Lanthanide-based luminescent assays for ligand-receptor interactions

The evaluation of receptor ligand interactions is important in the field of drug discovery and development. Currently these interactions are typically measured with cumbersome (low throughput) radiolabels. Higher throughput screens are available such as fluorescent measurements of G-protein coupled receptor-induced Ca2+ increases or fluorescence anisotropy, yet these have limited applicability and/or low signal to noise. Hence, there is a need to develop more widely applicable and more sensitive labels that can be used to monitor ligand-receptor interactions. Lanthanides provide an attractive alternative to the traditional labels used for monitoring ligand-receptor interactions. The incorporation of lanthanide labels into traditional assays used to assess receptor-ligand interactions can make these assays more affordable, less time consuming and amenable to automation. Lanthanides can be coupled to ligands and provide strong luminescent signals that can be detected using time-resolved fluorescence (TRF) methods. This approach takes advantage of the long fluorescence lifetime of the lanthanide and can detect less than one attomole of europium in a multiwell plate sample. This short review provides a basic introduction into lanthanides and TRF and describes some of the recent assays which have utilized lanthanides as labels to assess ligand-receptor interactions.     

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.     

Construction and calibration of an optical trap on a fluorescence optical microscope

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.     

Ecological fundamentals and methodology for selection and introduction of new species into an aquaculture

The present article gives an overview of problems related to the selection and introduction into the aquaculture of freshwater organisms that may be used for the purposes of fish farming, toxicological regulation and biotesting. The demands made on potential subjects of cultivation are discussed, and the reasons limiting the widening of such species are analyzed. The ecological fundamentals and methodology for selection and introduction into the aquaculture of new species are stated. The practical use of proposed methodology is demonstrated on the example of a cladoceran Bosmina longirostris O.F. Müller.     

Analysis of the Native Structure,Stability and Aggregation of Biotinylated Human Lysozyme

Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.     

微透析技术在猴脑递质研究中的应用

应用透析原理对脑内细胞外液中神经化学物质进行取样分析的想法已有２０多年历史,对脑内细胞外液进行分析可以更好地了解中枢神经系统的生理和病理情况,而微透析技术就是一种用来测定脑内细胞外液神经递质浓度的取样方法,随着探头设计的微型化和高灵敏度的分析方法的建立,微透析技术在许多实验室已成为一种常规实验手段,近年来,以猴脑的研究对象的微透析实验呈增长趋势,并获得了一些崭新的结果,本文介绍在猴脑上进行微透技术     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.     

Methodology for assaying recombinant interleukin-2 associated with liposomes by combined gel exclusion chromatography and fluorescence

A simple methodology based on fluorescence and gel exclusion chromatography (GEC) has been developed to assay recombinant Interleukin-2 (rIL-2) associated with vesicles. A Sephadex G75 column was used to separate the liposomes from non-entrapped rIL-2. The elution of the rIL-2 liposomes was monitored by coupling fluorescent and light scattering detection. The solubilisation of the vesicles with octylglucoside (OG) before the assay was necessary to avoid interference from light scattering. This methodology can be automated to yield an on-line system that can separate, solubilise and quantify rIL-2 in liposome samples. It can be extended to any protein associated with vesicles provided that the former can be detected by fluorescence.     

Use of Kaede fusions to visualize recycling of G protein-coupled receptors

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.     

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.     

A radiative transfer modeling approach for accurate interpretation of PAM fluorometry experiments in suspended algal cultures

The results of a numerical study on the simulation of pulse amplitude modulated (PAM) fluorometry within dense suspensions of photosynthetic microorganisms are presented. The Monte Carlo method was used to solve the radiative transfer equation in an algae‐filled cuvette, taking into account absorption, anisotropic scattering, and fluorescence, as well as Fresnel reflections at interfaces. This method was used to simulate the transport of excitation and fluorescence light in a common laboratory fluorometer. In this fluorometer, detected fluorescence originates from a multitude of locations within the algal suspension, which can be exposed to very different fluence rates. The fluorescence‐weighted fluence rate is reported, which is the local fluence rate of actinic light, averaged over all locations from which detected fluorescence originated. A methodology is reported for recovering the fluorescence‐weighted fluence rate as a function of the transmittance of measuring light and actinic light through the sample, which are easily measured with common laboratory fluorometers. The fluorescence‐weighted fluence rate can in turn be used as a correction factor for recovering intrinsic physiological parameters, such as the functional cross section of Photosystem II, from apparent (experimental) values. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1601–1615, 2016     

Genetic and individual assignment of tetraploid green sturgeon with SNP assay data

Polyploid organisms pose substantial obstacles to genetic analysis, as molecular assay data are usually difficult to evaluate in a Mendelian framework. Green sturgeon (Acipenser medirostris) is a tetraploid species and is facing significant conservation challenges, including bycatch in ocean fisheries. We present here novel molecular genetic assays and analytical methodology for green sturgeon that allow discrimination of fish from the two visually indistinguishable distinct population segments (DPSs), and also provide individual-specific genetic tags. We show how the relative fluorescence intensity data from a standard quantitative PCR assay, designed for a biallelic single nucleotide polymorphism, can be grouped into “genotype categories” using standard analytical software and post-processing manipulation. We then show how these genotype category data can be used to discriminate green sturgeon from the southern DPS, which is protected under the US Endangered Species Act, and the northern DPS, which is not. We also show how these data can be used to reliably identify individual green sturgeon, and can therefore be used in capture/recapture analyses. Both types of identification are extremely accurate even when fewer than half of the assays are successfully called. We then apply these new techniques to show that proportions of the two green sturgeon DPSs are extremely different in the two major fishery areas where they are encountered as bycatch. While these assays and methods do not provide data that can be used in pedigree-based analyses, they are an important advance in the application of genetic analysis to conservation and management of polyploid organisms.     

Enzymatic Reactions in Non-Conventional Media: Prediction of Solvent Water Content for Optimum Water Activity

Most enzymes provide their optimum performance at a given water activity (aw), which is generally solvent independent. For a given organic liquid solvent at a specific temperature or for a supercritical solvent at a specific temperature and pressure this corresponds to a water concentration in which water has the desired activity. We present here a methodology for predicting this water concentration thus reducing substantially the amount of experimental work needed to find the optimum solvent with respect to equilibrium conversion.

If the enzyme optimum water activity is known, the methodology predicts the required water content in the solvent to achieve this aw value. If, in addition, the enzyme water activity curve is available, this methodology provides the total water that must be added to the system (enzyme plus solvent) so that a specific water activity can be obtained.

The same methodology can also be applied to predict the effect of the total water content of the system (initial or initial plus produced) on the water activity values. It is shown that: (a) for esterification reactions taking place in hydrophobic organic solvents, the produced water can lead to a substantial change in water activity, but not for less hydrophobic solvents; (b) introduction of dry CO₂ into a system, pre-equilibrated to a certain water activity at atmospheric pressure, can lead to a substantial decrease in the water activity especially at temperatures just above the critical one of the solvent and pressures larger than that.     

Improved analytical methods for microarray-based genome-composition analysis

Background  

Whereas genome sequencing has given us high-resolution pictures of many different species of bacteria, microarrays provide a means of obtaining information on genome composition for many strains of a given species. Genome-composition analysis using microarrays, or 'genomotyping', can be used to categorize genes into 'present' and 'divergent' categories based on the level of hybridization signal. This typically involves selecting a signal value that is used as a cutoff to discriminate present (high signal) and divergent (low signal) genes. Current methodology uses empirical determination of cutoffs for classification into these categories, but this methodology is subject to several problems that can result in the misclassification of many genes.     

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.     

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.