Bioluminescence and Chemiluminescence

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First, I would like to thank the organizers for inviting me to give the Memorial lecture in honor of Professor Gordon Stewart who was taken away from us by brain cancer in the prime of his scientific career. I also would like to thank the Hamamatsu Corporation for supporting the Gordon Stewart Memorial Lecture. Today I have selected a topic, which is literally a Cinderella story. In what follows, I would like to describe for you how a small specialized area of biology, the study of bioluminescence, became a major contributor to molecular biology, molecular genetics, cell biology, developmental biology, molecular and cellular immunology, transplantation biology, medical, food, and environmental diagnostics, and even ex vivo gene therapy. In all these fields, the contribution of bioluminescence has the common theme of monitoring and imaging of biological processes based on light emission in living cells and organisms.¹ A byproduct of the contribution of the bioluminescence field is the gradual removal of the use of radioactivity from biomedical research and diagnostics, and the replacement of radioactivity with biological light-emitting proteins. Due to limited time, it is impossible to cover in depth all of the significant scientific contributions, which led to the transfer of light-emitting capabilities from naturally-occurring light-emitting organisms to non-light-emitting cells and organisms. Therefore I will try to point out some selected milestones achieved by curious, dedicated scientists during the past forty years, which made today's imaging science possible. I would like to outline in my lecture the milestones on the way to the application of light-emitting proteins in imaging. Of necessity I will concentrate on work done and data generated in our own laboratory using the Hamamatsu Argus 100 system, a Zeiss fluorescence microscope, and a Leica stereo fluorescence microscope…

The unicellular marine dinoflagellate Lingulodinium polyedra (Gonyaulax polyedra) is one of the classic circadian model organisms. It exhibits many different circadian rhythms, among others a circadian rhythm in bioluminescence.¹ Recent findings suggest circadian control of the input pathways of the L. polyedra circadian system.² A mathematical model was proposed by Roenneberg et al.³ implementing these new findings into the clockwork mechanism, but without reference to other proposed models. The aim of this study is to modify an existing algorithm for modelling circadian oscillators by adding an autoregulatory feedback loop as part of the input pathway and comparing the outcome of simulations with experimental data of L. polyedra bioluminescence rhythms…

Beetle luciferases are divided in two large groups according to their bioluminescence spectral sensitivity to pH¹: (I) the pH-sensitive luciferases (fireflies) which suffer red-shift upon decreasing pH and other denaturing conditions and (II) pH-insensitive (click beetle and railroad worms) luciferases which are not affected by these conditions. Most studies on the relationship between structure and emission spectra have focused on the pH-sensitive luciferases, much less is known about pH-insensitive luciferases. We cloned the cDNAs for four pH-insensitive luciferases: the red and green light-emitting luciferases of the Brazilian Phrixothrix hirtus (PxhRE) and P. vivianii railroad worms (PxvGR),² the green emitting luciferases of the Brazilian Pyrearinus termitilluminans larval click beetle (Pte)³ and the Japanese Ragophthalmus ohbai star worm (Rol).⁴ We found that the region before residue 344 determines the bioluminescence spectrum in railroad worm luciferases⁵ and that the residues R215 and T226 in pH-insensitive luciferases (N229 in pH-sensitive luciferases) are key residues for green light emission.^5,6 Through the construction of new chimerae using Phrixothrix luciferases we here show that the region between residues 220-344 is the main determinant of bioluminescence colors in railroad worm luciferases. We also investigated the effect of single and double substitutions of some conserved residues differing between pH-sensitive and pH-insenitive luciferases…

Fireflies communicate by emitting bioluminescent flashes in rapid and reproducible patterns that are species specific. Typically, the duration of a single flash is between 50 and 250 msec, and depending on the species the flash pattern may consist of repeated singles or repeated groups of 1-5 per second.¹ The biochemical basis of bioluminescence in fireflies involves the luciferase-catalyzed ATP-dependent activation of luciferin to a luciferyl-adenylate intermediate, which upon reaction with oxygen emits a photon.² While the biochemistry is well understood, the on-off mechanism that regulates luciferin-luciferase bioluminescence to produce precise flash patterns has been a long-standing and fascinating puzzle…

Although evident on land, bioluminescent organisms predominate in aquatic environments with only a few groups of terrestrial animals exhibiting the necessary components to generate visible light. These include worms, centipedes, millipedes, fungoidal gnats, but most prevalent of all are the bioluminescent beetles. Spanning three different families of coleopterans, Lampyridae, Elateridae and Phengodidae, these luciferase-luciferin systems are the most widely studied of all terrestrial metazoans. The biochemical and biological diversity of bioluminescent systems both on land and in the water suggests that the ability to generate light arose from many separate origins in evolution…

Firefly luciferase (FFL) catalyses the oxidation of a heterocyclic molecule called luciferin (LH₂) in the presence of ATP and oxygen. Reaction proceeds through the formation of a ternary complex followed by an adenylation step in which an intermediate product luciferyl-adenylate is formed. Different observations suggest that FFL undergoes significant conformational changes in the process of the bioluminescent reaction…

The crystal structure of firefly luciferase¹ shows the presence of several hydrophobic amino acids whose side chains are highly solvent-exposed. In a previous study, several such amino acids, which are non-conserved amongst beetle luciferases were selected to be mutated to alanine.² It was shown that none of these mutations affect the wavelength of bioluminescent emission and that several of these mutants exhibit an increase in thermo-stability…

Oxyluciferin in its singlet electronically excited state is the product of the firefly bioluminescent reaction. The examination of its fluorescent properties in solution and in complexes with firefly luciferase provides useful information about the mechanism of firefly bioluminescence.^1-3 Dimethyloxyluciferin (DMOL) is an oxyluciferin analog (Fig. 1), that exists only in a keto form, whereas oxyluciferin can be in the keto or enol form. That is why it was interesting to study the spectral and fluorescent properties of DMOL and its interaction with firefly luciferase…

The His433Tyr mutation in Luciola cruciata and Hotaria parvula firefly luciferases resulted in bioluminescence color changes from 570 to 610 nm.^1,2 It means that the highly conservative His433 residue located out of the luciferase active site plays a very important role in enzyme function. The goal of this work was to construct mutants His433Asn and His433Ser for Luciola mingrelica firefly luciferase, which has high homology with luciferases indicated above. We studied catalytic properties of the enzyme mutant forms and their bioluminescence spectra. Analysis of the data obtained permitted us to elucidate the mechanism of the influence of the His433 residue on the luciferase active site…

The firefly bioluminescence is currently of the great interest for researchers as an example of effective bioconversion of energy from a chemical reaction into light. Moreover, the firefly bioluminescent reaction is widely used as a very sensitive bioassay for ATP detection. Nevertheless, the mechanism of light-emitting species formation in firefly luciferase reactions has not been entirely understood yet. There is no unanimous opinion on the origin of colour differences in firefly bioluminescence. At least three alternative mechanisms of luciferin-luciferase interactions responsible for characteristics of luminescence have been under discussion: (1) enzyme provides basic vicinity for oxyluciferin, assisting its tautomerisation;¹ (2) the polarity of the active site influences on the electron-excited states of oxyluciferin;² (3) conformation of active site determines the twisted or flat structure of oxyluciferin molecule…

The biosynthetic pathway of beetle luciferin (LH₂) remains unresolved. It has been suggested that p-benzoquinone and cysteine are the in vivo precursors of LH₂.1 However, whilst LH₂ can be chemically synthesised from these compounds there is no evidence that an analogous enzyme-mediated reaction occurs in vivo (though studies using radio-labelled cysteine do seem to confirm it as a building block of the thiazoline rings of LH₂²). In fact the only known enzymatic activity associated with LH₂ synthesis is a recently reported ‘oxyluciferin recycling enzyme’.³ However, it is not known whether this enzyme has a role in the de novo synthesis of LH₂ in vivo. An understanding of LH₂ synthesis promises numerous benefits. For example, LH₂ is an expensive reagent that could potentially be produced more cheaply from bacteria/yeast containing the biosynthetic genes for LH₂. In addition, the biosynthetic genes for LH₂ could be used in conjunction with luciferase as a gene-reporter / imaging system so removing the need to extraneously supply luciferin. Further, enzymes involved in LH₂ synthesis could themselves form part of novel bioluminescent assays. Finally, the characterisation of the LH₂ biosynthetic pathway could supply important information on the basis of evolution of bioluminescence in Coleoptera…

Most firefly luciferases such as the luciferase from Photinus pyralis (Ppy) demonstrate pH dependent bathochromic shifts of their bioluminescent spectra in excess of 50 nm as the pH of the reaction is dropped from pH 8.0 to pH 6.0.¹ However luciferases from click beetles and railroad worms do not show such a bathochromic shift hence this effect is clearly protein mediated…

Beetle luciferases isolated from various insects are characterized by identical structures of both substrates and emitter. The main difference is in color of bioluminescence that is determined by the properties of microenvironment of the emitter in the enzyme active site. Several mechanisms were proposed to explain the bioluminescence color difference for native and mutant luciferases. According to White¹ bioluminescence spectral shifts are attributed to the changes in the polarity of oxyluciferin binding site similar to the effects of polarity of the solvent on spectral properties of fluorophore. However, this mechanism could not explain the existence of two forms of oxyluciferin (keton and enol) at different pH. According to other theory,² the changes in bioluminescent spectra, especially at different pH, could be the result of keto-enol tautomerization of oxyluciferin. However, this mechanism could not explain the spectral shifts without changes in the shape that were observed for click-beetles and mutant luciferases. According to McCapra³ excited oxyluciferin could exist in two stereo conformations that have different energy and provide different color of emission. However, this mechanism could not explain green fluorescence of enol forms of oxyluciferin and its analogues. Besides, the calculations of molecular structure of oxyluciferin in ground and excited state^4,5 revealed that planar conformations had minimal energy and thus were optimal for both ground and first singlet excited states. It seems that the mechanism of “twisted” conformation for color changes in firefly bioluminescence is non feasible. In this paper theoretical consideration of environment effect on emission spectra is used for analysis of spectral changes in different natural and mutant forms of luciferases…

The primary reaction step of firefly chemi- and bioluminescence is essentially the oxygenation of luciferin (Ln) at its C4 site to produce the electronically excited oxyluciferin (Oxyln*). We have already studied the optical absorptions of Ln and its deprotonated forms in a vacuum and in dimethyl sulfoxide (DMSO) in order to clarify the electronic properties of these compounds by molecular orbital (MO) calculations.¹ In the present work, the optical absorptions of firefly Oxyln, including its mono- and dianions (Oxyln^-1 ;I = 1,2) were obtained by ab initio calculations within the density functional approximation (B3PW91) using the 6-3 IG* basis set in the Gaussian 98 program (Gaussian Inc., Pittsburgh, PA, USA (1998)) and the INDO/S method in MOS-F (Fujitsu Ltd., Tokyo, Japan (1999)) prior to studying the formation process of the electronically excited Oxyln*. Furthermore, the emission spectrum of Oxyln was evaluated by optimizing its energy not in the ground state but in the excited state, using the semi-empirical MNDO-PM5 method in CAChe 5.0…

It was demonstrated previously that dark mutants of Vibrio harveyi, a marine luminescent bacterium, are more sensitive to UV irradiation than wild-type strains of this bacterium.¹ This difference in UV sensitivity was significantly reduced when after irradiation bacteria were cultured under a white fluorescent lamp.¹ On the basis of these results it was proposed that bacterial bioluminescence plays a role in DNA repair, most probably by stimulation of the photoreactivation process…

Immobilized enzymes are widely used for clinical and industrial purposes for the assay of different analytes and enzymes. At the same time, the continuing quest for sensitive non-radioactive methodologies, has contributed to a rapidly expanding repertoire of bioluminescent assays for metabolite, hormones, drugs, and even explosives. The immobilization of an enzyme generally results in increased stability and the additional property of reusability, while retaining specificity for substrates and effectors.¹ The immobilized enzyme can be used as an analytical tool in place of its soluble counterpart with the added advantages of easier handling and decreased cost. Bacterial luciferases catalyze the luminescence reaction utilizing FMNH₂, a long chain aliphatic aldehyde and O₂, to produce blue-green light at 490 nm. This light-emitting system is particularly attractive for use in bioassays as it is readily available, the substrates are very inexpensive and it can be readily coupled to formation of NAD(P)H generated on oxidation of a wide variety of different biological compounds…

Yellow emitting luminous bacterium Vibrio fischeri strain Y1 produces diverse fluorescent proteins. In particular, yellow fluorescent protein (YFP) is of functional importance to induce the yellow bioluminescence with a maximum around 540 nm in the luciferase reaction.^1-4 YFP exists as the two active forms, both of which are capable of being the secondary emitter.⁵ Besides YFP, V. fischeri Y1 has a blue fluorescent protein (Y1-BFP), whose cellular level may be comparable to the YFP level, but its function is unknown…

Streptococcus pyogenes is a Gram-positive cocci that frequently inhabits the nasopharynx. This Group A Streptococcus (GAS) has been implicaled in several severe systemic and invasive diseases such as scarlet fever, toxic shock syndrome, and necrotizing fasciitis^1,2 (also known in the media as flesh eating bacteria). In order to study GAS growth, infection and disease progression in animal models, a mechanism was sought that would allow in vivo monitoring of bacterial distribution in model systems…

To structural biochemists, the three-dimensional structure of the enzyme showing comprehensive ligand-protein binding interactions is extremely helpful in determining the function of specific amino acid residues in the protein by conducting chemical modification and mutagenesis experiments. The structure of bacterial luciferase has been solved recently,¹ but information concerning the flavin and fatty aldehyde binding sites are not available in the apo-luciferase structure. Although experimental results published over the past three decades have allowed researchers in the field to identify the location of the active site cavity lined by a number of residues implicated in modulating the catalysis and substrate binding, the specific interaction between these residues and the substrates could not be defined. By anchoring the phosphate moiety of flavin mononucleotide (FMN) and active flavin analogs to a phosphate binding site on the α subunit of luciferase and searching for a common orientation of the isoalloxazine ring, we have identified an unique binding conformation for the flavin substrate in the active site of luciferase,² and noticed that the catalytic isoalloxazine moiety of the FMN binds directly to a non-prolyl cis-peptide linkage between Ala 74 and Ala 75, which is an unique and extremely rare structural feature suspected to be involved in the reaction since its recognition in the crystal structure.¹ Among the potential residues binding to flavin, Cys 106 was of great interest as it has been extensively investigated, and its involvement in luciferase function and substrate interactions were recently under debate.³ In the flavin-luciferase model we proposed,² the thiol side chain closely abuts the re-face of FMN's isoalloxazine moiety, leaving a spacious tunnel-shaped cavity on the si-face side of isoalloxazine suitable for the entry and binding of the long chain fatty aldehyde. Our flavin-luciferase binary complex model is the only three-dimensional framework currently available for extending the investigation of the active site to understand the structural basis of light emission.² Here, we present results showing that the emission spectrum of the luminescence is shifted by a single point mutation at position 106 suggesting that the isoalloxazine chromophore and the side chain of Cys 106 are in close proximity, supporting the validity of the model…

The induction of luminescence in Vibrio harveyi is dependent on the production of autoinducers which partake in a complex system of signal transduction through a central regulator LuxO. SAM is one of the key metabolites required in biological systems and plays a central role in the formation of the two autoinducers (AI-1 and AI-2) in the quorum sensing systems controlling luminescence in Vibrio harveyi.^1,2 AI-1, N-hydroxybutanylhomoserine lactone is believed to arise from the reaction of SAM and N-hydroxybutanoyl-ACP catalyzed by LuxM. Strong evidence exists that AI-2 arises from cleavage of SAM to S-adenoysl homocysteine and then to Sribosylhomocysteine which is then converted by LuxS into a ribosyl derivative that cyclizes into a furanone. We therefore predicted that genes that limit the level of SAM should inhibit luminescence while genes that stimulate its synthesis should decrease luminescence. Recently we have found that proteins involved in the regulation and metabolism of SAM greatly affect the level of luminescence in autoinducer-deficient mutants of V. harveyi. As these proteins can be related to the synthesis and degradation of SAM and/or methionine (Met), elucidation of the mechanism by which these proteins affect luminescence including potential effects on the levels of autoinducers and other regulatory elements involved in the quorum sensing system is of primary interest. These studies should prove valuable in identifying the control and specific pathways responsible for the synthesis of the autoinducers…

In V. harveyi, the model for the regulation of luminescence¹ includes sensors 1 and 2, LuxN and LuxQ, that autophosphorylate within the sensor recognition kinase domain, then transphosphorylate to the downstream receiver domain. Phosphorelay to the hpt (histidine-containing phosphate transfer) domain in LuxU followed by transfer to the response domain of the regulator, LuxO, then ensues. LuxO is a σ⁵⁴-binding activator whose phosphorylated form at low cell densities represses luminescence. As autoinducers (AI) accumulate, AI-1 interacts with LuxN, and AI-2, the luxS product, associates with a periplasmic protein LuxP to interact with LuxQ. The sensors switch from kinase to phosphatase activity causing inactivation of LuxO. With the recent discovery that luxS is present in many bacteria and appears to have similar interspecies functions, it is of interest to see whether the signal transduction pathway to LuxO is also widely distributed…

Bioluminescent bacterial systems are used successfully for ecological monitoring.^1,2 Bioluminescent coupled enzyme system NAD(P)H:FMN-oxidoreductase-luciferase is currently used as a test system. The reactions of the coupled enzyme system are shown below…

Ca²⁺-activated photoproteins are promising candidates to be used as labels for a highly sensitive rapid immunoassay. A strict specificity of photoprotein for Ca²⁺ ions and a high quantum yield of bioluminescent reaction provide a low background signal and a high sensitivity of the assay. Successful photoprotein applications in immunoassay (aequorin from Aequorea Victoria¹ and obelin from Obelia longissima²) has been demonstrated. The photoprotein labels are stable and nontoxic, the reaction is rapid and simple, the linearity of light emission is observed over many log units of concentration…

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is widely used as a fluorescent marker in imaging applications to reveal the location of proteins in cells. However, such imaging of fluorescence intensity does not generally provide information about the biophysical environment of the protein. Fluorescence lifetime imaging (FLIM)^1,2 is a technique that, in addition to position and intensity, also captures the fluorescence lifetime. However, in order to interpret FLIM of GFP, the biophysical parameters that affect the GFP fluorescence lifetime have to be identified…

In the current era of proteomics and high throughput drug screening, rapid protein purification methods that do not compromise purity, yield, and sample volume are in great demand. We present a three-phase partitioning method which is highly applicable to the bioluminescent reporter protein GFP (green fluorescent protein).^1,2 Purifications in excess of 100-fold from whole, unlysed bacterial cells is achieved with nearly full GFP recovery in less than 10 minutes (on a micro scale). The method is highly scaleable, performed at a macro level in a matter of several hours…

Phototransformation of Aequorea victoria Green Fluorescent Protein (GFP) was discovered shortly after the cloning of the gfp gene.¹ UV/Vis light transforms the species absorbing at 398 nm (GFP₃₉₈), with a neutral, phenolic chromophore, into an ionic species absorbing at 483 nm (GFP₄₈₃), with a phenolate containing chromophore.^2,3 Besides its role as transient proton acceptor in the excited state, Glu 222 has also been suggested to function as the stable acceptor for the proton that is lost from the chromophore upon photoconversion. Conformational changes such as rotamer reorientation of a threonine side chain (Thr 203) and syn/anti-isomerisation of Glu 222 have been proposed to compete with reformation of the protonated ground state of the chromophore.⁴ However, a direct test of this model with FTIR spectroscopy turned out negative, indicating that Glu 222 is not the stable proton acceptor…

Each of us enters science with pre-conceived notions about our particular science and each picks up other notions from our mentors and our teachers. While some ideas begin as interesting hypotheses, such ideas have a way of self-solidifying. Some, by mere repetition, wind up being widely accepted. As we develop our own scientific careers, we create hypotheses to guide our work—ones that may unduly influence our students and the readers of our papers. Almost always, creators of hypotheses do so for the best of reasons. Almost always, those who repeat the hypotheses have equally noble intentions. All fields have to deal with inadvertent solidification of unproven hypotheses…

Dioxiranes, the smallest ring peroxides, are known as highly effective yet selective oxidants, which have been intensively studied over the last decades, mainly in oxygen-transfer processes.^1,2 During the last few years, however, attention has been focused on a new aspect of these oxidants, namely generation of singlet oxygen during the reaction of dioxiranes with nucleophilic anions.^3-6 One of the first anion-induced chemical liberation of singlet oxygen from dioxiranes has been the infra-red chemiluminescence (IR–CL) observed³ in the ketone-catalyzed decomposition of the monoperoxysulfate ion (Caroate). The key step in this catalytic process involves the nucleophilic attack of the HSO₅^- ion on the intermediary dioxirane with the release of ¹O₂. A similar pathway was proposed to account for the ¹O₂ formation by the ketone-catalyzed decomposition of a number of peroxycarboxylic acids.⁴ Moreover, it was suggested that ¹O₂ is generated in the reaction of the nitrite ion with the intermediary dioxirane formed in the ketone-catalyzed decomposition of peroxy nitrites; the latter are important oxidants in numerous biochemical processes.⁵ It was recently demonstrated⁶ that chloride, t-butoxide and hydroxide ions decompose dimethyldioxirane (DMD) with the production of singlet oxygen; however, no data on the efficiency of this process have been reported. Herein, we report the quantum yields of singlet oxygen generation for the catalytic decomposition of DMD, induced by a variety of nucleophilic anions…

The non-luminescent 2,3-dihydro-1,4-phthalazinedione (phthalic hydrazide) is converted upon hydroxylation of its aromatic ring into a light emitting species.¹ The sequence of reactions leading to the formation of the emitter is rather complex and well studied for 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol), the amino derivative of phthalic hydrazide. It includes the oxidation of the hydrazide moiety to a diazaquinone intermediate, the nucleophilic addition of the anion of hydrogen peroxide to the latter one with the formation of an α-hydroxy-hydroperoxide, its further conversion into an endoperoxide species and finally the repulsion of nitrogen that leads to derivatives of phthalic acid in the excited state.² Previous introduction of electron donating groups into the aromatic ring of non-substituted phthalic hydrazide is necessary to form molecules in the excited state.¹ An additional amino group leads to the well known luminescent derivatives of phthalic hydrazide, luminol and isoluminol. Also a hydroxylation causes luminescent molecules. The hydroxylation of phthalic hydrazide has been used by our group to investigate the hydroxylation of selected carbohydrates by a mixture of ferrous ions and hydrogen peroxide, the Fenton reagent…

The growing resistance of many microorganisms to antibiotic drugs has led to a revival interest in the photodynamic effect in this area (the photodynamic effect has been known to kill bacteria for almost 100 years)…

7-Amino-4-trifluoromethylcoumarin (ATFMC) is used in the synthesis of a substrate for the fluorimetric assay of proteolytic enzymes in biological fluids¹ and for use as a laser dye² and as a marker for proteinase.³ In this paper we report the first study of CL from reactions of peroxyoxalate esters, hydrogen peroxide and ATFMC…

The first stable and enzymatic triggerable 1,2-dioxetane was synthesized by the oxidation of (6-acetoxy-2-naphthyl) methoxymethyleneadamantane.¹ This 1,2-dioxetane utilized an arylesterase enzyme to catalyze the cleavage of the acetate to generate an unstable naphthoxide-substituted 1,2-dioxetane which on decomposition produces chemiluminescence in aqueous buffers at ambient temperature. The light producing efficiency of this 1,2-dioxetane in organic solvent and aqueous buffer was very poor. More efficient 1,2-dioxetanes were prepared by changing the size of the aromatic ring and position of the substitution in the aromatic ring.² The light producing efficiency of 4-(3-tert-butyldimethylsilyloxyphenyl)-4-methoxyspiro[1,2-dioxetane-3,2'-adamantane was found to be 25% when triggered with fluoride ion in dimethylsulfoxide. We wish to report on a new generation of 1,2-dioxetanes with π-electrons in a spiro-fused ring attached to the four-membered peroxide ring which can produce light in aqueous buffer…

Intracellular luminescent techniques requiring coelenterazine, such as bioluminescence resonance energy transfer (BRET), calcium detection, and intracellular reporter measurements, must accommodate the poor stability of this substrate in physiologically buffered solutions. Coelenterazine degradation leads both to loss of luminescence over time, and increased background luminescence caused by enzyme-independent oxidation (autoluminescence). Both conditions limit luminescence sensitivity by reducing the signal-to-noise ratio…

The intramicellar sensitization of phthalhydrazide chemiluminescent oxidation by energy transfer to selected xanthene as well as non-xanthene dyes has been investigated. The reactions were initiated using three different initiation systems: Sodium hypochlorite (1) hydrogen peroxide – sodium hypochlorite (2) and the alkaline copper sulphate – H₂O₂ system (3). In this work we report a significant difference in enhancement factors (ratio between sensitized chemiluminescence intensity and phthalhydrazide CL intensity) between the reactions initiated by systems 1 or 2 and 3. The mechanism of intramicellar sensitization based on Förster type energy transfer for the case (2) is proposed…

Recently, the electrogenerated chemiluminescence (ECL) has become an important detection technique in analytical chemistry. And Ru(bpy)₃²⁺ has become the most attractive ECL label because of its stability, regenerability, excellent luminescence properties and compatibility with a wide range of analyses…

Humus substances (HS) are amorphous, dark, partly aromatic and paramagnetic macromolecular anionic polyelectrolytes occurring in soil and water. They play a multifunctional role and are essential for life on Earth.¹ The presence in HS polyphenolic (PPh), quinoid (Q) and semiquinoid (SQ^˙, free radical) arrangements conjugated to aromatic and aliphatic backbones determines redox, optical and other properties of these biopolymers. The ratio of ortho- to para-Q and o-to-p-SQ^˙ may contribute to the antioxidative activity (AOA) and reactivity of HS towards environmental reactive oxygen species (ROS).² In order to verify this hypothesis we have determined the AOA and chemiluminescence (CL)-enhancing ability of selected PPh and Q. The results of the experiments and calculations of the electron density distribution and molecular parameters of certain o-and p-Q also confirm the hypothesis…

The effective application of horseradish peroxidase (HRP) in analytical biochemistry and biotechnology depends on its stability, especially when it is used repeatedly or in lengthy processes. HRP was shown to undergo irreversible time-dependent inactivation when catalysing different oxidation reactions. The peroxidation of some donor substrates results in the formation of highly reactive radical intermediates, which can in turn attack and modify the prosthetic group ^1,2 or apo-protein ³ causing the loss of enzymatic activity…

1,2-Dioxetanes are thermally labile substances, fragmenting on heating into electronically excited carbonyl products.¹ Our interest in this reaction stems not only from the experimental observation of “cold light” but also from the theoretical problem of understanding the mechanism of dioxetane decomposition.² To our knowledge, the activation parameters of thermal decomposition of 1,2-dioxetanes were always determined by isothermal methods.³ The used experimental technique is monitoring the rate of the dioxetane decomposition by chemiluminescence measurements in inert solvents. As a rule, the investigated temperature range is relatively small, frequently not larger man 15-20°K and the absolute temperature rarely exceeds 80°C. Surprisingly, up to now no experiment was published using the differential scanning calorimetry (DSC). If the decomposition is investigated in closed sample pans, the total part of the emitted radiation is completely converted into heat. Then, the instantaneous heat flow rate is a measure for the rate of the dioxetane decomposition. DSC measurements simultaneously allow the evaluation of both the activation parameters and the reaction heat. The investigation of the reaction with a number of different heating rates supplies more reliable kinetic results than isothermal scans in a relatively narrow temperature window. For this study we have used the 1,2-dioxetanes I – III. These dioxetanes were prepared by photooxygenation of the corresponding olefines at −20°C in methylene chloride…

Bioluminescent assay is one of the most efficient but yet insufficiently used tools of enlightenment and education. This paper will give a systematic review showing various applications of bioluminescence in teaching biology…

Being part of the EuroScience Week 2001, the Vinci-Darwin-Linnaeus event shared its major goal “to demonstrate and explain the impact of science, its uses, and its applications in the daily lives of European citizens.” The Florence science forum combined the efforts of three traditional partners from Italy, United Kingdom and Sweden within already established collaboration in a form of PUSH programme (Public Understanding of Science and Health). The three charismatic historical figures of Leonardo da Vinci, Darwin and Linnaeus were chosen by each country to symbolically represent and justify the motto of the Push 2001: “Toward a New Renaissance in Science.” In accordance with this motto the PUSH goal was to contribute in returning to science its integral holistic character and restoring its connection with the other disciplines studying life and natural world in general, as well as with art. To promote such a view of science in the perception of general public, teachers and young people, the wide variety of activities were designed and successfully carried out during three days of the Florence forum of science…

Bioluminescence measurements are reported in relative light units (RLU), which are dependent on the specific detector used and its operating conditions. In order to compare bioluminescent measurements from different detectors and assays, or to determine the quantum efficiency of a particular assay, photodetectors need to be calibrated such that measurements are reported in radiance (W/cm²/sr). This allows intensity measurements to be expressed in units normalized to the collection angle and independent of the device. We explore a calibration method using a diffused light emitting diode (LED) as a light source standard. The uniform light source is focused onto a silicon photodiode detector whose spectral calibration is traceable to a National Institute of Standards and Technology (NIST) standard. The intensity of the focused light source is calibrated in W/cm² as a function of the solid collection angle. The calibrated light source is then used to calibrate a photomultiplier tube (PMT)-based luminometer and a charge-coupled device (CCD) in W/cm²/sr. The method accounts for spectral properties of detector responsivities and light sources…

The knowledge about the mechanisms and the factors influencing protein-protein interactions including receptor oligomerisation,^1-3 G-protein coupled receptor functioning^2,3 and membrane reorganisation have become major targets in academic research and drug discovery. G-protein coupled receptors, also referred to as 7-transmembrane (7TM) receptors, comprise the largest and most diverse superfamily of proteins known. To a minor part only the ligands are known…

Peaks in bioluminescence are often associated with small spatial features including frontal boundaries, vertical thin-layers, and convergence/divergence zones.^1,2 Due to the highly variable nature of these features in the near shore environment, accurate assessment of bioluminescence potential is dependent upon the application of appropriate platforms and the use of these platforms at relevant time and space scales. Both traditional shipboard profiling techniques and moored vertical profiling instrumentation fail to adequately provide the appropriate spatial resolution to explain the dynamics of near shore bioluminescence. In an effort to resolve the mechanisms underlying the temporal and spatial variation in near shore bioluminescence, this study integrates an autonomous underwater vehicle (AUV) into a multi-platform adaptive sampling network. More specifically, we utilize the AUV to examine the effect of tides on the location and structure of optical, biological and physical signatures of a frontal zone in time and space. Furthermore, we examine the importance and significance of optimising the AUV flight paths to quantify small-scale structure in near shore environments…

Progress towards lower analytical detection limits is a key objective for many areas within proteomics and genomics, including clinical diagnosis, high throughput screening and drug target identification, fuelled by prospects of markedly reduced reagent costs, and ability to handle sample types or sample volumes containing inherently minute molar amounts of bioanalyte. Use of chemiluminescence, electrochemiluminescence and bioluminescence¹ as a means for quantitative analyte determination has formed the basis of many assays within the aforementioned areas, combining relatively intense luminescent phenomena with the instrumental and signal background advantages afforded by ‘excitationless’ emission. In particular, many proteomics approaches are taking advantage of chemiluminescent signal generation, making use of antibodies conjugated to enzymes such as Horse Radish Peroxidase (HRP) or Alkaline Phosphatase (AP) as a means for sandwich or competitive binding assays. For example, pre-deposited arrays of antibodies to known proteins may be used for clinical diagnosis or for protein expression profiling, the latter being of particular applicability in both drug target discovery and therapy monitoring. CCD cameras are ideally suited to the parallel quantitative determination of signal from each of the array test sites. Cameras with higher sensitivity and high dynamic range (16-bit) may be employed to extend the analytical capabilities of such platforms, markedly improving statistical performance as reflected in the assay Z-factors…

When the bioluminescent determination of ATP is performed by batch photometers, a computing procedure is necessary to exclude the mixing time of reagents from the global time of reaction because this produces a non statistic noise and, consequently, a poor analytical precision (please refer to Fig. 1 for details)…

Microarray technology allows multiple analyte detection in one assay, but requires very expensive instrumentation and highly trained personnel. We developed a simple, user-friendly system for multi-analyte detection in one well of a microtiter plate, requiring relatively inexpensive equipment, available in many laboratories. In brief, we designed a novel polystyrene microtiter plate where each of the 24 main wells is further divided into 7 subwells. This format enables separate immobilization of different bioprobes for multi-analyte binding assay in a single well. Chemiluminescent (CL) imaging detection allowed localization and quantification the signal in each subwell and provided high detectability and wide dynamic range…

There are three modes of light detection with photomultipliers (pmts). In pulse counting, information is contained in the shape and area of each pulse. In photon counting the intensity is given by the count rate, n, while in analogue detection it is proportional to the anode current, I_a. Pulse height encoding is uncommon in luminescence studies and choosing between photon counting or analogue detection is decided by signal levels. The spectrum of background counts contains an excess of small and large pulses which can be eliminated by a suitably set window.¹ In analogue detection, dark current is typically about twice the integrated dark counts.¹ Pmt gain changes with time and temperature and the stability of performance achieved by photon counting is at least twice that for analogue detection…

Chemiluminescence (CL) detection is a well-established detection principle, which has been successfully applied to several flow-through analytical techniques, including flow injection analysis (FIA) and separative methods such as pre- and post-column HPLC¹ and capillary electrophoresis.² A considerable increase in selectivity and analytical sensitivity was obtained, due to the high signal-to-noise ratio and specificity of CL…

Luminescence occurs when an electron relaxes from an excited state by photon emission. Electronic excitations require relatively high energies. Emission of a blue photon requires about 60 kcalmol^-1 of energy. This quantity of energy is significantly greater than the energies liberated in most biochemical reactions. For example, hydrolysis of ATP to ADP yields about 7 kcalmol…

The production of reactive oxygen species and the release of myeloperoxidase by stimulated polymorphonuclear leukocytes (neutrophils) causes an intense chemiluminescence in the presence of special luminophors. A very intense light emission is observed with Pholasin®, the photoprotein of the common piddock Pholas dactylus.^1,2 is a 34 kDa protein that is impermeable to cells. The amino acid sequence of Pholasin® was published recently.³ In model systems, Pholasin® is oxidised under light emission by superoxide anion radicals, peroxynitrite, hypochlorous acid as well as activated states of heme peroxidases.^4,5 Hydrogen peroxide at micromolar concentrations and heme peroxidases in the native ferric state did not oxidise Pholasin®…

Both singlet oxygen and superoxide are important in various biological and chemical processes. The chemiluminescence of Cypridina Luciferin Analogues (CLAs)¹ has been utilised as a versatile tool for detecting reactive oxygen species. Although there are many reports on the CL of CLAs, the properties and intermediates due to the reactions with O₂^- and ¹O₂ in water are not well characterised…

Inflammatory periodontal disease continues to be a major concern for dentists and patients. Indeed, over half of the subjects examined in the most recent UK Adult Dental Health survey had moderate periodontal disease on at least one tooth…

The potential toxic effects of industrial chemicals, pharmaceuticals, foods, and pesticides on cellular metabolism have been predicted using bacterial and tissue culture-based methods of testing. These in vitro techniques have proven to be useful in evaluating the potential mutagenic and carcinogenic effects of various chemicals, and for investigating the mechanisms of their toxic action. However, manipulation of cells in these techniques can alter their functional states and thus lead to divergent results when compared to in vivo data…

Travertine is a sedimentary rock rich of calcium carbonate that is present in the basin of Aniene and Tiber rivers near Rome (districts of Guidonia and Tivoli). It is largely used as building material and hundreds workers are normally employed in extractive activities. Some of these workers are subjected to professional diseases such as hand arm vibration syndrome, usually found in workers employing stone hammers, and respiratory illnesses caused by dust inhalation…

Luminol and lucigenin amplified chemiluminescence is widely used for the sensitive detection of reactive oxygen species (ROS) production in cells such as polymorphonuclear leukocytes and macrophages.¹ However, a few work in this field has been done on Kupffer cells (KC), the resident macrophages of the liver that appear to play an important role in host defence due to their ability to phagocyte bacteria and to detoxify endotoxins. Considerable evidence has shown that ROS production in liver during septic states can contribute to the deterioration of hepatic circulation² and that ROS are important mediators in liver injury.^3,4 Therefore, the study of ROS production by Kupffer cells during infectious states is important to elucidate the mechanisms of the onset of liver damage…

Oxidative stress, the imbalance between anti and pro-oxidant status, results in a loss of integrity of proteins,¹ lipids² and DNA,³ and is associated with a myriad of diseases related to accelerated and natural ageing processes. Pro-oxidants, reactive oxygen/nitrogen intermediates (RO(N)I), play a physiological role in normal cell function. They are formed both by inflammatory cells as part of the oxidative burst or by non-inflammatory cells as signalling molecules which operate through redox regulation. Antioxidants present within different cellular compartments protect cells from the toxic effects of RO(N)I. Excessive RO(N)I production imparts a demand on these antioxidants which if left unchecked leads to cell damage and eventual death. Of clinical interest, particularly in cardiovascular disease, is the plasma antioxidant status as this reflects oxidative stress originating from inflammatory cells,⁴ the endothelial lining⁵ and peripheral organ disorders…

Diabetes mellitus Type 2 (DM) is associated with excessive cardiovascular morbidity and mortality. Atherosclerotic changes start already in prediabetic states, e.g. in impaired glucose tolerance (IGT).¹ Oxidative stress has been suggested to significantly contribute to diabetic macrovascular complications. Several findings indicate that hyperglycemia provokes oxidative stress. Oral glucose challenge has been shown to increase generation of reactive oxygen species (ROS) by leukocytes and to decrease the antioxidant capacity of the blood.^2,3 The aim of the present study was to compare the magnitude of hyperglycemia-induced oxidative stress in subjects with normal glucose tolerance (NGT), IGT, and DM subjects to get further insights in the role of systemic oxidative stress in the development of diabetic complications…

The antiphospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis, recurrent pregnancy loss, increased cardiovascular risk, thrombocytopenia and the presence of circulating antiphosholipid antibodies (aPL). Many aPL are directed against neoepitopes of oxidized phospholipids suggesting that (1) oxidative events may be important in the pathophysiology of the antiphospholipid syndrome and (2) that oxidized phospholipids, present in oxidized LDL (oxLDL) as promoters of atherogenesis may give rise to common epitopes and common autoantibodies.^1,2 In this study we investigated the systemic oxidative/antioxidative balance in patients with primary antiphospholipid syndrome (PAPS), patients with systemic lupus erythematosus without secondary APS (SLE), and patients with SLE plus secondary antiphospholipid syndrome (SAPS). The parameters of oxidative stress were related to circulating levels of oxLDL and antibodies to oxidized LDL (anti-oxLDL)…

The production of reactive oxygen species (ROS) by phagocytic cells is one of the basic microbicidal mechanisms. Microbial invaders are phagocytosed and destroyed by ROS inside cells. However, ROS are also generated extracellularly (before a phagocytic vesicle is fully enclosed or during frustrated phagocytosis) or they can leak out of cells. Extracellular ROS induce a destruction of the surrounding cells and tissues. Thus, there is an effort to eliminate extracellular ROS and their effects pharmacologically, without disturbing the microbicidal functions of phagocytes. When compared to other methods used (spectrophotometry and flow cytometry) luminometric methods enable continual analysis of the respiratory burst of phagocytes. In this case, cells are incubated with a selected activator and a tested substance directly in the measuring chamber of a luminometer. Such a continual analysis reflects the total ROS production and could be employed to distinguish between its intra- and extracellular components. Two approaches are used for this purpose: I) using luminophores with different permeability through the cell membrane,¹ II) using antioxidant enzymes such as superoxide dismutase and catalase.² However, a certain amount of substances present in the cell environs can be ingested when the phagocytic vesicles are enclosed. Therefore, these approaches are not fully selective…

This report concerns the possible over-generation of active oxygen species in the heat- browned food materials ingested by humans. For the detection of minute amounts of active oxygen species in food materials, an extremely sensitive single photoelectron counting system was used. To correlate the photon emissions with the generation of active oxygens, certain scavengers and scavenging enzymes of active oxygen species were tested. The results indicated the increased formation of superoxide and singlet oxygen in the heat-browned foods. The possible role of these active oxygns in human carcinogenesis is of special interest…

Bovine blood and milk neutrophils (PMN) have a potential to produce a substantial amount of reactive oxygen species (ROS) to combat pathogens.^1-3 PMN ROS production can be measured following stimulation with soluble agents, e.g. phorbol 12–myristate 13–acetate (PMA) or with particles e.g. zymosan, bacteria, latex beads. The most widely used technique to quantify PMN ROS production is chemiluminescence (CL) assay, which was first described by Allen et al…

Peroxisome proliferators (PPs), such as fibrates and thiazolinediones may induce differentiation in tumour cells with a molecular mechanism whose action is not well known. Several PPs seem to induce changes in signal transduction pathways.¹ However, the effects of PPs are thought to be mainly mediated by three peroxisome proliferator-activated receptors (PPARs) that modulate the transcription of a large variety of genes involved in lipid metabolism and metabolic homeostasis…

Regulation of blood cells by histamine (His) has been extensively studied but its role in polymorphonuclear leukocyte (PMNL) function is not well understood as yet. A wide variety of responses have been described concerning the effects of His and His antagonists on phagocytosis, chemiluminescence (CL), reactive oxygen metabolites (ROM), etc.¹ Dithiaden (Dit), the selective histamine H₁-antagonist, decreased human blood platelet aggregation at the arachidonic acid pathway and significantly intensified the inhibitory effect of PMNL on platelet aggregation. In addition, Dit potentiated the suppressive effect of platelets on the generation of ROM by human PMNL.² We compared effects of Dit and His on: (i) CL in whole blood; (ii) CL of isolated human PMNL at the extra- and intracellular levels and (iii) PMNL aggregation…

Phospholipids (PLs) are the main constituents of biological membranes but may also represent many signaling molecules. Some PLs are produced by the action of phospholipase A₂ (PLA₂) on the sn-2 position of PLs. Main products of PL hydrolysis by PLA₂ are lysophospholipids (LPLs) and free fatty acids (FFA) that can be further metabolised. All of them – PLA₂, FFA and LPLs – are potent proinflammatory agents.¹ It has been demonstrated that LPLs influence the oxidative activity of human polymorphonuclear leukocytes (PMNs).² Lysophosphatidylcholine (LPC) influences the cell activity, such as growth and differentiation by stimulating various enzymes…

Stimulation of polymorphonuclear leukocytes (PMNs) results in the activation of several phospholipases that generate important lipid second messengers. Lipid-derived second messengers are known to be involved in the regulation of the PMN oxidative activity as well as in other PMN functions. It is generally accepted that during the PMN respiratory activity a continuous supply of the cells with diacylglycerols (DAGs) is provided and that all of them can stimulate protein kinase C (PKC) and subsequently the NADPH oxidase. Recent data of our group¹ imply that this hypothesis is questionable as DAGs with saturated fatty acids did not stimulate the respiratory burst response in contrast to unsaturated ones. Another lipid second messenger, phosphatide acid (PA) is also involved in regulating signalling pathways leading to the NADPH oxidase activation. The protein kinase activated with PAs, PAPK, is recently purified from PMNs.² Besides that, PAs are shown to play a role in intercellular communication and to be potent inflammatory agent.…

Constituents of several representative seaweeds, such as wakame Undaria pinnatifida; hijiki Hizikia fusifome; and kombu Laminaria japonica, were found to have fairly large reaction rates determined by quenching experiments of Cypridina chemiluminescence against superoxide (O₂^-) as well as against singlet oxygen (¹O₂^-). The results suggest a possibility that dietary fibers which are supposed to prevent the large-intestine cancer by their physical properties may prevent the cancer, at least in part, by their chemical, antioxidative activity…

Several cell components can be damaged by free radicals formed under biotic and abiotic stresses such as heavy metal pollution, salinity, pathogenic infections, heat shock etc. Plants have developed mechanisms to remove these free radicals (e.g., via enzymatic and non-enzymatic antioxidative systems such as glutathione, vitamins C and E, and hydroquinones) and it is known that levels of total glutathione and glutathione reductase activities increase with increasing metal uptake or other environmental stresses.¹ Only scarce data are available on antioxidant activity in extracts from Bryophyta…

Free radicals are reactive molecules that attack your cells, tearing through cellular membranes to react and create havoc with nucleic acids, proteins, and enzymes inside. These attacks by free radicals, collectively known as oxidative stress, are capable of causing cells to lose their structure, their function, and eventually destroying them. Sub-optimum dietary intakes of the phenolic antioxidant and vitamin E, are associated with the free radical-mediated destruction of cellular components and thus may promote the pathogenesis of many diseases.¹ There are many other phenolics in the diet such as catechins, flavonols, anthocyanins, coumarins and cinnamic acids which can act as antioxidants in vitro by readily donating hydrogen atoms or electrons from their hydroxyl groups to free radicals,^2,3 dietary sources include fruit, vegetables, wine and tea…

Living cells convert oxygen into the toxic intermediates via one electron reduction: superoxide radical, hydrogen peroxide, and hydroxyl radical. These reactive forms of oxygen are involved in ageing, and a number of diseases e.g. cancer. The defence against these intermediates works on three levels: firstly, the concentration of these intermediates is kept low (by e.g. superoxide dismutase, peroxidases); secondly, intermediates of reactions involved in the radical propagation step are scavenged (by antioxidants); thirdly, the damaged sites are repaired. Various parts of this defence are found intracellularly, in tissue fluid, in blood plasma, and in the external secretions…

Soon after the discovery of the chemiluminescent luminol reaction by Albrecht¹ in 1928, Specht² pointed out that the reaction could provide a very sensitive forensic test for the detection of traces of blood. Over the last 68 years, this test has been used for the detection of minute traces of blood at crime scenes by law enforcement agencies around the world…

Cigarette smoking is the main etiological factor in several respiratory and cardiovascular diseases.^1-2 Moreover, cigarette smoke exerts deleterious effects also in the oral cavity, where its products interact directly with oral structures…

Today bioluminescent analysis is one of promising techniques of biological research. Bacterial bioluminescence has high sensitivity to action of different inhibitors of biological activity: anesthetics, narcotics, factory poisons, insecticides, pesticides, poison and medicine substances.^1,2 Microbiosensor B17-677F bioassay designed at the Institute of Biophysics SB RAS on the basis of lyophilized luminous Photobacterium phosphoreum bacteria³ is used to detect integral toxicity of water samples.⁴ Analogous bioassays (Microtox, ToxAlert, LUMIStox) are used in many countries.^1,2 Luminescent genetically modified microorganisms (Pseudomonas, E. coli, etc.) as a test-object are widely used among luminescent bioassays now…

Three bioluminescent-based quorum sensing reporter strains were evaluated for detection of quorum sensing molecules in Pseudomonas fluorescens. The reporter strains studied were constructed for detection of cognate 3-oxo-C6-homoserine lactone (HSL), C4-HSL, and 3-oxo-C12-HSL using a bioluminescent Escherichia coli JM109 carrying plasmids pSB401, pSB536, and pSB1075, respectively…

Genetically engineered bacterial biosensors are obtained by introducing into a cell a reporter gene the expression of which is activated as a consequence of the interaction between the analyte and an operator/promoter (O/P) sequence, usually with the intervention of a regulatory protein.¹ Among reporter proteins, the green fluorescent protein (GFP) is available in various mutants with different spectral characteristics.² One main drawback encountered in the analytical application to real samples of such sensing devices is the non-specific effect of matrix components on the whole-cell biosensor response. In fact, being bacteria (complex biosystems) where hundreds of metabolic pathways influence one another, the modulation of the expression of the reporter protein and the related fluorescence is the result not only of the specific interaction with the analyte, but also of the overall metabolic activity of the cell. This requires the use of several controls for assessing bacterial viability and accordingly correct the analytical signal. In this context, the use of an internal reference signal control is highly desirable since it allows correcting the response, thus “cleaning” the analytical signal from non-specific interferences…

Determination of hydrogen peroxide (H₂O₂) is clinically requested for evaluation of oxidative stress ¹ and assay of substrates (e.g., glucose,² cholesterol³) of oxidase enzymes to produce H₂O₂. Chemilurninescent methods for determination of H₂O₂ are highly desirable for clinical analysis because they are simple, rapid and highly sensitive.² The chemilurninescent methods usually employ chemiluminescence molecules such as luminol,² isoluminol,⁴ lucigenin,⁵ or peroxyoxalate.³ In the course of the study using the flow cell reactor for chemilurninescent determination of H₂O₂, ² however, we have found the novel and quantitative light emission system that did not require a chemilurninescent molecule. It consisted of immobilised horseradish peroxidase (HRP), H₂O₂ and imidazole (1,3-diaza-2,4-cyclopentadiene) at alkaline pH at room temperature. We named this novel chemiluminescence as “imidazole chemiluminescence”, and we report here the “imidazole chemiluminescence” method used for quantitative assay of H₂O₂…

Field-flow fractionation (FFF) is a family of chromatographic-like techniques that are suited to separate and characterise supramolecular analytes in suspension, such as macromolecules, nanoparticles, colloids and micro-sized particles.¹ Separation is achieved, rather than by interaction with a stationary phase, by applying an external field perpendicular to the carrier flow. The origin of the field (i.e. gravitational, centrifugal, hydrodynamical, electrical, magnetical, thermal) characterises the different FFF sub-techniques…

The peroxyoxalate chemiluminescent reaction has been used for the detection of fluorescent molecules in liquid chromatography¹ and fluorophore-labeled protein and DNA bands on membranes.^2,3 In this nonenzymatic reaction, an oxalate ester reacts with hydrogen peroxide and gives rise to an intermediate capable of producing the excitation of different fluorophores.⁴ Several oxalate esters have been developed but TCPO [bis(2,4,6-triclorophenyl)oxalate] is the most used. The peroxyoxalate chemiluminescent reaction is very efficient in organic media, but the low solubility and instability of TCPO in aqueous media has limited the analytical applications of this system. In this work we have examined the properties of N,N'-di(trifluoromethanesulfonyl)-N,N'-di(4-sulfobenzylmethyl)oxamide, a new water-soluble peroxyoxalate reagent developed by Barnett et al.⁵ We describe the application of this oxamide reagent to the detection of specific protein bands on polyvinylidene difluoride (PVDF) membranes…

The use of wood as ecological material has a wide variety of applications. There is an increasing trend toward the use of wood for exterior applications. The surface of the wood exposed out of doors undergoes complex physico-chemical changes, which in most situations will result in a loss of original color, defibration and greying^1-3 described as weathering. The susceptibility to weathering of wood surfaces requires methods of enhancing the resistance of wood to photodegradation. Many factors are involved in the weathering process and the solar radiation is the most damaging component of the outdoor environment, serving to initiate a wide variety of chemical changes in wood.⁴ Photooxidation is the principal reaction in the outdoor weathering of wood. Although chemical constituents vary widely in their resistance to photooxidation, almost every wood constituent eventually deteriorates on continued exposure to solar radiation.⁵ Weathering is initiated by sunlight, though oxygen and moisture play important role as well as the contamination of atmosphere. Recently a new factor may become important, namely, changes of spectral distribution of sunlight, which reaches the surface of exposed wood. It is caused by two environmental factors: the ozone layer depletion and pollution of atmosphere.^{6, 7} Increase UV-B radiation in sunlight may accelerate the rate of wood degradation…

Conventional microbiological methods in the food industry take at least 2-3 days to provide results. Positive release is not possible for the majority of products. A technique allowing results to be obtained within one working day would enable even short shelf life products to be released to market with the assurance that they were contamination free. Previously Price et al have described a method for the detection of Listeria monocytogenes in rice,¹ by immunomagnetic separation, bacteriophage lysis and bioluminescent bacterial adenylate kinase (AK) detection, however this utilised a spike level of 10⁴ cfu mL^-1 of target pathogen. This level is unacceptably high for pathogens such as E. coli O157 and Salmonella spp possibly present in foodstuffs and is unrepresentative of natural contamination. We have previously described an improved method for the detection of pathogens in dairy products.^2,3 The approach combines immunomagnetic separation of target organisms from the food, selective resuscitation, bacteriophage-mediated cell lysis and bioluminescent detection of released target bacterial adenylate kinase. Here we describe a modified manual assay method for the detection of very low numbers of Escherichia coli O157 and Salmonella enteritidis PT4 in dairy products, achievable within 8 hours…

Listeria monocytogenes is a Gram⁺ intracytoplasmic pathogen. Like other intracellular bacterial pathogens Listeria can adhere to, invade, and release toxins into eukaryotic cells.¹ The basic virulence gene cluster of Listeria ← prfA ← plcA − hly → mpl → actA → plcB → is separated on the chromosome from another operon also involved in pathogenicity − inlA → inlB →.² Both clusters together form a regulon mostly activated by PrfA (pleiotropic activator protein). Internalins InlA and InlB mediate the entry of Listeria into the host cell by attaching to the Ecadherin receptor. The pore-forming Hly (lysteriolysin O) synergistically operates with PlcA (phosphatidylinositol-specific phospholipase C) to penetrate from the vacuole or phagosome into the cytoplasm. After actA is expressed, the protein (ActA) dimerizes and induces a ‘trail’ of actin-polymerization which apparently propels the bacterium at ∼0.3 μm/s through the cytoplasm into the adjacent host cell within a penetrating projection.² This projection pinches off into the adjacent cell to form a double membrane vacuole, which is lysed by PlcB, another phospholipase C, releasing the bacterium into the cytoplasm of the adjacent cell. Listeria infection thus spreads from cell to cell. The function of Mpl, a zinc-dependent metalloprotease is currently unknown…

Staphylococcus aureus is an opportunistic pathogen that occurs as part of the normal body flora in about a quarter of healthy adults. However, its methicillin-resistant variant (MRSA) has become endemic in hospitals worldwide,¹ causing wound sepsis and other infections, particularly post-operatively. Patients are tested for MRSA by taking swabs, usually from the nose, perineum, armpit or wound. Analysed using conventional microbiological techniques, presumptive negative results are obtained in two days and positive results in two to four days…

Staphylococcus aureus is a versatile pathogen, whose infections, due to the increasing emergence of antibiotic resistant strains, are causing considerable alarm within the medical community. The expression of many S. aureus virulence factors is under the control of the agr quorum sensing system.¹ A model was proposed for the function of agr mediated quorum sensing in staphylococcal invasion of cells, which suggested that arg related products were necessary for escape from the endosome and subsequent release and growth in the cytoplasm.² It was proposed that in an extracellular environment, agr was down-regulated due to dilution of the agr peptide in the surrounding fluids. Upon uptake, the agr peptide concentrations increased rapidly when the bacteria were confined within the endosome, and that lysis occurred due to the induction of agr related exoproteins.² Our previous studies³ with S. aureus RN6390 support the hypothesis that agr expression precedes host endosomal lysis…

The adenosine triphosphate (ATP) reaction with firefly luciferase to give bioluminescence is widely used in rapid methods for assessment of cleanliness in food processing plants. Most methods neither differentiate between bacterial and non-bacterial ATP, nor correlate with standard culture methods. Residual chemicals may cause a significant variation in the bioluminescence signal¹ thereby under estimating or overstating the actual amount of ATP signal and consequently affecting the decision tree…

Development of value-added convenience egg products is needed to meet consumer trends of increased spending on meals and snacks eaten away from home and increased consumption of eggs, possibly due to recent publicity of high protein diets and healthy attributes of eggs. Individually packaged, peeled, whole hard-boiled eggs (ready-to-eat) are a value-added version of whole shell eggs and could provide consumers the convenience of saving time. An important parameter in the development of value added food products is extended storage (shelf life) periods free of microbial contamination. To increase the shelf life of food products, a wide variety of preservatives are used to inhibit growth of both pathogenic and spoilage organisms. Preservative evaluation involves addition of the potential preservative to a food product followed by a challenge with a spoilage organism. Current assays used to determine effective concentrations of preservatives typically implement plate count methodology that is labor intensive, costly, time consuming, and requires days for detection. Evaluation of preservatives for hard-boiled eggs involves the removal of the test organism from the egg surface followed by enumeration. Bacterial bioluminescence is an alternative method for real-time monitoring of cell viability by cell light production. This study evaluates the use of a bioluminescent strain of Pseudomonas fluorescens in conjunction with a prototype bioluminescence detector for initial screening of preservatives for increasing the shelf life of hard-boiled eggs both in an aqueous and surface format…

Several studies have examined the vertical distribution of bioluminescence in the water column.^1-3 For example, Widder et al. examined the relationship between the number of bioluminescent organisms as a vertical profile using a bathyphotometer and a variety of physical factors, including temperature, to 250 m depth in the Gulf of Maine.¹ They showed that stimulable bioluminescence, temperature and salinity decrease exponentially from the surface layers…

Ecotoxicology is a unique field of biological sciences devoted to issues surrounding the release of countless man-made chemicals into the biosphere and potentially harmful effects they can exert, alone or in combination, toward biota of receiving environments.¹ Early studies of wastewater impact on the environment relied primarily on chemical analyses for identifying specific contaminants. The chemical data alone do not suffice for evaluation of global toxic effects because it is impossible to analyze all the relevant compounds including their degradation and transformation products. Biological systems may retain synergistic or antagonistic effects which are not easily assessed without biological tests…

The larval firefly {Photuris versicolor) possesses two tiny lanterns on the sternite of the 8^th abdominal segment. These lanterns are activated to produce a warning luminescence whenever the larva is attacked by a predator.¹ Projecting from the lateral, posterior edges of each abdominal segment are single, 500 to 600 micron long, setae that serve as mechanoreceptors and are likely to be bent during attack by a predator. Because the setae project backwards and would be pressed toward the larva's body during its movement through the underbrush, only anterior movement would likely signal a predatory attack. We have investigated movement of these setae to determine whether they play a role in activating the warning luminescence of the larvae…

Most deep-sea fish have retinae containing a single visual pigment absorbing maximally around 460-490 nm approximately matching the spectral composition of any residual downwelling sunlight and the bioluminescence produced by most mesopelagic animals.¹ The most striking exception are 3 genera of stomiid dragon fish, which have suborbital photophores producing far-red illumination.² While most inhabitants of the deep-sea will be unable to see this light, these stomiids have developed two different strategies to perceive their own illumination. Aristostomias³ and Pachystomias¹ have evolved at least three visual pigments that are significantly red-shifted in comparison to those of all other deep-sea fish. Malacosteus, on the other hand, has less red-shifted pigments linked to a longwave absorbing, chlorophyll-derived, photosensitizing pigment.⁴ These dragon fish thus produce wavelengths that it has hitherto been thought only they can see, affording them a ‘secret waveband’ they can use for communication or illuminating prey, immune from detection. Here we describe the retinal pigments of Bolinichthys longipes, a species not thought to produce far red bioluminescence, but that may nonetheless posses a system for detecting these ‘secret’ wavelengths…

Recently, rapid and direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these methods continue to suffer from limited sensitivity and a lack specificity. On the other hand, the detection method of strain using DNA diagnosis has higher specificity and sensitivity than the conventional immunoassay method and enables the detection of microorganisms through a simple operation. In this experiment, we attempted to develop a highly-sensitive and simple monitoring method for PCR products using bioluminescence. The allele specific PCRs for E. coli O 157 are conducted with primers specific to vero toxin genes, VT1 and VT2. VT is an important cause of haemorrhagic (HC) and haemolytic uraemic syndrome (HUS) worldwide. The method was carried out by the determination of pyrophosphoric acid (PPi) released during allele specific PCR. The released PPi is converted to ATP by ATP-sulfurylase and the concentration of ATP is determined by firefly luciferase reaction. As a result, VT1, VT2, and DNA with VT1/VT2 were clearly identified by this proposed method. This method does not require expensive equipment, can be used to rapidly monitor the PCR product, and also can be used as a high throughput method for the PCR product…

Luminescent bacteria and their enzymes are currently applied in bioassays for environmental monitoring.¹ Luminescent bacteria based assay is simple enough to be used by schoolchildren in their ecological activities. The results of the bioluminescent bioassay can be treated with modern statistical and geographical methods. The purpose of the work is to demonstrate the possibility of using of Bioluminescent Bioassay, Geographical Informational Systems and modern statistical methods of computer processing for practical ecological education of schoolchildren of 14-16 years old…

Immunoassays relying on enzyme label for antigens or antibodies have played a major role in biomedical science for more than three decades. The main limitation of traditional ELISA is that the methods developed are mostly single analyte methods, while in most cases it would be desirable to measure more than one analyte in a sample or discriminate the analytes within a group of structurally closed compounds. The aim of present study was to evaluate a chemiluminescent membrane multiassay in a format of a dipstick for the discrimination of several analytes in mixtures. Our approach for the multianalyte assay consisted in the simultaneous immobilization of antibodies (Abs) with different specificity towards analytes in separate spots of a membrane strip. The competitive immunoassay with a hapten labeled with horseradish peroxidase (HRP) as a tracer was combined with the enhanced chemiluminescence (ECL) reaction as it was established to be one of the most sensitive and expressive methods for medicine and analytical biochemistry.^1,2 Recently we showed its advantages as an effective detecting method for the purposes of ecological monitoring.³ The experimental data were processed with artificial neural network (ANN), which represents a new approach to the interpretation of multivariate data and for the pattern recognition…

Central to the future development of the whole cell biochip is a greater understanding of the natural phenomena that is bioluminescence, the conditions that facilitate it, and the physiological and biochemical parameters of biosensors…

Proteins separated by SDS-PAGE are often transferred to membranes for immunoprocesssing. This technique, known as Western blotting, is very useful for research and diagnostic procedures. However, Western blotting has its limitations, such as uneven transfer of proteins, and requires extra time for transfer and blocking steps.^1-3 To eliminate the problems associated with Western blotting, we have developed a procedure for detecting antigens directly within the gel…

CARLA SYSTEM (Captur Assay RADIM Liquid Allergen) is a Capture Assay-ELISA system for quantitative detection of specific IgE in human serum.¹ The samples are incubated with a biotinylated-allergen solution in a microplate coated with anti-human IgE. During the first incubation the sample IgE antibodies are bound to the solid phase and the biotinylated allergen are bound to the sample specific IgE. After removing the unbound material by an aspiration/washing cycle, the streptavidine HRP tracer is added to the wells to bind the biotinylated allergen. In order to quantify specific IgE, a standard curve with known amounts of total IgE antibodies is incubated in the same coated microplate with a biotinylated anti IgE antibody. The colorimetric reaction is evidenced by incubating the solid phase with the chromogen solution (tetramethylbenzidine, TMB) in a substrate buffer.² Horseradish Peroxidase (HRP) in a commonly used reporter enzyme in enzyme linked immunoassay and TMB and Lumigen PS-1 has been reported for visualization of peroxidase activity.³ Chemiluminescent detection offers potential advantages in terms of sensitivity and specificity of the assay. In particular the possibility to reduce the sample volume is an important aspect for the allergological pediatric investigations, as a reduced sample volume could allow to increase the number of allergen tests with the same blood sample. Our aim was to investigate the contribute of antiIgE and tracer concentration in order to increase the sensitivity on the Brio automatic System. Moreover a correlation with results obtained with an already assessed method commercially available has been studied…

“RADIM TSH IEMA WELL” is a kit based on an immunoenzymometric assay. Two different anti-TSH monoclonal antibodies are used, one adsorbed on the wells and the other conjugated to biotin. During incubation, the TSH present in the standard or samples is bound to both monoclonal antibodies at once by forming a “sandwich”. At the and of incubation the unbound material is removed by an aspiration /washing cycle. A solution of streptavidin conjugated with peroxidase (HRP) is added to all wells. This reagent will react with the biotinilated antibodies which are bound to the solid phase. After a further aspiration/washing cycle the residual enzyme activity found in the wells will thus be directly proportional to hTSH concentration in the standards or samples; this activity is evidenced by incubating the solid phase with the chromogen solution (tetramethylbenzidine, TMB) in a substrate buffer. Horseradish Peroxidase (HRP) in a commonly used reporter enzyme in enzyme linked immunoassay and TMB has been reported for visualization of peroxidase activity. Whereas colorimetric assays may be adequate for quantitation of analytes present in relatively high concentration, chemiluminescent detection offers potential advantages in terms of sensitivity, assay speed and can allow reduced usage of expensive antibodies. Our aim was to improve the hTSH timing performance and sensitivity in a completely automatic system evaluating the use of three different formulations of the Lumigen PS-1, a reagent for the ultrasensitive chemiluminescent detection of HRP…

Chemiluminescence has already become an essential tool in medical research and routine analysis. Acridinium (Ac) carboxamides have been utilized for highly sensitive immunoassays because of their stability and high chemiluminescence yield. The principle and reaction mechanism of using acridinium derivatives for chemiluminescent signal was reviewed recently.¹ Abbott PRISM^® is a high throughput, fully automated serological analyzer to screen plasma or sera for Hepatitis B surface antigen, antibodies to Hepatitis B core, HCV, HIV, and HTLV.² The instrument dispenses acridinium labeled antibodies or antigens to tag analytes captured on microparticles. Microparticles are washed with buffer to reduce nonspecific binding, alkaline peroxide is then dispensed to trigger chemiluminescence, and a photon multiplier detects light emission. Data are processed and aligned with the unique barcode on each sample tube. Reactive samples are identified by a computer algorithm that utilizes signals generated from calibrators and controls to calculate a cutoff value and to establish the run validity…

In recent years, although many chemiluminescent clinical immunoassay kits have been widely applied for detection of hormones, the kit for insulin is still not included in most commercial product lists. But it is very important to survey the function of B cells in pancreatic islets in order to diagnose insulinoma and hyperinsulinar obesity, and especially to distinguish the different types of diabetes, which are very common diseases.^1,2 One of the reasons for this lack is the difficulty in obtaining and selecting suitable pairs of antibodies because insulin itself is the product of hydrolysis of proinsulin and it is difficult to find an anti-insulin antibody that does not have cross-reaction to proinsulin. The insulin molecule is very small, and it is difficult to find two antibodies without binding inhibition for each other, while the competitive assay with single antibody is not sufficiently sensitive for clinical applications.¹ Another reason is the instability of insulin and its short lifetime in sera, and this means it is very difficult to keep the calibrators and samples stable.¹ With a carefully selected pair of antibodies, we have now established an enhanced chemiluminescent immunoassay to detect insulin in human sera…

For live bacterial cell assays, it is important to choose antibodies that provide high assay sensitivities. To accomplish this, the affinity constants of antibodies raised against target bacteria need to be determined and compared. Bacterial surfaces provide a vast array of antigenic determinants such as polysaccharides, proteins, and glycolipids. The affinity constants of antibodies raised against these small, extracted, molecular components can be easily derived, however it is uncommon to determine that against the bacterial cell as a whole. As a further complication, when antibodies are raised against bacteria, the cells are heat-treated and thus may have a different affinity constant when reacted to live cells. If the assay is used to detect live bacteria, it is pertinent to choose antibodies with high affinity constants that have been tested against bacteria with intact physiology…

Bacterial bioluminescence can be used as an enzymatic sensor to measure reduced nicotinamide adenine dinucleotide (NADH) as well as analytes that can be coupled to NADH. Lactate can be measured using lactate dehydrogenase (LDH) coupled to the bacterial bioluminescence system of flavin mononucleotide (FMN): NADH oxidoreductase (OR) and bacterial luciferase (BL):

Lactate sensors are useful for clinical analyses, food and beverage analyses, and in sports medicine. Lactate concentrations in blood (normal without exercise), tears, and sweat are approximately 0.5 mmol/L, 2.5 mmol/L, and 12 mmol/L, respectively. Some advantages of a bioluminescent lactate assay are sensitivity and small sample volumes. A previously reported aqueous homogeneous lactate assay¹ was enhanced with the additional of glutamic-alanine transaminase (GAT), which consumes pyruvate, thereby increasing NADH production by LDH…

We are developing devices using bioluminescence-based reactions to measure metabolites in bio fluids. Our NADH sensing platform involves NADH, FMN, RCHO, NADH-flavin mononucleotide (FMN) oxidoreductase (OR, Vibrio fischeri), and bacterial luciferase (BL, Vibrio harveyi). Here we present a practical NADH platform model (NPM) and its simulation:

The reaction coupling analyte to NADH is (where A and B are the reduced and oxidized form of the analyte respectively): Min used a common first order reaction to model FMNH₂ production.¹ Baldwin et al. showed a detailed model of the BL-catalyzed light emitting reaction.² Tu et al. presented a scheme for the OR catalysed reaction producing the needed FMNH₂ to “feed” the light emitting reaction.³ Based on Baldwin et al. and Min's model and rate constants, a simplified NPM was constructed using Gepasi…

Enzymatic triggerable 1,2-dioxetanes^1,2 are superior in immunoassays and other related applications compared to peroxidase substrates such as luminol³ and others. Stabilized 1,2-dioxetane substrates provide high signal, low background, wide dynamic range, rapid results and excellent reproducibility. These 1,2-dioxetanes provide substrates which are highly sensitive but can detect an enzyme concentration up to 10^-21M (6×l0² molecules of alkaline phosphatase) in solution as well as on a membrane.² Now we wish to report on the detection of a single molecule of alkaline phosphatase, in less than ten minutes, by using a combination of a stabilized 1,2-dioxetanes, an enhancer and an enzyme diluent…

Recently, we established a highly sensitive bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) using firefly luciferase and luciferin as a detection system. We succeeded in detecting 8.6 × 10^-21 mol/assay for AK and 1.4 × 10^-20 mol/assay for PPDK, respectively. These enzymes were applicable in bioluminescent enzyme immunoassay (BL-EIA) for various biological samples.^1-6 AK and PPDK are enzymes that generate ATP, and react in similar conditions. We have now developed simultaneous bioluminescent assays using AK and PPDK. The principle of the proposed assay is shown in Fig. 1. In first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the next step, the bioluminescent emission from AK is quenched by addition of glucose and ADP-dependent hexokinase (ADP-HK)⁷ that transforms ADP as a substrate for AK into AMP. At the same time, PPDK catalyzes the interconversion of AMP, pyrophosphate (PPi), and phosphoenolpyruvate (PEP) to ATP, phosphate and pyruvate. The ATP from PPDK is also determined by the firefly luciferase-luciferin reaction. Furthermore, we selected insulin and C-peptide as analytes, and applied the proposed method to BL-EIA for simultaneous measurement of these two analytes…

In any food-processing environment, food residues can encourage and facilitate the survival and growth of microorganism by providing a nutritious medium for growth and protection from disinfectants. In order to prevent contamination of the product, it is important to keep food contact surfaces clean. Several hygiene monitoring systems for determining the cleanliness of surfaces were developed. Above all, the adenosine triphosphate (ATP) method is recommended due to ATP being widely found in food residues and microorganisms. However, in some food residues the ATP method shows insufficient sensitivity, especially processed foods. In order to improve the sensitivity, we have developed a novel bioluminescent enzymatic cycling system using pyruvate orthophosphate dikinase (PPDK) (ATP-PPDK method) (Fig.l).¹ PPDK catalyses the formation of ATP from AMP, pyrophosphate (PPi), and phosphoenolpyruvate (PEP).² This system enables the detection of the combined total ATP and AMP with a high degree of consistency and higher bioluminescence when compared to existing ATP methods. We have applied this system to hygiene monitoring and have developed a novel hygiene monitoring device ‘LuciPac W’ and photodiode luminometer ‘Lumitester PD-10’…

Epithelial respiratory cells play a fundamental role in protecting the organism against aggressions, in particular oxidative stress. Little is known on their cytosolic regulation pathways, which can be assessed by optical techniques. The mitochondrial potential is of particular interest, since mitochondria deal with the main crossroads of antioxidant defences. Intracellular calcium is implied in many hormonal responses, and its relation to cellular energetics is largely unknown…

The enhanced chemiluminescence signal reagent based on acridan ester GZ-11 is a very versatile tool not only to quantify horseradish peroxidase (HRP), the most widely used enzyme label,¹ but also to determine total antioxidant capacity and activity (see this volume). Besides the acridan ester GZ-11, the signal reagent contains an enhancer such as p-phenylphenol (PPOH) and hydrogen peroxide. At a certain PPOH concentration range the chemiluminescence response increases linearly with increasing PPOH-concentrations. Therefore, this system also can be used to quantify PPOH. This provides a way to determine all kinds of enzymes which are capable to transform a derivative of PPOH (PPOX) into PPOH.² The derivative PPOX itself is not functional as an enhancer and may be called a proenhancer. In the corresponding luminol based enhanced chemiluminescence system several of such pro-enhancers have been developed including naphtol acetate that has been used to assay cholinesterase activity.³ Cholinesterase is inhibited by a number of pesticides and also by certain chemical warfare agents e.g. sarin and vx. A general assay for these chlolinesterase inhibitors can be used to screen for the presence of these agents e.g. after a chemical warfare attack. We decided to develop such a system based on GZ-11 enhanced chemiluminescence…

Milk components are known to have a variety of biological functions in addition to basic nutritional properties including opioid, antimutagenic, antimicrobial and immunomodulating activity. It has been reported that intake of milk fermented by lactic acid bacteria (LAB) produces a specific humoral immune response. In our previous research, it was shown that cell-free fractions of fermented milks enhanced cytokine production of macrophages in vitro.¹ However, such assays are expensive and difficult to perform. It is also difficult to extrapolate the results of these assays to events that occur in whole animals. Thus, to establish the biological relevance of this phenomenon, an in vivo model has been proposed to assess the effect of fermented milk and its components on the course of bacterial infections in mice…

The bioluminescent assay of TBC is one of the most rapid, simple and economically reasonable methods among «rapid microbiology» methods developed for assessment of hygiene quality of food samples and drinking water. Most samples analyzed contain excess of non-bacterial ATP (sum of somatic and free ATP) and/or a low number of bacteria, and therefore a special pretreatment of the sample is required and this is laborious and time consuming. To overcome this obstacle we used special luminometric polystyrene microcuvettes (h 13 mm, Ø 10 mm), Filtravette™, with the bottom made of bacterial membrane filter (pore size 0.45 µm). The design of Filtravette allows one to concentrate bacteria cells, extract bacterial ATP and measure bioluminescent signal in the same cuvette. This simplifies the analysis, enhances its accuracy and decreases detection limit of bacteria cells. The combined application of Filtravette™ and highly sensitive ATP-reagent developed in our laboratory permitted us to detect as low as ∼ 200 bacteria per Filtravette™…

Cyclodextrins are cyclic oligosaccharides formed by 6, 7 or 8 α-D(+)-glucopyranose units and described as α−, β-, and γ −cyclodextrin (α-CD, β-CD, γ-CD). Because of their structure, cyclodextrins are able to form internal and low polarized cavities. In this way many organic compounds can be included in cyclodextrins. Such kinds of ligand −receptor complexes can be used for different applications, e.g. for a transdermal therapeutic system (TTS). The aim of the present study was the investigation of the cytotoxicity of the different cyclodextrins using HaCaT keratinocytes, the influence on the cell proliferation and the determination of non-toxic cyclodextrin concentrations…

The epiphysial hormone melatonin is characterized by many pharmacological effects in biological systems. In vitro-investigations to characterize the growth of spontaneously immortal human HaCaT-keratinocytes (Deutsches Krebsforschungszentrum, Heidelberg, Germany) influenced by melatonin were performed by Nickel et al.¹ Evaluation of the cytotoxic effects after application of different substances was assessed by the DNA-synthesis rate which was determined by BrdU – insertion. The decrease of the DNA synthesis rate could be shown after treatment of keratinocytes with melatonin in high concentrations (1 nM). However, low concentrations of melatonin ( 10 μM -1 nM ) caused an increase of BrdU – insertion after 24h. The research laboratory of the Department of Dermatology (Friedrich-Schiller-University of Jena) found the inhibitory as well as the stimulating effects of melatonin depend on its concentration for growth of hair. A stimulation of hair growth could be detected in concentrations between 10 and 40 μmol in comparison to William's E medium. The maximum of the stimulating effect was found at 30 μmol.² The aim of the present study was to determine the optimal melatonin doses for increasing HaCaT cell proliferation…

ATP bioluminescence has been used as a measure of biomass since the sixties.¹ The current success of ATP bioluminescence as a hygiene-monitoring tool relies on the signals providing an indication of cleanliness levels as information for trend analyses, without there being an expectation of a direct correlation with colony plate counts, the traditional method of determining levels of microbial contamination…

The paper describes the use of tumor cell lines transfected with Photinus pyralis luciferase for both in vitro and in vivo ATP bioluminescence assays as a tool for drug development studies. Pre-clinical research and development of new chemotherapeutic drugs requires both in vitro and in vivo assay systems to test drug activity against different types of tumor cells. The ATP bioluminescence assay originally described for chemosensitivity testing tumor cell lines and clinical specimens has proven to be a useful technology for new drug development.^1-2 Dose-response and drug combination studies can be performed with a wide range of clinical specimens including needle biopsies, and good clinical correlation with patient benefit has been demonstrated for testing clinical specimens.^3-5 More recently, the ATP assay system used for in vitro chemosensitivity studies has been modified for testing luciferase (luc) gene transfected tumor cell lines…

Pre-clinical research and development of new chemotherapeutic drugs requires both in vitro and in vivo assay systems to test drug activity against different types of tumor cells. This includes screening and testing against tumor cell lines in vitro, testing against clinical specimens in vitro, and testing in vivo using xenograft model systems of tumors grown in nude mice…

Dioxin-like chemicals are a diverse group of widespread environmental contaminants, which include polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs). These polyhalogenated aromatic hydrocarbons (PHAHs) are very stable, thus persist in the environment and can be found in soil, sediment, water, air, food. They are highly toxic at low doses, accumulate in fatty tissues and milk of living organisms due to their lipophilicity and biomagnify in the food chain. Such compounds are also semi-volatile, which enables them to move long distances in the atmosphere before deposition occurs. They are known to produce a variety of species- and tissue-specific effects on humans and wildlife, such as tumor promotion, birth defects, hepatotoxicity, immunotoxicity, dermal toxicity, endocrine disruption…

Recent research in bioprocesses optimisation has focused on the maximisation of product formation. A fermentation process can lead to the production of high-value products and can be quite sensitive and expensive. Therefore, it is important to get as many measurements as possible about the state of the fermentation. Unfortunately, a lot of parameters cannot be measured on-line, because of the lack of sensors. Our objective is to use the measurement of an easy quantifiable macroscopic parameter, bacterial bioluminescence, to estimate the value of other parameters of the culture, like the production of a recombinant product. We constructed a recombinant Escherichia coli (E. coli) producing bioluminescence and a protein of interest under the regulation of the same promoter. Cultures were run in an automated fermentation system, and the correlation obtained between the bioluminescence and protein production was studied…

Recombinant firefly luciferases offer the potential to create novel bioluminescent reporter enzymes with enhanced properties for in vivo imaging. Increased thermostability and changes in the spectrum of emitted light are two parameters that could improve detection sensitivity in situations where signal attenuation is problematic e.g. in vivo imaging of mammalian tissues. Recently, we have identified mutations in the North American firefly Photinus pyralis that affect both thermostability and wavelength of emitted light. A library containing all possible amino acid permutations at these positions has previously been constructed, expressed in Escherichia. coli, and screened for thermostability and changes to in vivo spectra.¹ Here we describe the overexpression and purification of two mutant luciferases from this library. The in vitro properties of the purified enzymes are characterised and assessed for their suitability in both in vivo and in vitro assays…

Medakafish (Oryzias latipes) belongs to a group of small oviparous teleost fishes that inhabit fresh water in Asian countries including Japan.^1,2 Medakafish has been widely used to assess the biological effects of environmental chemicals,^3-5 due to its high sensitivity and ease of breeding. Also the transparency of the medakafish body enables us to investigate the expression of genes tagged by appropriate markers through its entire development, from very early in embryogenesis to a mature adult…

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