

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

PubMed

Dolz, María; O'Connor, José-Enrique; Lequerica, Juan L

2004-10-01

The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.

Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3).

PubMed

Pinne, Marija; Ponce, Elsa; Raucy, Judy L

2017-01-01

Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with screening tools incapable of detecting indirect activators could present liabilities. The aim of this study was to establish assays that identify indirect activators of human PXR and CAR. Commercially available human PXR and CAR transactivation assays were used for analyses. We show that transactivation assays containing full-length nuclear receptors with native promoters can identify indirect activators of human CAR and PXRwhen compared to those of commercially available assays containing only the LBD of PXR and CAR. Of these two assay systems, only human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors, phenobarbital and midostaurin were found to be direct and indirect activators of PXR while human CAR activation by phenobarbital occurs by indirect mechanisms only. Cell based transactivation assays employing the full-length receptors and native promoters identify both direct and indirect activators of either or both human PXR and CAR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation

PubMed Central

Gence, Rémi; Bouchenot, Catherine; Lajoie-Mazenc, Isabelle

2018-01-01

ABSTRACT The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains. PMID:29192060

Functional Assays and Metagenomic Analyses Reveals Differences between the Microbial Communities Inhabiting the Soil Horizons of a Norway Spruce Plantation

PubMed Central

Uroz, Stéphane; Ioannidis, Panos; Lengelle, Juliette; Cébron, Aurélie; Morin, Emmanuelle; Buée, Marc; Martin, Francis

2013-01-01

In temperate ecosystems, acidic forest soils are among the most nutrient-poor terrestrial environments. In this context, the long-term differentiation of the forest soils into horizons may impact the assembly and the functions of the soil microbial communities. To gain a more comprehensive understanding of the ecology and functional potentials of these microbial communities, a suite of analyses including comparative metagenomics was applied on independent soil samples from a spruce plantation (Breuil-Chenue, France). The objectives were to assess whether the decreasing nutrient bioavailability and pH variations that naturally occurs between the organic and mineral horizons affects the soil microbial functional biodiversity. The 14 Gbp of pyrosequencing and Illumina sequences generated in this study revealed complex microbial communities dominated by bacteria. Detailed analyses showed that the organic soil horizon was significantly enriched in sequences related to Bacteria, Chordata, Arthropoda and Ascomycota. On the contrary the mineral horizon was significantly enriched in sequences related to Archaea. Our analyses also highlighted that the microbial communities inhabiting the two soil horizons differed significantly in their functional potentials according to functional assays and MG-RAST analyses, suggesting a functional specialisation of these microbial communities. Consistent with this specialisation, our shotgun metagenomic approach revealed a significant increase in the relative abundance of sequences related glycoside hydrolases in the organic horizon compared to the mineral horizon that was significantly enriched in glycoside transferases. This functional stratification according to the soil horizon was also confirmed by a significant correlation between the functional assays performed in this study and the functional metagenomic analyses. Together, our results suggest that the soil stratification and particularly the soil resource availability impact the

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

PubMed

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

2015-01-01

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

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.

Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice.

PubMed

Schmidt, Katja; Butt, Julia; Mauter, Petra; Vogel, Klaus; Erles-Kemna, Andrea; Pawlita, Michael; Nicklas, Werner

2017-01-01

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.

A Rapid and Quantitative Recombinase Activity Assay

USDA-ARS?s Scientific Manuscript database

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

Anticancer and anti-inflammatory activities of some dietary cucurbits.

PubMed

Sharma, Dhara; Rawat, Indu; Goel, H C

2015-04-01

In this study, we investigated few dietary cucurbits for anticancer activity by monitoring cytotoxic (MTT and LDH assays), apoptotic (caspase-3 and annexin-V assays), and also their anti-inflammatory effects by IL-8 cytokine assay. Aqua-alcoholic (50:50) whole extracts of cucurbits [Lagenaria siceraria (Ls), Luffa cylindrica (Lc) and Cucurbita pepo (Cp)] were evaluated in colon cancer cells (HT-29 and HCT-15) and were compared with isolated biomolecule, cucurbitacin-B (Cbit-B). MTT and LDH assays revealed that the cucurbit extracts and Cbit-B, in a concentration dependent manner, decreased the viability of HT-29 and HCT-15 cells substantially. The viability of lymphocytes was, however, only marginally decreased, yielding a potential advantage over the tumor cells. Caspase-3 assay revealed maximum apoptosis with Ls while annexin V assay demonstrated maximum efficacy of Lc in this context. These cucurbits have also shown decreased secretion of IL-8, thereby revealing their anti-inflammatory capability. The results have demonstrated the therapeutic potential of dietary cucurbits in inhibiting cancer and inflammatory cytokine.

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

PubMed

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

2016-01-01

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

Assaying Oxidative Coupling Activity of CYP450 Enzymes.

PubMed

Agarwal, Vinayak

2018-01-01

Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products. © 2018 Elsevier Inc. All rights reserved.

ORAC and DPPH assay comparison to assess antioxidant capacity of tea infusions: relationship between total polyphenol and individual catechin content.

PubMed

Roy, Molay K; Koide, Motoki; Rao, Theertham P; Okubo, Tsutomu; Ogasawara, Yutaka; Juneja, Lekh R

2010-03-01

Commercially available tea infusions are the major source of catechins for preparing bottled tea beverages and tea supplements available in the market today. In the present study, we analyzed five tea infusions to measure the total antioxidant capacity (TAC) by oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (DRSC) assays, total polyphenol content by the colorimetric method and individual catechin content by high-performance liquid chromatography. Four major tea catechins were also analyzed for their TAC to reveal differential antioxidant behavior of the tea infusions, resulting in the ORAC and DRSC methods. The correlation coefficients between DRSC and the total polyphenol or total catechin content of the tea infusions were 1.0 and 0.99. However, the values fall to 0.73 and 0.69, respectively, while the ORAC activity was correlated with total polyphenol and total catechin content. Determining the TAC of individual tea catechins showed that ORAC of epicatechin was seven-fold higher than that of epigallocatechin gallate; on the contrary, epigallocatechin gallate showed significantly (P < 0.05) stronger DRSC activity than epicatechin. By evaluating the structure-activity relationship, this study further revealed that OH substitution at the 3' position in pyrogallol moieties contributes to the lower ORAC value of epigallocatechin and epigallocatechin gallate comparing with their non-3'-OH counterparts, such as epicatechin and epicatechin gallate, respectively. Also, numbers of OH substitutions were poorly correlated with the observed ORAC value unlike the DRSC. Overall, results of this study enabled us to hypothesize that substances having a lower TAC value in the ORAC assay compared with that in DPPH assays may pertain to a pro-oxidant effect by generating reactive oxygen species in an aqueous buffer, at a physiological pH. We also propose that substances exhibiting lower TAC value in the ORAC assay

Carbohydrate active enzymes revealed in Coptotermes formosanus transcriptome

USDA-ARS?s Scientific Manuscript database

A normalized cDNA library of Coptotermes formosanus was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. Sequencing of this library generated 131,637 EST and 25,939 unigenes were assembled. Carbohydrate active enzymes (CAZymes) revealed in this library we...

Open chromatin reveals the functional maize genome

USDA-ARS?s Scientific Manuscript database

Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay

PubMed Central

Amakura, Yoshiaki; Tsutsumi, Tomoaki; Yoshimura, Morio; Nakamura, Masafumi; Handa, Hiroshi; Matsuda, Rieko; Teshima, Reiko; Watanabe, Takahiro

2016-01-01

The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay. PMID:28231110

Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

PubMed

De Yan, Hong; Zhang, Yin Jun; Liu, Hong Cai; Zheng, Jian Yong; Wang, Zhao

2013-01-01

p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Crude and purified proteasome activity assays are affected by type of microplate.

PubMed

Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2014-02-01

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate. Copyright © 2013 Elsevier Inc. All rights reserved.

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

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology.

PubMed

Macarrón, R; Mensah, L; Cid, C; Carranza, C; Benson, N; Pope, A J; Díez, E

2000-09-10

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods. Copyright 2000 Academic Press.

An optimized whole blood assay measuring expression and activity of NLRP3, NLRC4 and AIM2 inflammasomes.

PubMed

Grinstein, Lev; Endter, Kristin; Hedrich, Christian M; Reinke, Sören; Luksch, Hella; Schulze, Felix; Robertson, Avril A B; Cooper, Matthew A; Rösen-Wolff, Angela; Winkler, Stefan

2018-06-01

The proinflammatory protease caspase-1 plays pivotal roles in central pathways of innate immunity, thereby contributing to pathogen clearance. Beside its physiological role, dysregulated activity of caspase-1 is known to contribute to an increasing number of diseases. In this study, we optimized and validated a low-volume human whole blood assay facilitating the measurement of caspase-1 activation and inflammasome-related gene expression upon stimulation of the NLRP3, NLRC4 or AIM2 inflammasome. Using the NLRP3 inflammasome specific inhibitor MCC950, we were able to measure the activity of canonical or alternative NLRP3 pathways, AIM2 and NLRC4 inflammasomes in whole blood. Based on our data we assume a superposition of NLRP3 and NLRC4 inflammasome activities in human whole blood following stimulation with S. typhimurium. The optimized whole blood assay may be suitable for diagnostic and research purposes for pediatric patients who can only donate small amounts of blood. Copyright © 2017 Elsevier Inc. All rights reserved.

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 ...

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

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

Chen, Aiqiong; Du, Dan; Lin, Yuehe

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

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

PubMed

Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

2016-03-01

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. Copyright © 2015 Elsevier Inc. All rights reserved.

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair

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

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

Production and assay of forskolin antibodies

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

Ho, L.T.; Ho, R.J.

1986-05-01

Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracermore » and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.« less

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

2017-03-01

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

PubMed

Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

2017-10-01

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Development of a sensitive enzyme-linked immunosorbent assay for the measurement of biologically active etanercept in patients with ankylosing spondylitis.

PubMed

Wang, Lei; Wang, Xiaoxia; Li, Ying; Cheng, Zeneng

2016-01-01

Etanercept is the first tumor necrosis factor inhibitor to be approved for rheumatic disease treatment. Its in vivo concentration is usually detected with commercial enzyme-linked immunosorbent assay (ELISA) kits; specifically, previous researchers have mostly used double-antibody sandwich ELISA technology. Double-antibody sandwich ELISA is employed to detect the total etanercept rather than biologically active etanercept, which is more relevant in terms of therapeutic drug monitoring. In this work, a sensitive ELISA that employed its antigen TNF-α to capture biologically active etanercept for concentration detection was established and validated for etanercept pharmacokinetic (PK) study in patients with ankylosing spondylitis (AS). The proposed assay was demonstrated to be precise and accurate over the linear range of 12.5-400pg/mL. The intra- and inter-assay relative standard deviation ranged from 3.9 to 12.2% and 6.2 to 11.1%, respectively, and recovery varied between 90.1 and 99.7%, confirming the assay's reliability. The effectiveness and accuracy of the assay was also validated according to quality samples containing etanercept with different TNF-α concentrations, and with plasma samples from patients with AS. To complete the study, both the proposed assay and double-antibody sandwich ELISA were applied to the PK study of etanercept in patients and compared. The multiple-dose results of both analytical methods were consistent, while the drug exposure of the first dose as-detected by the proposed assay was lower than that detected by double-antibody sandwich ELISA. In conclusion, the proposed ELISA was shown to provide more accurate concentration data for therapeutic drug monitoring in comparison to commercial ELISA kits. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

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

1997-01-01

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

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

PubMed Central

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-01-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

PubMed

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-03-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

The Spheroplast Lysis Assay for Yeast in Microtiter Plate Format

PubMed Central

Ovalle, Rafael; Spencer, Moyah; Thiwanont, Monthiwa; Lipke, Peter N.

1999-01-01

A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes. PMID:10427014

Development of reproducible assays for polygalacturonase and pectinase.

PubMed

Li, Qian; Coffman, Anthony M; Ju, Lu-Kwang

2015-05-01

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30min and substrate concentration is 5g/L. For polygalacturonase, the sample should be adjusted to have 0.3-0.8U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor×0.687U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA-protective activities of hyperforin and aristoforin.

PubMed

Ševčovičová, A; Šemeláková, M; Plšíková, J; Loderer, D; Imreová, P; Gálová, E; Kožurková, M; Miadoková, E; Fedoročko, P

2015-04-01

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activation of Rho GTPase Cdc42 promotes adhesion and invasion in colorectal cancer cells.

PubMed

Gao, Lei; Bai, Lan; Nan, Qing zhen

2013-07-25

The purpose of this study was to investigate the role of activated Rho GTPase cell division control protein 42 homolog (Cdc42) in colorectal cancer cell adhesion, migration, and invasion. The constitutively active form of Cdc42 (GFP-Cdc42L61) or control vector was overexpressed in the colorectal cancer cell line SW480. The localization of active Cdc42 was monitored by immunofluorescence staining, and the effects of active Cdc42 on cell migration and invasion were examined using an attachment assay, a wound healing assay, and a Matrigel migration assay in vitro. Immunofluorescence staining revealed that constitutively active Cdc42 predominately localized to the plasma membrane. Compared to SW480 cells transfected with the control vector, overexpression of constitutively active Cdc42 in SW480 cells promoted filopodia formation and cell stretch and dramatically enhanced cell adhesion to the coated plates. The wound healing assay revealed a significant increase of migration capability in SW480 cells expressing active Cdc42 compared to the control cells. Additionally, the Matrigel invasion assay demonstrated that active Cdc42 significantly promoted SW480 cell migration through the chamber. Our results suggest that active Rho GTPase Cdc42 can greatly enhance colorectal cancer cell SW480 to spread, migrate, and invade, which may contribute to colorectal cancer metastasis.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

NASA Astrophysics Data System (ADS)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

A multiwell format assay for heparanase.

PubMed

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Identification of formaldehyde as the metabolite responsible for the mutagenicity of methyl tertiary-butyl ether in the activated mouse lymphoma assay.

PubMed

Mackerer, C R; Angelosanto, F A; Blackburn, G R; Schreiner, C A

1996-09-01

Methyl tertiary-butyl ether (MTBE), which is added to gasoline as an octane enhancer and to reduce automotive emissions, has been evaluated in numerous toxicological tests, including those for genotoxicity. MTBE did not show any mutagenic potential in the Ames bacterial assay or any clastogenicity in cytogenetic tests. However, it has been shown to be mutagenic in an in vitro gene mutation assay using mouse lymphoma cells when tested in the presence, but not in the absence, of a rat liver-derived metabolic activation system (S-9). In the present study, MTBE was tested to determine if formaldehyde, in the presence of the S-9, was responsible for the observed mutagenicity. A modification of the mouse lymphoma assay was employed which permits determination of whether a suspect material is mutagenic because it contains or is metabolized to formaldehyde. In the modified assay, the enzyme formaldehyde dehydrogenase (FDH) and its co-factor, NAD+ are added in large excess during the exposure period so that any formaldehyde produced in the system is rapidly converted to formic acid which is not genotoxic. An MTBE dose-responsive increase in the frequency of mutants and in cytotoxicity occurred without FDH present, and this effect was greatly reduced in the presence of FDH NAD+. The findings clearly demonstrate that formaldehyde derived from MTBE is responsible for mutagenicity of MTBE in the activated mouse lymphoma assay. Furthermore, the results suggest that the lack of mutagenicity/clastogenicity seen with MTBE in other in vitro assays might have resulted from inadequacies in the test systems employed for those assays.

Complementing in vitro screening assays with in silico ...

EPA Pesticide Factsheets

High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive under assay conditions but that can generate active metabolites under in vivo conditions. To address this potential issue, a case study will be presented to demonstrate the use of in silico tools to identify inactive parents with the ability to generate active metabolites. This case study used the results from an orthogonal assay designed to improve confidence in the identification of active chemicals tested across eighteen estrogen receptor (ER)-related in vitro assays by accounting for technological limitations inherent within each individual assay. From the 1,812 chemicals tested within the orthogonal assay, 1,398 were considered inactive. These inactive chemicals were analyzed using Chemaxon Metabolizer software to predict the first and second generation metabolites. From the nearly 1,400 inactive chemicals, over 2,200 first-generation (i.e., primary) metabolites and over 5,500 second-generation (i.e., secondary) metabolites were predicted. Nearly 70% of primary metabolites were immediately detoxified or converted to other metabolites, while over 70% of secondary metabolites remained stable. Among these predicted metabolites, those that are most likely to be produced and remain

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...

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

PubMed

Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

1992-03-15

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Antioxidant activity of methanol extract of Helichrysum foetidum Moench.

PubMed

Tirillini, Bruno; Menghini, Luigi; Leporini, Lidia; Scanu, Nadia; Marino, Stefania; Pintore, Giorgio

2013-01-01

Methanol extract of Helichrysum foetidum Moench (Asteraceae) was investigated for antioxidative properties. The antioxidant activities were investigated by 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, ß-carotene/linoleic acid assay, scavenging of hydrogen peroxide (HRPO test), superoxide anion scavenging (S.A.S. test) and hypochlorous acid scavenging (taurine test). The antioxidant activity was reported as IC50 and reveals Trolox-like antioxidative effects.

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

PubMed

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

2015-07-01

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations. Copyright © 2015 Elsevier Inc. All rights reserved.

Application of the Attagene FACTORIAL™ assay to ...

EPA Pesticide Factsheets

Bioassays can be used to evaluate the integrated effects of complex mixtures from both known and unidentified contaminants present in environmental samples. However, such bio-monitoring approaches have typically focused only on one or a few pathways (e.g. estrogen receptor, androgen receptor) despite the fact that the chemicals in a mixture may exhibit a range of biological activities. High-throughput screening approaches that can rapidly assess samples for a broad diversity of biological activities offer a means to provide a more comprehensive characterization of complex mixtures. The Attagene FactorialTM platform is a high-throughput, cell based assay utilized by US EPA’s ToxCast Program, which provides high-content assessment of over 90 different gene regulatory pathways and all 48 human nuclear receptors (NRs). This assay has previously been used in a preliminary screening of surface water extracts from sites across the Great Lakes. In the current study, surface waters samples from 38 sites were collected, extracted, and screened through the Factorial assay as part of a USGS nationwide stream assessment. All samples were evaluated in a six point, 3-fold dilution series and analyzed using the ToxCast Data Pipeline (TCPL) to generate dose-response curves and corresponding half-maximal activity concentration (AC50) estimates. A total of 27 assay endpoints responded to extracts from one or more sites, with up to 14 assays active for a single extract. The four

Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

PubMed Central

2015-01-01

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. PMID:25188731

Topological structure dynamics revealing collective evolution in active nematics

PubMed Central

Shi, Xia-qing; Ma, Yu-qiang

2013-01-01

Topological defects frequently emerge in active matter like bacterial colonies, cytoskeleton extracts on substrates, self-propelled granular or colloidal layers and so on, but their dynamical properties and the relations to large-scale organization and fluctuations in these active systems are seldom touched. Here we reveal, through a simple model for active nematics using self-driven hard elliptic rods, that the excitation, annihilation and transportation of topological defects differ markedly from those in non-active media. These dynamical processes exhibit strong irreversibility in active nematics in the absence of detailed balance. Moreover, topological defects are the key factors in organizing large-scale dynamic structures and collective flows, resulting in multi-spatial temporal effects. These findings allow us to control the self-organization of active matter through topological structures. PMID:24346733

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.

PubMed

Lehman, Nicholas; Cutrone, Rochelle; Raber, Amy; Perry, Robert; Van't Hof, Wouter; Deans, Robert; Ting, Anthony E; Woda, Juliana

2012-09-01

Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

HPLC-Based Activity Profiling for GABAA Receptor Modulators in Extracts: Validation of an Approach Utilizing a Larval Zebrafish Locomotor Assay.

PubMed

Moradi-Afrapoli, Fahimeh; Ebrahimi, Samad Nejad; Smiesko, Martin; Hamburger, Matthias

2017-05-26

Gamma-aminobutyric acid type A (GABA A ) receptors are major inhibitory neurotransmitter receptors in the central nervous system and a target for numerous clinically important drugs used to treat anxiety, insomnia, and epilepsy. A series of allosteric GABA A receptor agonists was identified previously with the aid of HPLC-based activity profiling, whereby activity was tracked with an electrophysiological assay in Xenopus laevis oocytes. To accelerate the discovery process, an approach has been established for HPLC-based profiling using a larval zebrafish (Danio rerio) seizure model induced by pentylenetetrazol (PTZ), a pro-convulsant GABA A receptor antagonist. The assay was validated with the aid of representative GABAergic plant compounds and extracts. Various parameters that are relevant for the quality of results obtained, including PTZ concentration, the number of larvae, the incubation time, and the data analysis protocol, were optimized. The assay was then translated into an HPLC profiling protocol, and active compounds were tracked in extracts of Valeriana officinalis and Magnolia officinalis. For selected compounds the effects in the zebrafish larvae model were compared with data from in silico blood-brain barrier (BBB) permeability predictions, to validate the use for discovery of BBB-permeable natural products.

Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens.

PubMed

Marrero, Allison; Lawrence, Scott; Wilsker, Deborah; Voth, Andrea Regier; Kinders, Robert J

2016-08-01

Multiplex pharmacodynamic (PD) assays have the potential to increase sensitivity of biomarker-based reporting for new targeted agents, as well as revealing significantly more information about target and pathway activation than single-biomarker PD assays. Stringent methodology is required to ensure reliable and reproducible results. Common to all PD assays is the importance of reagent validation, assay and instrument calibration, and the determination of suitable response calibrators; however, multiplex assays, particularly those performed on paraffin specimens from tissue blocks, bring format-specific challenges adding a layer of complexity to assay development. We discuss existing multiplex approaches and the development of a multiplex immunofluorescence assay measuring DNA damage and DNA repair enzymes in response to anti-cancer therapeutics and describe how our novel method addresses known issues. Copyright © 2016 Elsevier Inc. All rights reserved.

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen.

PubMed

Glogowski, J; Demianowicz, W; Piros, B; Ciereszko, A

1998-10-15

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.

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

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

Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel

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 ofmore » 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.« less

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics.

PubMed

Sun, Kaiwen; Zheng, Yuyu; Zhu, Ziqiang

2017-11-20

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Functional Assays for Ricin Detection

NASA Astrophysics Data System (ADS)

Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.

PubMed

Welsh, J D; Colace, T V; Muthard, R W; Stalker, T J; Brass, L F; Diamond, S L

2012-11-01

Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 μm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF μm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients. © 2012 International Society on Thrombosis and Haemostasis.

Comparison of Antioxidant Evaluation Assays for Investigating Antioxidative Activity of Gallic Acid and Its Alkyl Esters in Different Food Matrices.

PubMed

Phonsatta, Natthaporn; Deetae, Pawinee; Luangpituksa, Pairoj; Grajeda-Iglesias, Claudia; Figueroa-Espinoza, Maria Cruz; Le Comte, Jérôme; Villeneuve, Pierre; Decker, Eric A; Visessanguan, Wonnop; Panya, Atikorn

2017-08-30

The addition of antioxidants is one of the strategies to inhibit lipid oxidation, a major cause of lipid deterioration in foods leading to rancidity development and nutritional losses. However, several studies have been reported that conventional antioxidant assays, e.g., TPC, ABTS, FRAP, and ORAC could not predict antioxidant performance in several foods. This study aimed to investigate the performance of two recently developed assays, e.g., the conjugated autoxidizable triene (CAT) and the apolar radical-initiated conjugated autoxidizable triene (ApoCAT) assays to predict the antioxidant effectiveness of gallic acid and its esters in selected food models in comparison with the conventional antioxidant assays. The results indicated that the polarities of the antioxidants have a strong impact on antioxidant activities. In addition, different oxidant locations demonstrated by the CAT and ApoCAT assays influenced the overall antioxidant performances of the antioxidants with different polarities. To validate the predictability of the assays, the antioxidative performance of gallic acid and its alkyl esters was investigated in oil-in-water (O/W) emulsions, bulk soybean oils, and roasted peanuts as the lipid food models. The results showed that only the ApoCAT assay could be able to predict the antioxidative performances in O/W emulsions regardless of the antioxidant polarities. This study demonstrated that the relevance of antioxidant assays to food models was strongly dependent on physical similarities between the tested assays and the food structure matrices.

Open innovation for phenotypic drug discovery: The PD2 assay panel.

PubMed

Lee, Jonathan A; Chu, Shaoyou; Willard, Francis S; Cox, Karen L; Sells Galvin, Rachelle J; Peery, Robert B; Oliver, Sarah E; Oler, Jennifer; Meredith, Tamika D; Heidler, Steven A; Gough, Wendy H; Husain, Saba; Palkowitz, Alan D; Moxham, Christopher M

2011-07-01

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

Development of fluorescent methods for DNA methyltransferase assay

NASA Astrophysics Data System (ADS)

Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

PubMed

Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

2012-12-01

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride.

PubMed

Li, Xue; Zhou, Yunlei; Xu, Yan; Xu, Huijie; Wang, Minghui; Yin, Huanshun; Ai, Shiyun

2016-08-31

Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity.

PubMed

Huang, Christine S; Oberbeck, Nina; Hsiao, Yi-Chun; Liu, Peter; Johnson, Adam R; Dixit, Vishva M; Hymowitz, Sarah G

2018-05-01

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cytokinin activity of N6-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico.

PubMed

Savelieva, Ekaterina M; Oslovsky, Vladimir E; Karlov, Dmitry S; Kurochkin, Nikolay N; Getman, Irina A; Lomin, Sergey N; Sidorov, Georgy V; Mikhailov, Sergey N; Osolodkin, Dmitry I; Romanov, Georgy A

2018-05-01

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N 6 -benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N 6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking

Active medulloblastoma enhancers reveal subgroup-specific cellular origins

PubMed Central

Lin, Charles Y.; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J.; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C.; Ju, Bensheng; Orr, Brent A.; Zeid, Rhamy; Polaski, Donald R.; Segura-Wang, Maia; Waszak, Sebastian M.; Jones, David T.W.; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V.; Millen, Kathleen J.; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O.; Pfister, Stefan M.; Bradner, James E.; Northcott, Paul A.

2016-01-01

Summary Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-Seq, that are responsible for subgroup divergence and implicate candidate cells-of-origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins. PMID:26814967

Correlation of liquid chromatographic and biological assay for potency assessment of filgrastim and related impurities.

PubMed

Skrlin, Ana; Kosor Krnic, Ela; Gosak, Darko; Prester, Berislav; Mrsa, Vladimir; Vuletic, Marko; Runac, Domagoj

2010-11-02

In vivo and in vitro potency assays have always been a critical tool for confirmation of protein activity. However, due to their complexity and time consuming procedures, it remains a challenge to find an alternative analytical approach that would enable their replacement with no impact on the quality of provided information. The goal of this research was to determine if a correlation between liquid chromatography assays and in vitro biological assay could be established for filgrastim (recombinant human granulocyte-colony stimulating factor, rhG-CSF) samples containing various amounts of related impurities. For that purpose, relevant filgrastim related impurities were purified to homogeneity and characterized by liquid chromatography and mass spectrometry. A significant correlation (R(2)>0.90) between the two types of assays was revealed. Potency of oxidized filgrastim was determined to be approximately 25% of filgrastim stated potency (1 x 10(8)IU/mg of protein). Formyl-methionine filgrastim had potency of 89% of the filgrastim stated potency, while filgrastim dimer had 67% of filgrastim stated potency. A mathematical model for the estimation of biological activity of filgrastim samples from chromatography data was established and a significant correlation between experimental potency values and potency values estimated by the mathematical model was obtained (R(2)=0.92). Based on these results a conclusion was made that reversed phase high performance liquid chromatography could be used as an alternative for the in vitro biological assay for potency assessment of filgrastim samples. Such an alternative model would enable substitution of a complex and time consuming biological assay with a robust and precise instrumental method in many practical cases. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

PubMed

Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

2017-12-01

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

PubMed

Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

2017-03-15

Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel cerium oxide nanoparticles-based colorimetric sensor using tetramethyl benzidine reagent for antioxidant activity assay.

PubMed

Ozdemir Olgun, F Ayca; Üzer, Ayşem; Ozturk, Birsen Demirata; Apak, Reşat

2018-05-15

Antioxidant activity (AOA) assays using nanotechnology are recently developed utilizing nanoparticles of transition metal oxides, especially nanoceria that can switch between trivalent and tetravalent oxidation states of cerium. Cerium oxide nanoparticles (CeO-NPs) may act as both an oxidant and an antioxidant, depending on the preparation method and particle size. A novel colorimetric sensor for AOA assay is proposed with the use of poly(acrylic acid) sodium salt (PAANa)-coated CeO-NPs. PAANa-coated CeO-NPs oxidized tetramethyl benzidine (TMB), a peroxidase substrate, in a slightly acidic solution at pH 4.0 to a blue charge-transfer complex. Antioxidants decreased the color intensity of the nanoceria suspension, and were indirectly determined by absorbance difference. Detection limits, linearity, additivity and precision were calculated, e.g., quercetin quantification with the proposed assay showed a detection limit of 8.25 × 10 -9 mol L -1 . The trolox equivalent antioxidant capacities of hydrophilic and lipophilic antioxidants were compatible with those of conventional antioxidant assays. Potential interferents such as glucose, citric acid, mannitol, sorbitol and benzoic acid did not adversely affect AOA determination. The developed sensor is more sensitive and selective than similar colorimetric sensors relying on the intrinsic color change of nanoceria. The measurement wavelength is sufficiently red-shifted, preventing possible interferences from plant pigments. Copyright © 2018 Elsevier B.V. All rights reserved.

Activity-Based Detection and Bioanalytical Confirmation of a Fatal Carfentanil Intoxication.

PubMed

Cannaert, Annelies; Ambach, Lars; Blanckaert, Peter; Stove, Christophe P

2018-01-01

Carfentanil, one of the most potent opioids known, has recently been reported as a contaminant in street heroin in the United States and Europe, and is associated with an increased number of life-threatening emergency department admissions and deaths. Here, we report on the application of a novel in vitro opioid activity reporter assay and a sensitive bioanalytical assay in the context of a fatal carfentanil intoxication, revealing the highest carfentanil concentrations reported until now. A 21-year-old male was found dead at home with a note stating that he had taken carfentanil with suicidal intentions. A foil bag and plastic bag labeled "C.50" were found at the scene. These bags were similar to a sample obtained by the Belgian Early Warning System on Drugs from a German darknet shop and to those found in the context of a fatality in Norway. Blood, urine and vitreous, obtained during autopsy, were screened with a newly developed in vitro opioid activity reporter assay able to detect compounds based on their μ-opioid receptor activity rather than their chemical structure. All extracts showed strong opioid activity. Results were confirmed by a bioanalytical assay, which revealed extremely high concentrations for carfentanil and norcarfentanil. It should be noted that carfentanil concentrations are typically in pg/mL, but here they were 92 ng/mL in blood, 2.8 ng/mL in urine, and 23 ng/mL in vitreous. The blood and vitreous contained 0.532 and 0.300 ng/mL norcarfentanil, respectively. No norcarfentanil was detected in urine. This is the first report where a novel activity-based opioid screening assay was successfully deployed in a forensic case. Confirmation and quantification using a validated bioanalytical procedure revealed the, to our knowledge, highest carfentanil concentrations reported in humans so far.

Activity-Based Detection and Bioanalytical Confirmation of a Fatal Carfentanil Intoxication

PubMed Central

Cannaert, Annelies; Ambach, Lars; Blanckaert, Peter; Stove, Christophe P.

2018-01-01

Carfentanil, one of the most potent opioids known, has recently been reported as a contaminant in street heroin in the United States and Europe, and is associated with an increased number of life-threatening emergency department admissions and deaths. Here, we report on the application of a novel in vitro opioid activity reporter assay and a sensitive bioanalytical assay in the context of a fatal carfentanil intoxication, revealing the highest carfentanil concentrations reported until now. A 21-year-old male was found dead at home with a note stating that he had taken carfentanil with suicidal intentions. A foil bag and plastic bag labeled “C.50” were found at the scene. These bags were similar to a sample obtained by the Belgian Early Warning System on Drugs from a German darknet shop and to those found in the context of a fatality in Norway. Blood, urine and vitreous, obtained during autopsy, were screened with a newly developed in vitro opioid activity reporter assay able to detect compounds based on their μ-opioid receptor activity rather than their chemical structure. All extracts showed strong opioid activity. Results were confirmed by a bioanalytical assay, which revealed extremely high concentrations for carfentanil and norcarfentanil. It should be noted that carfentanil concentrations are typically in pg/mL, but here they were 92 ng/mL in blood, 2.8 ng/mL in urine, and 23 ng/mL in vitreous. The blood and vitreous contained 0.532 and 0.300 ng/mL norcarfentanil, respectively. No norcarfentanil was detected in urine. This is the first report where a novel activity-based opioid screening assay was successfully deployed in a forensic case. Confirmation and quantification using a validated bioanalytical procedure revealed the, to our knowledge, highest carfentanil concentrations reported in humans so far. PMID:29867491

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...

Assessing the impact of nano- and micro-scale zerovalent iron particles on soil microbial activities: particle reactivity interferes with assay conditions and interpretation of genuine microbial effects.

PubMed

Cullen, Laurence G; Tilston, Emma L; Mitchell, Geoff R; Collins, Chris D; Shaw, Liz J

2011-03-01

The effects of nano-scale and micro-scale zerovalent iron (nZVI and mZVI) particles on general (dehydrogenase and hydrolase) and specific (ammonia oxidation potential, AOP) activities mediated by the microbial community in an uncontaminated soil were examined. nZVI (diameter 12.5 nm; 10 mg g⁻¹ soil) apparently inhibited AOP and nZVI and mZVI apparently stimulated dehydrogenase activity but had minimal influence on hydrolase activity. Sterile experiments revealed that the apparent inhibition of AOP could not be interpreted as such due to the confounding action of the particles, whereas, the nZVI-enhanced dehydrogenase activity could represent the genuine response of a stimulated microbial population or an artifact of ZVI reactivity. Overall, there was no evidence for negative effects of nZVI or mZVI on the processes studied. When examining the impact of redox active particles such as ZVI on microbial oxidation-reduction reactions, potential confounding effects of the test particles on assay conditions should be considered. Copyright © 2010 Elsevier Ltd. All rights reserved.

Optimization of antifungal activity of Aeollanthus heliotropioides oliv essential oil and Time Kill Kinetic Assay.

PubMed

Ngo Mback, M N L; Agnaniet, H; Nguimatsia, F; Jazet Dongmo, P-M; Hzounda Fokou, J-B; Bakarnga-Via, I; Fekam Boyom, F; Menut, C

2016-09-01

The limitations encountered in the management of fungal infections are due to the resistance, high toxicity, and overuse of conventional antifungal drugs. For bringing solutions, the antifungal activity of Aeollanthus heliotropioides essential oil will be evaluated and optimized. The aerial parts of A. heliotropioides were harvested and essential oil extracted by hydrodistillation. The chemical composition was determined using gas chromatography and gas chromatography coupled with mass spectrometry and nuclear magnetic resonance. The sensitivity of fungal strains was determined using broth microdilution method. The fungicidal parameters were checked by viability assay using methylene blue dye. The Fractional Inhibitory Concentration Index was determined according the two-dimensional checkboard methods. The efficiency of the simulated optimum concentrations confirmed experimentally on American type culture collection strains, through the Time Kill Kinetic Study. The yield of extraction of essential oil was 0.1%. The major compounds were linalool (38.5%), Z-α-farnesene (25.1%), 9-hexa-decen-1-ol (13.9%) saturated/unsaturated massoia and γ-lactones (4.5%). The MIC of extract on yeast isolates ranged from 0.6mg/mL to 5mg/mL. The combination of essential oil with thymol leads mainly to synergistic effects (0.5≤FICI). The optimums of essential oil (1.6±0.4μl/mL) and thymol (0.6±0.1mg/mL) revealed a total inhibition of yeast after 120 and 180minutes according to the yeasts strains used. This study highlights the in vitro antifungal activity of A. heliotropioides essential oil and it synergistic effect with thymol. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Development of a microplate coagulation assay for Factor V in human plasma.

PubMed

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

2011-06-28

Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement

Development of a microplate coagulation assay for Factor V in human plasma

PubMed Central

2011-01-01

Background Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. Methods The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. Results The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Conclusions The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide

EPA Science Inventory

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosph...

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

PubMed

Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

2015-12-01

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

CPTAC Assay Portal: a repository of targeted proteomic assays

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

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2014-06-27

To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigatorsmore » to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.« less

[Evaluation of serum PIVKA-II by Lumipulse PrestoII assay].

PubMed

Hiramatsu, Kumiko; Tanaka, Yasuhito; Takagi, Kazumi; Kani, Satomi; Goto, Takaaki; Takasaka, Yoshimitsu; Matsuura, Kentaro; Sugauchi, Fuminaka; Moriyama, Kazushige; Murakami, Hiroshi; Kitajima, Sachiko; Mizokami, Masashi

2009-03-01

Measurements of serum concentrations of Des-gamma-carboxy Prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, in Lumipulsef assay, it was reported that antibodies against alkaline phosphatase (ALP) derived from anti bleeding sheets led false high values of PIVKA-II in the patients with HCC resection. To improve the previous issue, newly developed Lumipulse PrestoII assay was examined. (1) The assay was reliable and positively correlated with the previous assays (Lumipulse f and Picolumi, R = 0.997 and 0.994 (n=115), respectively). (2) Eleven cases, which had false high values of PIVKA-II by the Lumipulsef assay, were examined by the PrestoII assay with excess of inactive ALP. The false high values of 10 cases were improved, but only one was still high. False reactivity of this case was stronger than other cases, more effective adsorption was required. (3) Comparing the absorbent activity of inactive ALP among 6 different kinds, we found inactive ALP with much higher adsorbent activity. When this inactive ALP was applied to assay, false high values of PIVKA-II were improved in all 11 cases. In conclusion, the PrestoII assay, which applies the inactive ALP with high activity, is reliable and useful for clinical screening.

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination.

PubMed

Silva, Melissa T G; Simas, Sonia M; Batista, Terezinha G F M; Cardarelli, Paola; Tomassini, Therezinha C B

2005-11-01

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

PubMed

Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

2014-02-20

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Gold nanoclusters-Cu(2+) ensemble-based fluorescence turn-on and real-time assay for acetylcholinesterase activity and inhibitor screening.

PubMed

Sun, Jian; Yang, Xiurong

2015-12-15

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Fast automated online xylanase activity assay using HPAEC-PAD.

PubMed

Cürten, Christin; Anders, Nico; Juchem, Niels; Ihling, Nina; Volkenborn, Kristina; Knapp, Andreas; Jaeger, Karl-Erich; Büchs, Jochen; Spiess, Antje C

2018-01-01

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

PubMed

Mesnage, Robin; Phedonos, Alexia; Arno, Matthew; Balu, Sucharitha; Corton, J Christopher; Antoniou, Michael N

2017-08-01

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in "BPA-free" products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10-11-10-4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.

Optimization of a UDP-glucuronosyltransferase assay for trout ...

EPA Pesticide Factsheets

An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

PubMed

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

2009-12-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation

PubMed Central

Tang, Matthew Y.; Vranas, Marta; Krahn, Andrea I.; Pundlik, Shayal; Trempe, Jean- François; Fon, Edward A.

2017-01-01

Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy. PMID:28276439

Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation.

PubMed

Tang, Matthew Y; Vranas, Marta; Krahn, Andrea I; Pundlik, Shayal; Trempe, Jean-François; Fon, Edward A

2017-03-09

Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy.

Multiplex tandem mass spectrometry assay for newborn screening of X-linked adrenoleukodystrophy, biotinidase deficiency, and galactosemia with flexibility to assay other enzyme assays and biomarkers.

PubMed

Hong, Xinying; Kumar, Arun Babu; Ronald Scott, C; Gelb, Michael H

2018-03-29

All States screen for biotinidase deficiency and galactosemia, and X-linked adrenoleukodystrophy (X-ALD) has recently been added to the Recommended Uniform Screening Panel (RUSP).We sought to consolidate these tests by combining them into a single multiplex tandem mass spectrometry assay as well as to improve the current protocol for newborn screening of galactosemia.A 3 mm punch of a dried blood spot (DBS) was extracted with organic solvent for analysis of the C26:0-lysophosphatidylcholine biomarker for X-ALD.An additional punch was used to assay galactose-1-phosphate uridyltransferase (GALT) and biotinidase.All assays were combined for a single injection for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (2.3 min per sample).The GALT LC-MS/MS assay does not give a false positive for galactosemia if glucose-6-phosphate dehydrogenase is deficient.The multiplex assay shows acceptable reproducibility and provides for rapid analysis of X-ALD, biotinidase deficiency, and galactosemia.The throughput and ease of sample preparation are acceptable for newborn screening laboratories.We also show that the LC-MS/MS assay is expandable to include several other diseases including Pompe and Hurler diseases (enzymatic activities and biomarkers).Because of consolidation of assays, less manpower is needed compared to running individual assays on separate platforms.The flexibility of the LC-MS/MS platform allows each newborn screening laboratory to analyze the set of diseases offered in their panel. Copyright © 2018 Elsevier Inc. All rights reserved.

Biochemical Assays of Cultured Cells

NASA Technical Reports Server (NTRS)

Barlow, G. H.

1985-01-01

Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

Evaluation of antioxidant activity of chrysanthemum extracts and tea beverages by gold nanoparticles-based assay.

PubMed

Liu, Quanjun; Liu, Haifang; Yuan, Zhiliang; Wei, Dongwei; Ye, Yongzhong

2012-04-01

A gold nanoparticles-based (GNPs-based) assay was developed for evaluating antioxidant activity of chrysanthemum extracts and tea beverages. Briefly, a GNPs growth system consisted of designated concentrations of hydrogen tetrachloroaurate, cetyltrimethyl ammonium bromide, sodium citrate, and phosphate buffer was designed, followed by the addition of 1 mL different level of test samples. After a 10-min reaction at 45°C, GNPs was formed in the reduction of metallic ions to zero valence gold by chrysanthemum extracts or tea beverages. And the resultant solution exhibited a characteristic surface plasmon resonance band of GNPs centered at about 545 nm, responsible for its vivid light pink or wine red color. The optical properties of GNPs formed correlate well with antioxidant activity of test samples. As a result, the antioxidant functional evaluation of chrysanthemum extracts and beverages could be performed by this GNPs-based assay with a spectrophotometer or in visual analysis to a certain extent. Our present method based on the sample-mediated generation and growth of GNPs is rapid, convenient, inexpensive, and also demonstrates a new possibility for the application of nanotechnology in food science. Moreover, this present work provides some useful information for in-depth research of involving chrysanthemum. Copyright © 2011 Elsevier B.V. All rights reserved.

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

PubMed

Fernández-Sierra, Mónica; Quiñones, Edwin

2015-03-15

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect. Copyright © 2015 Elsevier Inc. All rights reserved.

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow-injection chemiluminescence assay.

PubMed

Zhang, Qingfeng; Zhang, Cuiyun; Yang, Meiding; Yu, Donghong; Yu, Cong

2017-11-01

A sensitive and convenient flow-injection chemiluminescence (FI-CL) turn-on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu 2+ can catalyze the luminol-H 2 O 2 CL reaction. Pyrophosphate (PPi) can chelate Cu 2+ and therefore the Cu 2+ -mediated luminol-H 2 O 2 CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu 2+ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained. Copyright © 2017 John Wiley & Sons, Ltd.

Assaying Ornithine and Arginine Decarboxylases in Some Plant Species 1

PubMed Central

Birecka, Helena; Bitonti, Alan J.; McCann, Peter P.

1985-01-01

A release of 14CO2 not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-14C]- or [U-14C]ornithine. The total 14CO2 produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH4)2SO4 did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant 14CO2 release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris·HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard 14CO2-based method by assessing labeled Put and/or Agm—formed in the presence of added aminoguanidine and/or ornithine when needed—is recommended especially when crude or semicrude plant extracts are assayed. When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids. PMID:16664441

h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

PubMed

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-03-01

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

Performance evaluation and multicentre study of a von Willebrand factor activity assay based on GPIb binding in the absence of ristocetin.

PubMed

Patzke, Juergen; Budde, Ulrich; Huber, Andreas; Méndez, Adriana; Muth, Heidrun; Obser, Tobias; Peerschke, Ellinor; Wilkens, Matthias; Schneppenheim, Reinhard

2014-12-01

The functional activity of von Willebrand factor (VWF) is most frequently measured by using the ristocetin cofactor assay (VWF:RCo). However, the method's drawbacks include unsatisfactory precision, sensitivity and availability of automated system applications. We have developed an alternative assay (INNOVANCE VWF Ac) that is based on the binding of VWF to recombinant glycoprotein Ib (GPIb). Two gain-of-function mutations were introduced into a GPIb fragment, allowing an assay format without ristocetin. Fully automated assay applications are available for the BCS/BCS XP systems and the Sysmex CS-2000i, Sysmex CA-7000, Sysmex CA-1500 and Sysmex CA-560 systems.The INNOVANCE VWF Ac assay measuring range extends from 4 to 600% VWF for all systems except the Sysmex CA-560 system. Within-device precision values were found to be between 2 and 7%. The limit of detection was below 2.2% VWF. In a study on the BCS XP system, a total number of 580 sample results yielded a correlation to the VWF:RCo assay of r equal to 0.99 (slope = 0.96). Very similar results were observed when von Willebrand disease samples type 1, 2A, 2B, 2M, 2N and 3 were investigated with the new assay and the VWF:RCo assay. The excellent performance data and comparability to VWF:RCo, together with the ease of use, led us to the conclusion that the ristocetin cofactor assay can be replaced by the new GPIb-binding assay to reliably diagnosing patients with von Willebrand disease.

Bivalent O-glycoside mimetics with S/disulfide/Se substitutions and aromatic core: Synthesis, molecular modeling and inhibitory activity on biomedically relevant lectins in assays of increasing physiological relevance.

PubMed

Kaltner, Herbert; Szabó, Tamás; Fehér, Krisztina; André, Sabine; Balla, Sára; Manning, Joachim C; Szilágyi, László; Gabius, Hans-Joachim

2017-06-15

The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships (MCBIOS)

EPA Science Inventory

Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships Jie Liu1,2, Richard Judson1, Matthew T. Martin1, Huixiao Hong3, Imran Shah1 1National Center for Computational Toxicology (NCCT), US EPA, RTP, NC...

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

PubMed

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

PubMed

Misra, Rajeev; Morrison, Keith D; Cho, Hyun Jae; Khuu, Thanh

2015-08-01

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance

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

PubMed

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

2016-09-21

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

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.

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

Luciferase reporter assay in Drosophila and mammalian tissue culture cells

PubMed Central

Yun, Chi

2014-01-01

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

PubMed

Wiggers, Helton J; Rocha, Josmar R; Fernandes, William B; Sesti-Costa, Renata; Carneiro, Zumira A; Cheleski, Juliana; da Silva, Albérico B F; Juliano, Luiz; Cezari, Maria H S; Silva, João S; McKerrow, James H; Montanari, Carlos A

2013-01-01

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound

A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

PubMed

Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

2016-12-15

DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

PubMed

Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

2016-01-01

The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

PubMed

Ho, Pei-i; Yue, Kimberley; Pandey, Pramod; Breault, Lyne; Harbinski, Fred; McBride, Aaron J; Webb, Brian; Narahari, Janaki; Karassina, Natasha; Wood, Keith V; Hill, Adam; Auld, Douglas S

2013-05-17

Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.

Replacing antibodies with modified DNA aptamers in vaccine potency assays.

PubMed

Trausch, Jeremiah J; Shank-Retzlaff, Mary; Verch, Thorsten

2017-10-04

Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM 197 . Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM 197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM 197 , a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

A fluorescence assay for peptide translocation into mitochondria.

PubMed

Martinez-Caballero, Sonia; Peixoto, Pablo M V; Kinnally, Kathleen W; Campo, María Luisa

2007-03-01

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

A simple microplate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method).

PubMed

Visvanathan, Rizliya; Jayathilake, Chathuni; Liyanage, Ruvini

2016-11-15

For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of α-amylase inhibitory activity using the glucose assay kit was developed. The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The test is done in microtitre plates with a total volume of 260μL and an assay time of 40min including the pre-incubation steps. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining α-amylase activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Automated reagent-dispensing system for microfluidic cell biology assays.

PubMed

Ly, Jimmy; Masterman-Smith, Michael; Ramakrishnan, Ravichandran; Sun, Jing; Kokubun, Brent; van Dam, R Michael

2013-12-01

Microscale systems that enable measurements of oncological phenomena at the single-cell level have a great capacity to improve therapeutic strategies and diagnostics. Such measurements can reveal unprecedented insights into cellular heterogeneity and its implications into the progression and treatment of complicated cellular disease processes such as those found in cancer. We describe a novel fluid-delivery platform to interface with low-cost microfluidic chips containing arrays of microchambers. Using multiple pairs of needles to aspirate and dispense reagents, the platform enables automated coating of chambers, loading of cells, and treatment with growth media or other agents (e.g., drugs, fixatives, membrane permeabilizers, washes, stains, etc.). The chips can be quantitatively assayed using standard fluorescence-based immunocytochemistry, microscopy, and image analysis tools, to determine, for example, drug response based on differences in protein expression and/or activation of cellular targets on an individual-cell level. In general, automation of fluid and cell handling increases repeatability, eliminates human error, and enables increased throughput, especially for sophisticated, multistep assays such as multiparameter quantitative immunocytochemistry. We report the design of the automated platform and compare several aspects of its performance to manually-loaded microfluidic chips.

Prospective Comparison of QFT-GIT and T-SPOT.TB Assays for Diagnosis of Active Tuberculosis.

PubMed

Du, Fengjiao; Xie, Li; Zhang, Yonghong; Gao, Fei; Zhang, Huibin; Chen, Wei; Sun, Bingqi; Sha, Wei; Fang, Yong; Jia, Hongyan; Xing, Aiying; Du, Boping; Zheng, Li; Gao, Mengqiu; Zhang, Zongde

2018-04-12

T-SPOT.TB and QuantiFERON-TB Gold In-Tube (QFT-GIT) tests, as two commercial blood assays for diagnosing active tuberculosis (ATB), are not yet fully validated. Especially, there are no reports on comparing the efficacy between the two tests in the same population in China. A multicenter, prospective comparison study was undertaken at four hospitals specializing in pulmonary diseases. A total of 746 suspected pulmonary TB were enrolled and categorized, including 185 confirmed TB, 298 probable TB and 263 non-TB. Of 32 patients with indeterminate test results (ITRs), age and underlying disease were associated with the rate of ITRs. Furthermore, the rate of ITRs determined by T-SPOT.TB was lower than QFT-GIT (0.4% vs. 4.3%, P < 0.01). When excluding ITRs, the sensitivities of T-SPOT.TB and QFT-GIT were 85.2% and 84.8%, and specificities of 63.4% and 60.5%, respectively in the diagnosis of ATB. The two assays have an overall agreement of 92.3%, but exhibited a poor linear correlation (r 2  = 0.086) between the levels of interferon-γ release detected by the different assays. Although having some heterogeneity in detecting interferon-γ release, both the QFT-GIT and T-SPOT.TB demonstrated high concordance in diagnosing ATB. However, neither of them showed suitability in the definitive diagnosis of the disease.

Short-term effect of aniline on soil microbial activity: a combined study by isothermal microcalorimetry, glucose analysis, and enzyme assay techniques.

PubMed

Chen, Huilun; Zhuang, Rensheng; Yao, Jun; Wang, Fei; Qian, Yiguang; Masakorala, Kanaji; Cai, Minmin; Liu, Haijun

2014-01-01

The accidents of aniline spill and explosion happened almost every year in China, whereas the toxic effect of aniline on soil microbial activity remained largely unexplored. In this study, isothermal microcalorimetric technique, glucose analysis, and soil enzyme assay techniques were employed to investigate the toxic effect of aniline on microbial activity in Chinese soil for the first time. Soil samples were treated with aniline from 0 to 2.5 mg/g soil to tie in with the fact of aniline spill. Results from microcalorimetric analysis showed that the introduction of aniline had a significant adverse effect on soil microbial activity at the exposure concentrations ≥0.4 mg/g soil (p < 0.05) and ≥0.8 mg/g soil (p < 0.01), and the activity was totally inhibited when the concentration increased to 2.5 mg/g soil. The glucose analysis indicated that aniline significantly decreased the soil microbial respiratory activity at the concentrations ≥0.8 mg/g soil (p < 0.05) and ≥1.5 mg/g soil (p < 0.01). Soil enzyme activities for β-glucosidase, urease, acid-phosphatase, and dehydrogenase revealed that aniline had a significant effect (p < 0.05) on the nutrient cycling of C, N, and P as well as the oxidative capacity of soil microorganisms, respectively. All of these results showed an intensively toxic effect of aniline on soil microbial activity. The proposed methods can provide toxicological information of aniline to soil microbes from the metabolic and biochemical point of views which are consistent with and correlated to each other.

Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

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

Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P

2012-08-01

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-Smore » cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k cat/K M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k cat/K M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.« less

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

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

PubMed

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

2016-01-27

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. A cohort-based longitudinal study. Two antiretroviral therapy (ART) centres in Karnataka state, South India, providing treatment under the Indian AIDS control programme. A cohort of 327 HIV-1-infected Indian adult patients initiating first-line ART. 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. 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. 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. ISRCTN79261738. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Assessment of the microscreen phage-induction assay for screening hazardous wastes (1989)

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

Houk, V.S.; DeMarini, D.M.

1989-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage Lambda in Escherichia coli WP2s(Lambda), was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that weremore » mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.« less

AKT1, LKB1, and YAP1 revealed as MYC interactors with NanoLuc-based protein-fragment complementation assay. | Office of Cancer Genomics

Cancer.gov

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. Here we report the development of a NanoLuc®-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells.

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

PubMed

Klein, Karen Oerter

2015-07-01

There are many questions which cannot be answered without a very sensitive estradiol assay. A recombinant cell bioassay (RCBA) for estradiol was developed in 1994. The sensitivity of the bioassay is 0.02-0.2 pg/ml (0.07-0.7 pmol/L), more than 20 times more sensitive than commercial RIAs and 10 times more sensitive than newer mass spectrometry assays. The RCBA for estradiol opened the door to study low levels of estradiol equivalents (EE) across the physiological spectrum of life from prepubertal children through menopause and across the spectrum from normal physiology, in boys as well as girls, to pathology, including: premature thelarche; estradiol suppression in children treated with GnRH analogues for precocious puberty; aromatase inhibition in boys with growth hormone deficiency; the differences between oral and transdermal routes of estrogen administration in girls with Turner's syndrome; women with breast cancer treated with aromatase inhibitors; and women with urogenital atrophy treated with low dose vaginal estrogen. A bioassay also allows study of endocrine disruptors, like phytoestrogens and other environmental compounds, which are relevant to public health and alternative medicine options. This paper reviews the assay and the last 20 years of applications. A bioassay for estrogen has a role because measuring biological effect is theoretically useful, increasing the understanding of physiology in addition to biochemical levels, giving different information than other assays, and opening the door to measure very low levels of estrogen activity in both humans and the environment. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

PubMed

Coppola, Julia M; Hamilton, Christin A; Bhojani, Mahaveer S; Larsen, Martha J; Ross, Brian D; Rehemtulla, Alnawaz

2007-05-01

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

PubMed Central

Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

2007-01-01

Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

Biomass conversion determined via fluorescent cellulose decay assay.

PubMed

Wischmann, Bente; Toft, Marianne; Malten, Marco; McFarland, K C

2012-01-01

An example of a rapid microtiter plate assay (fluorescence cellulose decay, FCD) that determines the conversion of cellulose in a washed biomass substrate is reported. The conversion, as verified by HPLC, is shown to correlate to the monitored FCD in the assay. The FCD assay activity correlates to the performance of multicomponent enzyme mixtures and is thus useful for the biomass industry. The development of an optimized setup of the 96-well microtiter plate is described, and is used to test a model that shortens the assay incubation time from 72 to 24h. A step-by-step procedure of the final assay is described. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

PubMed Central

Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

2010-01-01

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165