The coupling of NAD(P)+-producing reactions to a semiautomated bioluminescent reaction

Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P)+. Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests that NAD(P)+ bioluminescence has a wide potential for application.     

A review of bioluminescent ATP techniques in rapid microbiology

Use of firefly luciferase to assay adenosine triphosphate (ATP) extracted from microorganisms provides an easy means to enumerate microbes within minutes. The small amount of light produced is proportional to ATP and thus microbial number. The average bacterium contains around 10^?15 g ATP per cell. Present reagents permit detection of 10³ cells per tube. Luminometers currently on the market detect about 10^?12 g ATP. Proper extraction of ATP from the microbes is an essential part of any protocol, as is the removal of non-microbial ATP from, for example, somatic cells also present in samples. The technique may be applied to a wide range of samples, for example food and beverages and clinical samples such as urine. The ATP assay gives a global measure of microbial numbers, i.e. it is not species specific unless a species separation step is included in the protocol.     

Potential applications of laccase-mediated coupling and grafting reactions: a review

Many industries are currently pursuing enzymatic approaches for developing green chemistry technologies mainly due to shortcomings of physico-chemical methods, growing environmental concerns, legal restrictions, and increasing scientific knowledge. Laccase-assisted reactions, in particular, are being intensively investigated as they are generally eco-friendly and have wide application potential. Laccases only require oxygen as co-substrate, they release water as the only by-product and have a wide substrate range which can be further extended by use of laccase-mediator systems. Consequently, research covering various applications of laccase has been rapidly increasing in recent years, particularly in the areas of coupling and grafting reactions. This review summarizes the advances that have been made in developing technologies based on laccase-mediated coupling and grafting reactions for potential application in areas such as environmental pollution control, modification of lignocellulose materials, food industry, biosensors, textile industry, pharmaceutical industry, and in organic synthesis.     

Animal reactions to oncoming vehicles: a conceptual review

Animal–vehicle collisions (AVCs) are a substantial problem in a human‐dominated world, but little is known about what goes wrong, from the animal's perspective, when a collision occurs with an automobile, boat, or aircraft. Our goal is to provide insight into reactions of animals to oncoming vehicles when collisions might be imminent. Avoiding a collision requires successful vehicle detection, threat assessment, and evasive behaviour; failures can occur at any of these stages. Vehicle detection seems fairly straightforward in many cases, but depends critically on the sensory capabilities of a given species. Sensory mechanisms for detection of collisions (looming detectors) may be overwhelmed by vehicle speed. Distractions are a likely problem in vehicle detection, but have not been clearly demonstrated in any system beyond human pedestrians. Many animals likely perceive moving vehicles as non‐threatening, and may generally be habituated to their presence. Slow or minimal threat assessment is thus a likely failure point in many AVCs, but this is not uniformly evident. Animals generally initiate evasive behaviour when a collision appears imminent, usually employing some aspect of native antipredator behaviour. Across taxa, animals exhibit a variety of behaviours when confronted with oncoming vehicles. Among marine mammals, right whales Eubalaena spp., manatees Trichechus spp., and dugongs Dugong dugon are fairly unresponsive to approaching vehicles, suggesting a problem in threat assessment. Others, such as dolphins Delphinidae, assess vehicle approach at distance. Little work has been conducted on the behavioural aspects of AVCs involving large mammals and automobiles, despite their prevalence. Available observations suggest that birds do not usually treat flying aircraft as a major threat, often allowing close approach before taking evasive action, as they might in response to natural predators. Inappropriate antipredator behaviour (often involving immobility) is a major source of AVCs in amphibians and terrestrial reptiles. Much behavioural work on AVCs remains to be done across a wide variety of taxa. Such work should provide broad phylogenetic generalizations regarding AVCs and insights into managing AVCs.     

发光细菌法测定环境中金属毒性的研究进展

在环境污染物的毒性评价和监测中,发光细菌法是一种具有快速、灵敏和廉价等优点的直接生物测试方法.本文简要回顾了发光细菌法的测定原理及其在水环境中的应用;总结了发光细菌法测定水环境中金属毒性的主要影响因素(如pH、有机无机配体和交互作用);重点评述了发光细菌法在土壤样品金属毒性测定方面的应用、不同提取方法的优缺点以及土壤金属毒性与形态之间的关系;提出今后应加强土壤中金属对发光细菌的毒害机理、土壤环境中发光细菌法标准化以及发光细菌法与其他测试方法的关系等研究.快速、廉价、标准化发光细菌法的建立对土壤环境中金属风险评价和监测具有重要的意义.     

Engineering of a bioluminescent antigen-binding protein

A gene fusion plasmid was constructed by inserting the structural gene of the firefly luciferase into a vector which allows expression of a functional recombinant scFv. The fusion protein which is expressed in the periplasm of bacteria, retains dual activity and functions in bioluminescent immunoassays. Such a genetic approach offers many advantages over chemical cross-linking of antibodies to colorimetric enzymes and may be adaptable to clinical immuno-assays and high through-put drug screening assays.     

The zona pellucida: a coat of many colors

The zona pellucida is an extracellular coat that surrounds all mammalian eggs. It is a porous matrix of interconnected filaments that are assembled from glycoproteins synthesized and secreted by growing oocytes. The zona pellucida is responsible both for species-specific binding of sperm to unfertilized eggs and inducing bound sperm to undergo the acrosome reaction. The latter enables sperm to penetrate the extracellular coat and fertilize the egg. The zona pellucida also aids in prevention of polyspermy following fertilization and in protection of preimplantation embryos. In mice, several of these important functions can now be ascribed to specific zona pellucida glycoproteins that have been purified and characterized. Furthermore, the enzyme responsible for hatching of embryos from the zona pellucida, just prior to implantation, has been identified and characterized.     

Energy transfer in a bioluminescent system

Many (but not all) of the bioluminescent systems in coelenter-ates involve energy transfer from an excited product molecule of the calcium activated photoprotein to a second species, the green fluorescent protein, with emission at 508 nm from its excited state. Although all the luminescent coelen-terates studied possess photoproteins, not all of them have the green fluorescent protein. This green fluorescent molecule is localized in the luminescent cells; they can thus be easily distinguished by fluorescence microscopy. The active components occur in subcellular particles; these have been isolated in an active form by homogenization in isotonic (to sea water) salt solutions.     

Method for reducing the rate of light emission from chemiluminescent and bioluminescent reactions using frozen reagents

Frozen assay reagents have been used to reduce the rate of light emission from the rapid chemiluminescent acridinium ester and the bioluminescent firefly luciferase reactions. Melting of the assay reagent delays the initiation of the light emission, thus eliminating the need to initiate these rapid reactions by injection of the assay reagents in front of the photodetector.     

Infusion reactions in natural killer cell immunotherapy: a retrospective review

Background aimsThe use of natural killer (NK) cells as a cellular immunotherapy has increased over the past decade, specifically in patients with hematologic malignancies. NK cells have been used at the authors’ institution for over 15 years. Most patients have a reaction to NK cell infusion. The authors retrospectively analyzed the reactions associated with NK cell infusions to characterize the types of reactions and investigate why some patients have higher-grade reactions than others.MethodsA retrospective chart review of NK cell infusions was performed at the authors’ institution under nine clinical protocols from 2008 to 2016. An infusion reaction was defined as any symptom from the time of NK cell infusion up to 4 h after infusion completion. The severity of infusion reactions was graded based on Common Terminology Criteria for Adverse Events, version 4. Two major endpoints of interest were (i) infusion reaction with any symptom and (ii) grade ≥3 infusion reaction. Multivariable logistic regression models were used to investigate the association between variables of interest and outcomes. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained for each variable.ResultsA total of 130 patients were receiving NK cell infusions at the authors’ institution. The most common reported symptom was chills (n = 110, 85%), which were mostly grade 1 and 2, with only half of patients requiring intervention. There were 118 (91%) patients with infusion reactions, and only 36 (28%) were grade 3. There was one life-threatening grade 4 reaction, and no death was reported due to infusion reaction. Among grade ≥3 reactions, cardiovascular reactions (mainly hypertension) were the most common, and less than half of those with hypertension required intervention. NK cell dose was not associated with any of the grade 3 infusion reactions, whereas monocyte dose was associated with headache (grade ≤3, OR, 2.17, 95% CI, 1.19–3.97) and cardiovascular reaction (grade ≥3, OR, 2.13, 95% CI, 1.13–3.99). Cardiovascular reaction (grade ≥3) was also associated with in vitro IL-2 incubation and storage time. Additionally, there was no association between grade ≥3 infusion reactions and overall response rate (OR, 0.75, 95% CI, 0.29–1.95).ConclusionsThe majority of patients who receive NK cell therapy experience grade 1 or 2 infusion reactions. Some patients experience grade 3 reactions, which are mainly cardiovascular, suggesting that close monitoring within the first 4 h is beneficial. The association of monocytes with NK cell infusion reaction relates to toxicities seen in adoptive T-cell therapy and needs further exploration.     

Neonothopanus gardneri: a new combination for a bioluminescent agaric from Brazil

The bioluminescent agaric, Agaricus gardneri Berk., was rediscovered recently in central Brazil. The new combination, Neonothopanus gardneri, is proposed for this long-forgotten taxon supported by morphological and molecular data.     

Calcium and bioluminescent probes

Ca2+ is an universal second messenger in numerous cell physiological processes. Aequorin, a bioluminescent calcium-binding protein is used today as a cellular probe to measure and image variations in calcium concentrations. The paper describes the characteristics and the use of aequorin as a luminescent calcium probe, and the future in the use of this protein for calcium imaging.     

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5‐disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.     

Homogeneous, bioluminescent protease assays: caspase-3 as a model

Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease substrate and a firefly luciferase that has been molecularly evolved for increased stability. By combining the proluminescent caspase-3 substrate, Z-DEVD-aminoluciferin, with a stabilized luciferase in a homogeneous format, the authors developed an assay that is significantly faster and more sensitive than fluorescent caspase-3 assays. The assay has a single-step format, in which protease cleavage of the substrate and luciferase oxidation of the aminoluciferin occurs simultaneously. Because these processes are coupled, they rapidly achieve steady state to maintain stable luminescence for several hours. Maximum sensitivity is attained when this steady state occurs; consequently, this coupled-enzyme system results in a very rapid assay. The homogeneous format inherently removes trace contamination by free aminoluciferin, resulting in extremely low background and yielding exceptionally high signal-to-noise ratios and excellent Z' factors. Another advantage of a luminescent format is that it avoids problems of cell autofluorescence or fluorescence interference that can be associated with synthetic chemical and natural product libraries. This bioluminescent, homogeneous format should be widely applicable to other protease assays.     

N-Alkylated 6'-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase: new potential scaffolds for bioluminescent assays

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.     

Two simple and generic antibody-independent kinase assays: comparison of a bioluminescent and a microfluidic assay format

In this study, the authors have compared the performance of 2 high-throughput screening assays for a serin/threonine kinase: a microplate-based, bioluminescent assay that uses the luciferin/luciferase system to monitor ATP consumption, and a microfluidic assay that measures the change in mobility in an electric field of a fluorescently labeled peptide upon phosphorylation. Both assays are homogeneous, nonradioactive, antibody independent and could be miniaturized to a reaction volume of 4 microl. The robustness of both formats was demonstrated by Z' values > 0.8. Screening of a small library (2133 compounds) showed that the results obtained with both technologies correlate very well. Although the threshold for hits was set to a comparably low value-22.2% and 13.7% inhibition for the ATP consumption and microfluidic assay, respectively, corresponding to mean plus 3 standard deviations-the overlap of active compounds identified with the 2 assay formats was greater than 94%. Thus, both assays allow the identification of even low potency inhibitors with a high level of confidence.