Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.     

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.     

Effect of biogenic amines on oxidation of farmorubicin by Fenton-like reagents

The aim of this study was to investigate the effect of dopa and catecholamines on the generation of oxygen active species during oxidation of farmorubicin by "Fenton-like" reagents using chemiluminescent and spectrophotometric techniques. The tested catechols were found to reduce the light emission accompanying oxidation of farmorubicin by Co(II) + H2O2 and Cu(II) + H2O2 mixtures. The quenching effect was followed by their rapid oxidation to aminochromes possessing toxic activities.     

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities

The optimal experimental conditions of the enzyme assay described by Mulder and Van Doorn (1975, Biochem J. 151, 131-140) for the measurement of UDP-glucuronosyltransferase activities were tested towards structurally different aglycones. This assessment of this assay revealed that addition of Triton X-100 as enzyme activator was necessary because of its apparent inhibitory effects on interfering reactions. Under these conditions, accordance of the data with results published in the literature was obtained. We present for the first time an UDP-glucuronosyltransferase assay adapted on a fast analyser centrifuge which allows a rapid and sensitive measurement of enzyme activity that is very useful for kinetic constant determination, without consuming a large volume of reagents.     

Rate of acclimation of the capacity for isoprene emission in response to light and temperature

Isoprene emission from plants accounts for nearly half of all non‐methane hydrocarbons entering the atmosphere. Light and temperature regulate the instantaneous rate of isoprene emission but there is increasing evidence that they also affect the capacity for isoprene emission (i.e. the rate measured under standard conditions). We tested the rate of acclimation of the capacity for isoprene emission following step changes in growth conditions. Acclimation to new growth temperatures was very rapid, with most of the change occurring within a few hours and complete adjustment occurring within a day. Acclimation to new light levels was more complicated. Following a switch from low‐light growth conditions to standard assay conditions (30 °C and 1000 µmol photons m^?2 s^?1), there was a rapid (5–10 min) and a slightly slower (10–50 min) acclimation of the capacity for isoprene emission. After accounting for these short‐term changes, there was also a small, long‐term (4–6 d) acclimation of the isoprene emission capacity to the light level of growth conditions. We found no effect of growth conditions on the coefficients used to describe the instantaneous light and temperature response of isoprene emission. Therefore, current models of isoprene emission will only need to be altered to account for changes in the capacity for isoprene emission.     

Reporter enzymes for the study of promoter activity

This article describes the use of three reporter enzymes used to study promoter activity in transgenic animals. Chloramphenicol acetyl transferase may be assayed by a nonchromatographic method that is rapid and sensitive. β-Galactosidase is measured by a photometric assay and luciferase is assayed by measuring the emission of light using a luminometer. The relative merits of each enzyme is discussed. Ths use of reporter enzymes provides a rapid and sensitive method for analysis of transgene expression.     

A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.     

Parallel synthesis of peptide libraries using microwave irradiation

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-beta-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer alpha- and beta-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).     

A nonradioisotope chemiluminescent assay for evaluation of 2-deoxyglucose uptake in 3T3-L1 adipocytes. Effect of various carbonyls species on insulin action

We have developed a rapid nonradioisotope chemiluminescent assay adapted to high-throughput screening experiments, to evaluate glucose uptake activity in cultured cells. For chemiluminescence quantification of 2-deoxyglucose, we used a luminol oxidation reaction after an enzymatic dephosphorylation of 2-deoxyglucose-6-phosphate. All reactions were performed at 37 °C by consecutive addition of reagents, and the assay is able to quantify 2DG in picomole per well. To confirm the reliability of this method, we have evaluated the dose–effect of insulin, GLUT4 inhibitors and insulin-sensitizing agent on 2DG uptake into 3T3-L1 cells. The results obtained with the assay for 2DG uptake in vitro in the absence or presence of insulin stimulation, were similar to those obtained by the previous radioisotopic and enzymatic methods. We have also used this assay to evaluate the effect of various reactive carbonyl and oxygen species on insulin-stimulated 2DG-uptake into adipocytes. All reactive carbonyl species tested decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner without affecting basal glucose uptake in 3T3-L1 cells. 4-hydroxynonenal was found to be the most potent in the impairment of glucose uptake. This new enzymatic chemiluminescent assay is rapid and useful for measurement of 2DG uptake in insulin-responsive in cultured cells.     

Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays. Received: 11 March 2000 / Accepted: 16 May 2000     

Analytical applications of bioluminescence--a matter of proper kinetic design and recording

The way bioluminescence analysis employs photometric technique is illustrated in relation to the resulting demands on signal processing and detectability. The analytical reaction may be regarded as composed of a supply reaction providing the excited species followed by a decay reaction in which light is emitted. Bioluminescence analysis implies the recording of a velocity, hence rate regulation forms the basis of the development of an analytical set-up. In principle two means of design are used, either the application of a pulse technique or the monitoring of a durable emission. Both methods have their respective pros and cons but operating in the intermediate time range is sometimes favorable. The pulse technique is an arrangement where the entire emission develops out during a limited amount of time usually as a flash of light in the subminute range. It is accompanied by demands for rapid mixing and initiation of the analytical process as well as fast recording techniques. Durable emission measurements are based on a slowing down of the process, e.g., by reducing the concentration of enzyme or by the addition of inhibitors, so that the light intensity may be regarded as constant during the measurements. This facilitates the measuring procedure and provides for simplified handling, but occurs at a cost of emission intensity and sensitivity. Bioluminescence analysis mimics metabolic routes yielding great possibilities for coupling with other metabolic pathways. Such coupled systems are suited for analysis of a wide variety of metabolites and enzymes. By proper kinetic design it is possible to make the analyses largely insensitive to variations in activity of the reagents.     

Rapid and direct microRNA quantification by an enzymatic luminescence assay

A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay uses miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated high cross-platform consistency between this assay and the TaqMan real-time polymerase chain reaction (PCR) assay with R(2)=0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than 1h. Compared with the real-time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.     

Rapid tool for cell nanoarchitecture integrity assessment

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.     

Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system

An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents. The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min. Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized. Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B. subtilus. Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria.

The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.     

Novel Microarrays for Simultaneous Serodiagnosis of Multiple Antiviral Antibodies

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79–0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.     

Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin

Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage 1 to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. α-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation.     

Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction

We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.