bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo bioluminescence: A cellular reporter for research and industry
Authors: Jassim S. A. A. , Ellison A. , Denyer S. P. , Stewart G. S. A. B.
Published: May 2005

The detection of specific bacterial pathogens, indicator microorganisms and antimicrobial substances, and the recovery of microorganisms from sub‐lethal injury, are all aspects of importance to industry which are currently being targeted using in vivo bioluminescence. In all instances, a key requirement for the application of bioluminescence is the establishment of a strict correlation between in vivo bioluminescence and cell viability, as determined by colony counting on agar plates. Comparative studies for biocides (phenol, chlorhexidine diacetate, phenol thioether), for a virucide (hypochlorite) and for cellular recovery of S. typhimurium from sub‐lethal injury, all indicate that such a correlation is valid. Furthermore, real‐time measurements of in vivo bioluminescence reveal a major population of bacterial cells that retain functional intracellular biochemistry, but are defective in their ability to replicate post of freeze injury.
CCD imaging of luciferase gene expression in single mammalian cells
Authors: Hooper Claire E. , Ansorge R. E. , Browne Helena M. , Tomkins Patricia
Published: May 2005

Quantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooper et al., 1990). A brief review of low‐light‐level imaging is presented, with particular reference to charge‐coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photoncounting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular highsensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguez et al., 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non‐infected cells. The rapid detection of luciferase‐expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles.

This direct imaging technique is simple and versatile. It offers a rapid, non‐invasive method for the sensitive detection of luciferase activity in single, luciferase‐expressing cells. One can envisage the use of luciferase as a sensitive and convenient co‐selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and cellular biology.
Whole blood luminol‐enhanced chemiluminescence: Statistical analysis of the responses of different subjects
Authors: Ristola M. , Repo H.
Published: May 2005

Whole blood luminol‐enhanced chemiluminescence (CL) was studied using N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or opsonized zymosan particles as stimuli. The peak and the integral responses of CL were recorded. In the FMLP‐induced CL the initial activation (1‐minute values) was also studied, because it coincides with the extracellular production of oxy radicals. Correction factors based on neutrophil count and on haemoglobin concentration were found to decrease dispersion and shape the distributions of the CL responses close to normal in a study of 50 healthy adults. One‐minute values were significantly lower in women than in men but there were no significant differences for peak or integral values between sexes. Depressed reaction is in accordance with the previous findings that phagocytic oxy radical production is depressed in female plasma. Thus, our results suggest that 1‐minute value is a variable more sensitive than peak or integral value of the CL response.
Enhanced chemiluminescence in the measurement of proteins and haptens: Evaluation of choriogonadotropin (hCG) and free thyroxin
Authors: Bonini P. A. , Banfi G. , Murone M.
Published: May 2005

We evaluated the Amerlite system (Amersham, Bucks, UK) for hCG and FT4. The within‐run imprecision (CV%) for hCG was 4.05 at 19.6 U/I (n = 10), 6.28 at 43.45 U/I (n = 10) and 4.62 at 298.57 U/I (n = 10). The between‐run imprecision (five replicates for ten days) was 4.8%, 15% and 11%, respectively. The system was linear up to 200 U/I. A good correlation between Amerlite hCG and an IRMA assay (Becton Dickinson, r = 0.91), Delfia (Pharmacia, r = 0.91) and an automated ELISA assay on ES 600 (Boehringer, r = 0.92) was observed on 70 samples.

Within‐run imprecision for FT4 was 3.8% at 0.7 ng/dl (n = 10), 3.3% at 1 ng/dl (n = 10) and 4.32% at 5.15 ng/dl (n = 10), and between‐run was 5.95%, 4.4% and 8.2%, respectively. The comparison with a commercial direct RIA (Becton Dickinson) showed good correlation (r = 0.90, n = 100 samples). The diagnostic value of the association of thyrotropin and FT4, in comparison with the traditional thyroid tests (T3, T4, thyrotropin, FT4, FT3) has been assessed in various thyroid diseases.
Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell
Authors: Watanabe Haruo , Suzuki Sohkichi , Kobayashi Masaki , Usa Masashi , Inaba Humio
Published: May 2005

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.
Simultaneous measurement of endothelial cell damage, elastase release and chemiluminescence response during interaction between polymorphonuclear leukocytes and endothelial cells
Authors: Jonas Eleonore , Dwenger Alexander , Lueken Britta , Boehme Ulrich
Published: May 2005

Using cultured human umbilical cord vein endothelial cells and human blood neutrophils, the interaction between neutrophils and endothelial cells, in vitro, was studied. The aim of the study was to examine whether a respiratory burst stimulation by neutrophils would be observed by neutrophil/endothelial cell interaction and whether the respiratory burst stimulation of neutrophils by endothelial cells could be enhanced by lipopolysaccharide stimulation of neutrophils. The second aim was whether such an effect, or secretion of elastase, could cause an endothelial cell damage in vitro.

Chemiluminescence as an indicator of oxygen‐derived metabolites produced by neutrophils, elastase release by neutrophils, and endothelial cell damage, based on111 In‐oxine release from labelled endothelial cells, were measured simultaneously. The present investigation demonstrates that neutrophils can be directly stimulated by endothelial cells. A further amplification of this process following lipopolysaccharide priming up to 10 ng/ml blood could be demonstrated. A slight endothelial cell damage occurs following neutrophil stimulation, although elastase secretion does not increase during interaction between neutrophils and endothelial cells. These results raise the possibility that oxygen‐derived metabolites rather than elastase contribute to an endothelial cell damage which might occur in conditions such as endotoxin‐induced adult respiratory distress syndrome.
Effect of different inhibitors on the intracellularly and extracellularly generated chemiluminescence induced by formylmethionyl‐leucyl‐phenylalanine in polymorphonuclear leukocytes. Cellular response in the presence of mannitol, benzoate, taurine, indomethacin and NDGA
Authors: Dahlgren Claes
Published: May 2005

When polymorphonuclear leukocytes (PMNL) interact with the soluble stimulus formylmethionyl‐leucyl‐phenylalanine (FMLP), the cells increase their production of oxidative metabolites. This increased production can be measured as lumino‐amplified light emission or chemiluminescence (CL). In the present report, experimental systems which allow a quantitation of extracellularly and intracellularly generated metabolites have been used, and the effect of mannitol, benzoate, taurine, indomethacin and nordihydroguaiaretic acid has been investigated. The presence of the hypochlorous acid scavenger taurine had no effect on the intracellular response, whereas the extracellular response was reduced with around 50%. The hydroxyl radical scavenger mannitol had only minor effects on the response, whereas benzoate, another hydroxyl radical scavenger, reduced the extracellular response with around 50% and the intracellular response with more than 90%. Indomethacin, an inhibitor of arachidonic acid metabolism, did not influence the response, whereas NDGA, also an inhibitor of the arachidonic acid metabolism, totally abolished both the extracellular and the intracellular response. The use of scavengers/inhibitors as a means of determining the mechanisms of light emission, and the origin of chemiluminescence produced by neutrophils stimulated by FMLP is discussed.
The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations In Vitro
Authors: Kantorski J. , Tchórzewski H.
Published: May 2005

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor‐dependent and receptor‐independent stimuli were studied, e.g. opsonized zymosan, formyl‐methionyl‐leucyl‐phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose‐dependent manner after treatment of cells with the various stimuli. Non‐specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator‐induced CL. We suggest that cell‐bound chymotrypsin‐like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor‐dependent and receptor‐independent stimulation.
A survey of more than 90 commercially available luminometers and imaging devices for low‐light measurements of chemiluminescence and bioluminescence, including instruments for manual, automatic and specialized operation, for HPLC, LC, GLC and microtitre plates. Part 1: Descriptions
Authors: Stanley Philip E.
Published: May 2005

This survey was compiled in January and February 1992 from information available in public domain literature requested by and supplied to the author by numerous companies in the previous two months. More than 90 luminometers (manual, automatic, microtitre plate, HPLC, LC, GLC, imaging and specials) from more than 60 companies are included. Each company was invited to supply company brochures, technical details, user manual and information about software and any other information concerning their product(s). The response varied from a single information sheet to promotional material and up to full product information and specification with technical details, user manuals and scientific publications. Where an instrument is dedicated to a single task the company may have only provided details relevant to accomplishing that task. Part 2 of this survey will contain photographs of some of the luminometers. It is intended that updates to this review will be published at least annually in this journal and suppliers are invited to provide full technical details of new luminometric equipment to the author.
Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis Subsp. diacetylactis
Authors: Simpson William J.
Published: May 2005

High levels of constitutive aldehyde‐dependent light emission were obtained from non‐growing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with luxA/B when they were suspended in buffered solutions. Inductions of light emission was time‐dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission.
Chemiluminescence of mononuclear cells is enhanced during antigen recognition
Authors: Tengler Rita S. , Furukawa Kazuhito , de Weck Alain L. , Maly Friedrich E.
Published: May 2005

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen‐activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin‐dependent chemiluminescence. Mononulcear cells which underwent antigen‐induced proliferation showed a delayed rise in lucigenin‐dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20‐fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di‐phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon‐gamma completely abrogated antigen‐induced chemiluminescence. Recombinant interferon‐gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen‐induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon‐gamma generated by activated lymphocytes.
Flow injection determination of glucose, bile acid and ATP using immobilized enzyme reactor and chemiluminescent assay of NAD(P)H
Authors: Maeda Masako , Tsuji Akio , Ohshima Nobuo , Hukuoka Masayoshi
Published: May 2005

We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1‐methoxy‐5‐methylphenazinium methyl sulphate (1‐MPMS)/isoluminol(IL)/microperoxidase (m‐POD) system and immobilized enzyme reactors such as 3α‐hydroxysteroid dehydrogenase, glucosedehydrogenase, hexokinase and glucose‐6‐phosphate dehydrogenase. The standard curves were obtained in the range of 5 ∼ 100 pmol for bile acid, 0.5 ∼ 5.0 nmol for glucose and 10−7 ∼ 10−5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.
Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients
Authors: Jonas Eleonore , Dwenger Alexander , Jonas Michael , Nerlich Michael , Tscherne Harald
Published: May 2005

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen‐derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group.

Production of oxygen‐derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post‐trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post‐trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen‐derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.
Bioluminescence and chemiluminescence literature—Luciferase reporter genes—Lux and Luc
Authors: Hill P. J. , Stewart G. S. A. B. , Stanley P. E.
Published: May 2005

The following references encompass a review of recent literature where the in vivo expression of eukaryotic and/or prokaryotic luciferases provide for sensitive reporters of cellular activity. The list is subdivided into prokaryotic and eukaryotic applications. We have included the uses of luciferases in elucidating the control of gene expression or for monitoring cell viability. We have not included papers cited by Stanley and Stewart (J Biolumin Chemilumin 1990; 5:141–52) nor have we included papers on the structure and regulation of luciferases as this now substantial literature will be the subject of a future review. References cited in both this review and previous ones are referred to by the number assigned to them in the earlier review.
Growth and luminescence of luminous bacteria promoted by agents of microbial origin
Authors: Rodicheva E. K. , Trubachev I. N. , Medvedeva S. E. , Egorova O. I. , Shitova L. Yu.
Published: May 2005

The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis. These agents are nucleic components (nucleotides, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen‐oxidizing, ironoxidizing and carboxydobacteria. The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two‐ to three‐fold compared to a control.
Luminescence of Ca2+‐activated photoprotein obelin initiated by NaOCl and MnCl2
Authors: Vysotski Eugene S. , Trofimov Konstantin P. , Bondar' Vladimir S. , Gitelson Josef I.
Published: May 2005

The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn2+ enhances the light emission >300‐fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl‐activated obelin luminescence in the presence or absence of Mn2+. This suggests that the addition of NaOCI to the mixture causes singlet oxygen formation (stimulated by Mn2+ ions), and singlet oxygen initiates the light‐emitting reaction.
Chemiluminescence of diglycidyl ether of bisphenol A — Norbornene anhydride networks
Authors: Tcharkhtchi A. , Audouin L. , Verdu J.
Published: May 2005

Networks were prepared by curing diglycidyl ether of bisphenol A (DGEBA) with variable concentrations of norbornene anhydride (NA). Almost completely cured samples with anhydride/epoxide (A/E) inolar ratios of respectively 0.8, 0.9, 1.0 and 1.1, and one incompletely cured sample with A/E = 1.0, were studied by chemiluminescence in the temperature range 135–220°C, using isothermal stationary or non‐stationary (atmosphere change) exposures. The comparison of kinetic curves of intensity variation reveals: the importance of unreacted epoxide groups as sources of highly emissive radical species, the lowering effect of oxidation products, and the increasing effect of the decrease of macromolecular mobility due to crosslinking in the case of the incompletely cured sample. Most of the features of kinetic curves obtained in non‐stationary experiments are explained in terms of radical formation mechanisms during exposure in inert atmosphere. The results show clearly that chemiluminescence is due to reactions of peroxy radicals rather than hydroperoxide groups.
Acridinium ester labelled cytokines: Receptor binding studies with human interleukin‐1α, interleukin‐1β and interferon‐γ
Authors: Joss U. R. , Towbin H.
Published: May 2005

As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6,‐dimethyl‐4‐(N‐succinimidyloxycarbonyl)phenyl 10‐methylacridinium‐9‐carboxylate methosulphate (DMAE‐NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE‐moiety decays with emission of a short‐lasting chemiluminescent flash.

With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin‐1α (125I‐IL‐1α), interleukin‐1β (125I‐IL‐1β) and interferon‐γ (125I‐IFN‐γ) with the corresponding DMAE‐labelled ligands. The luminescence signal was assessed in a single‐tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE‐N‐hydroxy‐succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE‐labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label. Thus, the method could be used to replace the standard 125I label in receptor screening assays as well as other applications.
Active oxygen generation in γ‐irradiated whole seedlings of Cicer arietinum L.
Authors: Mathew B. G. , Kumar S.
Published: May 2005

Whole seedlings of Cicer arietinum L. when exposed to 819 Gy γ‐radiation, were found to emit ultraweak intensity luminescence. The phenomenon was oxygen‐dependent, and the intensity of emission could be suppressed by post‐irradiation treatment with catalase, superoxide dismutase, and ascorbic acid. Deuterium oxide and luminol amplified the emission intensity. These results suggest that singlet oxygen is a cause of the emission during the post‐irradiation phase.
Enhanced chemiluminescent assays for acetylcholine
Authors: Ternaux Jean‐Pierre , Chamoin Marie‐Claude
Published: May 2005

Acetylcholine and choline chemiluminescent assays have limitations when these compounds are detected in small areas of mammalian nervous tissue. Use of 7‐dimethyl‐aminonaphthalene‐1,2‐dicarbonic acid hydrazide (7‐DMAN), instead of luminol, gives a threefold increase in emitted light in the chemiluminescent assay for acetylcholine based on the coupled choline oxidase‐peroxidase reaction. Addition of light enhancers, such as para‐iodophenol or D‐luciferin, to luminol or 7‐DMAN further increased the light emission. Under these conditions the detection limit for acetylcholine was 650 femtomoles. This enhanced chemiluminescent assay should be convenient for the detection of in vivo and in vitro acetylcholine release from mammalian neurons.
Increased native chemiluminescence in granulocytes isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis
Authors: Arnhold J. , Sonntag K. , Sauer H. , Häntzschel H , Arnold K.
Published: May 2005

Polymorphonuclear leukocytes (PMNs) isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis and from peripheral blood of volunteers were stimulated with 12‐Phorbol‐13‐myristate acetate (PMA). No significant differences in luminol‐amplified chemiluminescence were found between different patients and control groups. However, two distinct patterns of native chemiluminescence were observed. Type I showed no, or only a small, increase in native chemiluminescence with integral counts over 30 min less than 3 × 105 cpm, and the majority of samples from volunteers were of this type. Type II was characterized by a burst of native chemiluminescence starting 8 to 15 min after cell stimulation. It was found in most PMN samples from patients with rheumatoid arthritis. Integral counts over 30 min were always higher than 106 cpm and as high as 108 cpm in some cases. A strong inhibition of the Type II native chemiluminescence was caused by desferal, catalase, thiourea, and glutathione. However, the luminol‐amplified chemiluminescence remained unchanged or was only slightly decreased under the same experimental conditions. Sodium azide strongly inhibited both kinds of luminscence. Hydroxyl radicals, formed in a Fenton reaction, may be important intermediates in the Type II native chemiluminescence.
Commercially available luminometers and imaging devices for low‐light level measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update 3
Authors: Stanley Philip E.
Published: May 2005

This survey was compiled in February 1994 and includes products not covered in the luminometer survey (Jan 1992: Stanley PE, J Biolumin Chemilumin 1992; 7:77–108 and 7:157–69), kits and reagent survey (Nov 1992: Stanley PE, J Biolumin Chemilumin 1993; 8:51–63), update 1 (June 1993 luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1993; 8:237–40) and update 2 (Dec 1993: luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1994; 9:51–3). Technical details are provided together with company addresses and contact information.
Imaging of chemiluminescent signals with cooled CCD camera systems
Authors: Martin Chris S. , Bronstein Irena
Published: May 2005

We investigated imaging of chemiluminescent signals from 1,2‐dioxetanes with cooled CCD cameras. Non‐radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compred to film‐based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to 32P detection methods utilizing storage phosphor screen imaging systems.
Stable luciferase transfected cells for studying steroid receptor biological activity
Authors: Gagne Didier , Balaguer Patrick , Demirpence Ediz , Chabret Claude , Trousse Françoise , Nicolas Jean‐Claude , Pons Michel
Published: May 2005

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in Intact cells using a photon‐counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time.

Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.
Spatial and dye correlation analysis of intracellular Ca2+ distribution
Authors: Mironov S. L. , Hermann A.
Published: May 2005

Intracellular Ca2+ is an important regulator of many cellular processes. Besides ion channels and transporters in the plasmalemma, changes in [Ca]i can be mediated by uptake and release mechanisms of internal organelles. Theoretical and experimental procedures are developed aiming to reveal the distribution of internal Ca2+ pools and their role in generating complicated spatial patterns of [Ca]i gradients. Cultured pyramidal neurons from rat hippocampus were loaded with Ca2+‐sensitive fluorescent dyes, fura‐2 and fluo‐3. Cell images were partitioned according to pixel amplitude and highlighted pictures were characterized by their intensity, relative area and connectivity. This approach facilitates the localization of the sites of Ca2+ release from internal stores induced by application of different agents. After each trial, neurons were stained with dyes, acridine orange or DiOC6, which bind preferentially to nucleus and endoplasmic reticulum. A correlation between images confirmed the spatial localization of Ca2+ release sites. Application of the partition procedure also gave a clear evidence for the importance of Ca2+ influx in the mechanism of [Ca]i oscillations.
Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells
Authors: Gandelman Olga , Allue Isidro , Bowers Keith , Cobbold Peter
Published: May 2005

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphate greatly depresses the signal in vitro and that micromolar concentrations of inoragnic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP‐binding to luciferase. Luciferse‐injected cells exposed to extracellular luciferin concentrations above about 100 μmol/1 (corresponding to a cytoplasmic level of c. 5–10 μmol/1 because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumlation of a luminescently inactive, irreversible, luciferase‐oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100μmol/1 extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2‐4 μmol/1.
TCA‐100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies
Authors: Andreotti Peter E. , Linder Dee , Hartmann Diana M. , Cree Ian A. , Pazzagli Mario , Bruckner Howard W.
Published: May 2005

The BATLE LE TCA‐100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.
A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay
Authors: Kitamura Mitsuko , Maeda Masako , Tsuji Akio
Published: May 2005

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP+ or glycerol‐3‐phosphate as substrate. The detection limits for ALP using each substrate were 1.25 × 10−19 mol/assay and 2.5 × 10−19 mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP+ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK‐8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimtric EIA (ALP label). The correlation between CCK‐8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time‐resolved fluoroimmunoassay (TR‐FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x − 0.04, r = 0.997 (n = 51) and y = 1.00x − 0.03, r = 0.999 (n = 10), respectively.
Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase
Authors: Jockers Ralf , Ziegler Torsten , Schmid Rolf D.
Published: May 2005

The interaction of trizine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s‐triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite‐based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C‐10 and C‐11, respectively. Only the substrate activity of 10‐(4‐chloro‐6‐methyithio‐s‐triazine‐2‐yl)aminodecanal (5) was as high as n‐decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 × 10−30 m3. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 × 10−30 m3 or greater. All triazine aldehydes showed approximately 10‐fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n‐decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.
Optimization of a phagocyte microplate chemiluminescent assay
Authors: Kournikakis Bill , Simpson Maureen
Published: May 2005

Chemiluminescent assays have been used to quantify phagocytic activity since 1972. In recent years these assays have been adapted to the 96‐well microplate format as new luminometers have been developed. In this report we describe the optimization of a lucugenin enhanced phagocyte chemiluminescent assay using a Titertek Luminoskan. Factors such as cell concentration, serum concentration in the opsonization of the zymosan used and lucigenin concentration were all optimized in our assay. In addition we have found that some of the unique features of the Luminoskan, continuous microplate agitation during the assay and microplate temperature control up to 43°C, also significantly enhanced the chemiluminescent response.
A new approach for analysis of chemiluminescent kinetics of activated phagocytes in blood
Authors: Magrisso Moni J. , Bechev Blagovest G. , Bochev Petyo G. , Markova Vania I. , Alexandrova Margarita L.
Published: May 2005

Luminol chemiluminescence (LCL) is a simple, sensitive and time‐saving tool to elucidate the oxidative activity of polymorphonuclear leukocytes (PMNL). In this study, a new approach for analysing the LCL kinetics, recorded from stimulated PMNL, and for a more accurate elucidation of their functional state, is proposed. This approach is based on the proposal that the LCL kinetics of stimulated PMNL is a result of time‐probabilistic nature of the processes, leading to light emission. On this basis the LCL response was described by the parameters of a Poisson‐type distribution.

Relationships between these parameters and some factors influencing LCL kinetics (number of PMNL and erythrocytes, temperature, stirring) were investigated. Their development in time was different. It was suggested, that the two phases of the model LCL response were connected with extracellular and intracellular LCL. The terms and the advantages of this approach for analysis of phagocyte oxidative capacity are discussed.
Ticlopidine augments luminol‐dependent chemiluminescence in human neutrophils
Authors: Bednar Martin M. , Dooley Richard H. , Tapanes Ricardo , Lublin Jennifer C. , Gross Cordell E.
Published: May 2005

Neutrophils contribute to the pathophysiology of various ischaemic states. Since many agents thought to be antiplatelet have also been shown to affect neutrophil function, it was of interest to examine the effect of ticlopidine (250 mg, p.o., b.i.d. for three doses), an antiplatelet agent, on fMLP (formyl‐methionyl‐leucyl‐phenylalanine) stimulated neutrophil aggregation and luminol‐dependent chemiluminescence in whole blood. Neutrophil aggregation did not significantly change from baseline values during ticlopidine administration. However, luminol‐dependent chemiluminescence, an index of respiratory burst metabolism, was noted to be markedly increased during ticlopidine administration. Two hours following the final dose of ticlopidine, the chemiluminescent response (mean ± SEM, n = 5) was significantly increased from 6.27 ± 1.88 to 12.66 ± 2.19 units (p < 0.05). A return to baseline (6.68 ± 2.24 units) five days following the administration of ticlopidine was noted. It is concluded from this study that the acute oral administration of ticlopidine may affect neutrophil function as demonstrated by the significant increase in stimulated luminol‐dependent chemiluminescence.
Electrogenerated chemiluminescence from violanthrone
Authors: Quickenden T. I. , Hansongnern K.
Published: May 2005

Violanthrone, an emitter of exceptionally bright chemiluminescence, was examined in dimethylformamide solution to determine whether it also emits particularly bright electrogenerated chemiluminescence (ECL). The ECL measurements were carried out using a cycled potential which was applied to platinum electrodes. At the maximum sweep rate of 80 Vs−1 available, the intensity of the violanthrone ECL was still increasing with sweep rate and was c. 56% of that from rubrene, a bright, commonly used emitter of ECL. Furthermore, assuming that the emission arises from radical anioncation recombination, the sweep rate dependence showed that the least stable radical ion (probably the cation) decays with a half‐life shorter than 0.2s.
The development of Luminomaster™, a fully automated chemiluminescent enzyme immunoassay system
Authors: Ikegami Takashi , Yamamoto Masatoshi , Sekiya Koichi , Sato Yoshihiro , Saito Yukio , Maeda Masako , Tsuji Akio
Published: May 2005

We have developed a fully automated discrete chemiluminescent heterogeneous enzyme immunoassay system called Luminomaster™. The characteristics of this analyser are: 120 test/h throughput, 14 test menu, wide dynamic range, automated sample dilution, automatic retest, communication with a host central processing unit (CPU) and connection with sample transfer system.
A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence
Authors: Yu Hao , Bruno John G. , Cheng Tu‐chen , Calomiris Jon J. , Goode Michael T. , Gatto‐Menking Deborah L.
Published: May 2005

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)32+)‐end‐labelled primers. In this way, biotin for capture and Ru(bpy)32+ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin‐ and Ru(bpy)32+‐labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)‐coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi‐automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA‐based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.
Comparison of seven bio‐ and chemiluminescent reagents for in situ detection of antigens and nucleic acids
Authors: Lamarcq L. , Lorimier P. , Negoescu A. , Labat‐Moleur F. , Durrant I. , Brambilla E.
Published: May 2005

Bio‐ and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio‐or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, aikaline phosphatase, β‐galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission.

An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears.

Among the enzyme‐luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.
Action of phenolic antioxidants on various active oxygen species
Authors: Cynshi Osamu , Takashima Yoshiaki , Katoh Yoshiaki , Tamura Kunio , Sato Mitsuo , Fujita Yuzo
Published: May 2005

The action of phenolic antioxidants, such as probucol, on various active oxygen species was investigated using luminol chemiluminescence and spin trapping with 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). The various active oxygen species, including hydroxyl radicals (Fenton reaction), superoxide anions, singlet oxygen and hypochlorite ions were examined with phenolic antioxidants under aqueous and nonaqueous conditions. Probucol showed a quenching effect on both superoxide anions and hypochlorite ions in nonaqueous solution. However, it had no effect on hydroxyl radicals. α‐Tocopherol, a natural phenolic antioxidant, showed a stronger quenching effect on superoxide anions and hypochlorite ions than probucol, and quenched hydroxyl radicals in nonaqueous solution. Furthermore, Trolox showed a quenching effect on all active oxygen species in both aqueous and nonaqueous solution. The antioxidants were studied under comparable conditions in a series of test systems and the reactivity profiles depicted as ‘radar charts’ which are helpful for characterizing antioxidant action.