bluuebunny/crossref_metadata_2025 huggingface dataset:
Luminol‐amplified chemiluminescence activity in human monocytes: A comparison with the activity induced in granulocytes
Authors: Johansson Agneta , Dahlgren Claes
Published: May 2005

The characteristics of the luminol‐amplified chemiluminescence (CL) induced in mononuclear phagocytes interacting with PMA, FMLP or ionomycin were determined. Azide reduced the CL activity by more than 80%, while superoxide dismutase and catalase had minor effect on the monocyte CL response. The sensitivity of the monocyte CL response, to the addition of extra peroxidase differed depending on the stimulus used. Furthermore, no direct correlation was obtained between the CL response and superoxide anion or hydrogen peroxide production. In comparison with the response in granulocytes, minor quantitative differences were observed. The mechanism for the light‐generating reaction, seems to be the same in both cell types.
Enhancement of granulocyte luminescence by murine macrophage secretory products: Suggestive evidence for the release of a new activator
Authors: Willems J. , Leclercq G. , Joniau M.
Published: May 2005

The incubation of macropages (MΦ) in the presence of lipopolysaccharides (LPS) usually results in the release of a variety of immunoregulatory cytokines such as interleukins (IL), tumour necrosis factor (TNF) and colony stimulating factors (CSF). We recently observed that conditioned media (CM) from LPS‐treated murine MΦ lines probably contain another protein endowed with granulocyte stimulatory activity. This cytokine, which has an apparent MW of about 55 kDa enhances the PMA‐induced luminescence of granulocytes and also stimulates their degranulation as measured by lactoferrin release. In contrast to IL1 and IL6 this factor is destroyed by brief treatment at pH 2, but is stable for 60 minutes at 65°C. Unlike CSF, its activity is unchanged by reducing agents such as beta‐mercaptoethanol. Furthermore, pretreatment of the MΦ with dexamethasone, in order to reduce the release of IL1 and TNF, hardly reduces the effect on granulocyte activation. Finally, treatment with a neutralizing polyclonal anti‐murine TNF antiserum only partly abolishes its activity.

These results show that, in addition to the already well‐described cytokines, LPS‐treated murine MΦ lines most probably secrete another granulocyte activator.
Luminol chemiluminescence: Chemistry, excitation, emitter
Authors: Merényi Gábor , Lind Johan , Eriksen Trygve E.
Published: May 2005

The mechanism of luminol chemiluminescence is a special case of nucleophilic addition to carbonyl compounds. The breakdown of the key intermediate, an alpha hydroxy hydroperoxide, produces a peracid ortho to an acyl diazene group. After intramolecular addition of the peracid, the energy from nitrogen expulsion is utilized in the formation of an anti‐aromatic endoperoxide. Rupture along the O,O bond leaves a substantial part of the ensuing phthalate in its excited state. The emitter is shown to be a mono‐protonated phthalate unaccessible by photoexcitation. The dark reaction is a concerted decomposion of the alpha hydroxy hydroperodixe to yield ground‐state phthalate.
Fibre‐optic biosensor based on luminescence and immobilized enzymes: Microdetermination of sorbitol, ethanol and oxaloacetate
Authors: Gautier Sabine M. , Blum Loïc J. , Coulet Plerre R.
Published: May 2005

We have investigated highly selective and ultrasensitive biosensors based on luminescent enzyme systems linked to optical transducers. A fibre‐optic sensor with immobilized enzymes was designed; the solid‐phase bioreagent was maintained in close contact with the tip of a glass fibre bundle connected to the photomultiplier tube of a luminometer. A bacterial luminescence fibre‐optic sensor was used for the microdetermination of NADH. Various NAD(P)‐dependent enzymes, sorbitol dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, were co‐immobilized on preactivated polyamide membranes with the bacterial system and used for the microdetermination of sorbitol, ethanol and oxaloacetate at the nanomolar level with a good precision.
The luminescent bacteria toxicity test: Its potential as anin vitroalternative
Authors: Bulich Anthony A. , Tung Ker‐Kong , Scheibner Grace
Published: May 2005

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–‐the L‐929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC50values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.
From Luc and Phot genes to the hospital bed
Authors: Campbell Anthony K. , Sala‐Newby Graciela , Aston Paul J. , Kalsheker Noor , Kishi Yoshito , Shimomura Osamu
Published: May 2005

The Luc gene from the firefly Photinus pyralis has been isolated by cloning it in pcDV1 PL plasmid primer and Honjo linker and the Phot gene isolated from Aequorea victoria using the polymerase chain reaction. A method has been established using SP6 RNA polymerase for transcribing and translating bioluminescent genes in vitro. It should now be possible to engineer these genes to measure intracellular ATP and the covalent modification of proteins in single, live cells, providing unique insights into the molecular basis of disease.
Chemiluminescence properties of human, canine and rat polymorphonuclear cells
Authors: Lindena Joachim , Burkhardt Hannelore
Published: May 2005

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two‐step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol‐enhanced assays after stimulation with various agents: non‐opsonized zymosan (3.5 g/I), phorbol myristate acetate (PMA, 2.8 × 10−6 mol/I), calcium ionophore A 23187 (10−5 mol/l) and N‐formylmethionyl‐leucyl‐phenylalanine (FMLP, 3.5 × 10−6 mol/l). The isolated cells of the three species responded to all of the various stimuli. Species‐related sensitivity could be ordered: human > canine > rat. Response to the various agents in the human cells can be ranked: PMA ⩾ A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan ⩾ PMA > FMLP ⩾ A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.
Different influences of cytochalasin B on the activation of human neurophils settled onto petri dishes displayed by simultaneously detected native and luminol‐dependent luminescence
Authors: Roschger Paul , Graninger Wolfgang , Klima Herbert
Published: May 2005

Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells. The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli. PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N‐formylmethionyl‐phenylalaline. The production of reactive oxygen species were monitored by simultaneous detection of native, luminol‐independent, luminescence (NL) and luminol‐dependent luminescence (LDL) using a method of spectral discrimination. Different influences of CB on NL with respect to LDL as well stimuli‐dependent influences of CB on the luminescence response of PMNL were observed. Especially phagocytosis‐associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL. A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged.
Chemiluminescence (CL) in blood samples reflects leukocyte activation (LA) during anaphylactoid reactions in dogs
Authors: Wilhelms O.‐H. , Lipponer L. , Seilnacht W.
Published: May 2005

Inflammation is accompanied by leukocyte activation (LA). We decribe a simple ex vivo technique for studying LA that might help to find new LA inhibitors for the treatment of pathologic events related to LA.

Arterial and venous blood samples obtained from six permanently catheterized beagle dogs −60, 0, +15 min and +23 h after i.v. challenge with C 48/80, and also blood samples from six normal beagles, were minimally diluted 1:2.5 with buffer. Total leukocyte counts (LC), and luminol amplified CL, induced by opsonized zymosan (C3‐Z), were estimated. Blood samples from dogs elicited CL responses of almost 1/10 the magnitude of erythrocyte‐free human leukocytes, whereas blood samples from rats reacted three orders of magnitude less. Obviously quenching of CL by accompanying erythrocytes in blood samples from dogs is not important, for CL correlated almost linearly with the CL in differently diluted samples. In arterial, but not in venous samples from catheterized dogs, absolute CL and LC, both were significantly depressed (p < 0.05) 15 min after C 48/80 challenge. CL/106 leukocytes was augmented twofold. All leukocyte deviations returned to pre‐values 23 h post‐challenge.
A new fluorescence enhancement solution for Europium‐based time‐resolved fluoroimmunoassays
Authors: Degan Paolo , Abbondandolo Angelo , Montagnoli Giorgio
Published: May 2005

A cationic detergent is proposed as suitable insulating agent for the preparation of a fluorescence enhancement solution useful for europium‐based time‐resolved fluoroimmunoassays. Luminescence from europium ions at concentration as low as 0.5 pmol/l can be detected in solution containing 1.6 mmol/l thenoyltrifluoroacetone, 110.5 μ mol/l Adogen 464 and 0.1% Tween 20, in the presence of 0.5 mol/l NaCl. A competitive TR‐FIA for rabbit lgG is described, showing that the new enhancement solution allows a sensitivity comparable to that of the high performance LKB Wallac DELFIA.
Bioluminescence decay kinetics in the reaction of bacterial luciferase with different aldehydes
Authors: Ismailov Anvar D. , Sobolev Alexander Yu. , Danilov Vadim S.
Published: May 2005

At 22°C the bioluminescence decay kinetics in the in vitro reaction catalysed by Vibrio harveyi luciferase in the presence of different aldehydes–‐nonanal, decanal, tridecanal and tetradecanal did not follow the simple exponential pattern and could be fitted to a two‐exponential process. One more principal distinction from the first‐order kinetics is the dependence of the parameters on aldehyde concentration. The complex bioluminescence decay kinetics are interpreted in terms of a scheme, where bacterial luciferase is able to perform multiple turnovers using different flavin species to produce light. The initial phase of the bioluminescent reaction appears to proceed mainly with fully reduced flavin as the substrate while the final one results from the involvement of flavin semiquinone in the catalytic cycle.
Effect of propagators and inhibitors on the ultraweak luminescence from maize roots
Authors: Radotić K. , Redenović Č. , Jeremić M. , Vučinić Ž.
Published: May 2005

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide‐radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.
A chemiluminescence assay for erythrophagocytosis
Authors: Downing I. , Templeton J. G. , Mitchell R. , Fraser R. H.
Published: May 2005

A luminol‐dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti‐D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes‐luminol‐erythrocytes (either anti‐D IgG sensitized or unsensitized controls) was 500 μl, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35–45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra‐ and inter‐assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.
Activation of human monocytes by interleukin 2: Role of T lymphocytes
Authors: Ennen J. , Ernst M. , Flad H.‐D.
Published: May 2005

The effect of interleukin 2 (IL 2) on the capability of human monocytes to secrete reactive oxygen species triggered via Fc‐γ receptor (Fc‐γ R) function had been investigated by measurement of chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to Fc‐γ R mediated phagocytic stimulation with an enhanced respiratory burst activity unless low numbers of T cells had been co‐cultured with hp monocytes. Supernatants from IL 2 treated PBMC contained interferon‐γ (IFN‐γ) and monocyte activating factor (MAF) activity. The secretion of both cytokine activities was strongly enhanced by cooperative function of monocytes. The correlation of IL 2 induced secretion of IFN‐γ and MAF activity was striking, however, monoclonal antibody (mAb) anti‐human IFN‐γ failed to abrogate IL 2 stimulated and lymphocyte dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes with IL 2 led to monocyte priming: subsequent co‐culture with autologous control T cells enhanced the monocyte Fc‐γ R mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor as shown by inhibition experiments with anti IL 2 R monoclonal antibody. Thus the IL 2 driven monocyte/T‐cell interaction leads to an increased Fc‐γ R mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN‐γ.
Extra‐weak chemiluminescence of drugs XI. Quenching effects of purine and pyrimidine derivatives on the extra‐weak chemiluminescence derived from the maillard reaction
Authors: Kurosaki Yuichiro , Sato Hideaki , Mizugaki Michinao
Published: May 2005

Quenching effects of purine and pyrimidine derivatives on the extra‐weak chemiluminescence (CL) derived from the Maillard reaction of L‐lysine with D‐arabinose were investigated The pyrimidine derivatives 2′‐deoxy cytidine, uridine, and uracil quenched the CL. Cytidine did not quench the CL. Purine derivatives, e.g. uric acid and 1‐methyl adenosine were particularly effective in quenching the CL. 5‐Methyl adenine and xanthine also quenched the CL, but adenosine had no effect. A comparison of the CL‐quenching abilities of compounds that have common basic structure was made; those with ribose at the 5‐position were the strongest quenchers. A linear relationship between CL‐quenching activity and the HOMO energy of the pi orbital for the various compounds was shown.
Nucleophilic addition to the 9 position of 9‐phenylcarboxylate‐10‐methylacridinium protects against hydrolysis of the ester
Authors: Hammond Philip W. , Wiese Wendy A. , Waldrop Alex A. , Nelson Norman C. , Arnold Lyle J.
Published: May 2005

The chemiluminescent reaction of an acridinium ester (AE) requires addition of peroxide to the 9 position of the acridinium ring. The addition of a hydroxide ion to the 9 position of an acridinium ester to form the carbinol adduct has also been well documented. We have observed a similar addition of other nucleophiles to the acridinium ring to form an acridan adduct. The adduct formed with bisulphite has been particularly well‐characterized for rate of formation, rate of reversion, and reaction equilibrium. The formation of an adduct (other than H2O2) has been demonstrated to decrease significantly the reactivity of the adjacent ester bond to alkaline hydrolysis. The resulting, more stable adduct is very useful when the acridinium ester is used as a label in DNA probe‐based assays. The adduct is highly resistant to hydrolysis under the conditions often desired for DNA probe‐based assays (high temperature, elevated pH, extended storage).
Measurement of rate constants for quenching singlet oxygen with a Cypridina luciferin analog (2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one) and sodium azide
Authors: Mashiko Shinro , Suzuki Nobutaka , Koga Satoshi , Nakano Minoru , Goto Toshio , Ashino Toshihiko , Mizumoto Iwao , Inaba Humio
Published: May 2005

The rate constants for [1O2] [MCLA] and [1O2][NaN3] were measured by quenching the near‐infrared emission (1Δg→3∑g) in steady state with MCLA and NaN3, respectively. 1O2 was constantly generated by energy transfer to O2 from Ar laser‐excited Rose Bengal. The Stern—Volmer plots yielded the second‐order rate constants of 2.94 × 109 M−1 S−1 and 3.83 × 108 M−1 S−1 for quenching 1O2 with MCLA and NaN3 in water at pH 5.4, respectively. The 1O2 + MCLA reaction emitted light with maximum at 465 nm at pD 4.5 identical to the O2− + MCLA reaction.
Direct chemiluminescence immunoassay (CLIA) for muramyl tripeptide phosphatidyl‐ethanolamine in plasma
Authors: Gay B. , Towbin H. , Schnell C. , Einsle K. , Graf P. , Gygax D.
Published: May 2005

A competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl‐ethanolamine (MTP‐PE) in plasma has been developed. The assay is based on the use of an acridinium ester‐labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1‐5 nmol/l, and the inter‐assay CVs are ⩽ 10%. Using up to 6000‐fold sample dilutions, a wide working range (0.1‐30 000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP‐PE. Following infusion, the concentrations in plasma declined multiphasically. Half‐life time was 0.37 h ± 0.03 (mean ± SD, alpha phase) and 1.76 h ± 0.08 (mean ± SD, beta phase), clearance and volume of distribution were 0.09 ± 0.02 l/h × kg (mean ± SD) and 0.06 ± 0.01 l/kg (mean ± SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with reagents of limited shelf‐life.
The effects of N‐ethylmaleimide on extracellularly and intracellularly generated chemiluminescence in neutrophils indicate that the rate of deactivation of NADPH‐oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane
Authors: Dahlgren Claes , Sundqvist Tommy
Published: May 2005

Sustained generation of reactive oxygen metabolites following respiratory burst activation in neutrophils is a result of continued replenishment of a pool of active NADPH‐oxidase. The sulphydryl‐modifying reagent N‐ethylmaleimide (NEM) has been shown to be without effect on the turnover of activated NADPH‐oxidase but to inhibit the replenishment of active oxidase molecules (Akard et al., 1988). NEM was thus used to determine the rate of deactivation of extracellularly and intracellularly generated chemiluminescence in human neutrophils. We have shown that deactivation is more rapid when activation leads to a release of oxygen metabolites (extracellular chemiluminescence) than when the metabolites are generated intracellularly. The results indicate that the rate of deactivation of NADPH‐oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane.
Extraction and analysis of superoxide free radicals (·O−2) from whole mammalian liver
Authors: Shoaf Antony R. , Shaikh Ali U. , Harbison Raymond D. , Hinojosa Oscar
Published: May 2005

Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N2 gives extracts which contain 5—10 μmol/l·O−2 (50‐100 nmol·O−2 per 10 ml extract per 4 g liver; 1.25‐2.50 nmol·O−2 per millilitre per gram liver). Evidence for ·O−2 in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O−2. Extraction of ·O−2 is enhanced by tetrabutyl ammonium ion, and is maximal at 1‐3 min. These results raise the possibility that substantial amounts of ·O−2 are normally sequestered in protective membranous sites in vivo.
Weak chemiluminescence of bilirubin and its stimulation by aldehydes
Authors: Watanabe Haruo , Usa Masashi , Kobayashi Masaki , Agatsuma Shin‐Ichi , Inaba Humio
Published: May 2005

Bilirubin in an alkaline solution exhibits a weak chemiluminescence (CL) under aerobic conditions. This spontaneous CL was markedly enhanced by the addition of various aldehydes. The fluorescent emission spectrum of bilirubin, excited by weak intensity light at 350 nm, coincided with its CL emission spectrum (peak at 670 nm). CL emission from bilirubin was not quenched by active oxygen scavengers. This suggests that triplet oxygen reacts with bilirubin, and forms an oxygenated intermediate (hydroperoxide) as a primary emitter (oxidative scission of tetrapyrrole bonds in bilirubin is not involved in this CL). The Ehrlich reaction (test for monopyrroles) and hydrolsulphite reaction (test for dipyroles) on the CL reaction mixture and unreacted bilirubin showed no differences. When the CL was initiated by singlet oxygen, rather than superoxide anion, monopyrrole, was detected in the reaction products by gel chromatography. The inhibitory effect of a scavenger of singlet oxygen on CL was eliminated in the presence of formaldehyde. Therefore, triplet carbonyl, formed by singlet oxygen through the dioxetane structure in bilirubin, is not an emitter. The reaction mechanism of bilirubin CL and the formation of a hydroperoxide intermediate is discussed in relation to the chemical structure of luciferin molecules from bioluminescent organisms.
Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles
Authors: Metelitza D. I. , Eryomin A. N. , Shibaev V. A.
Published: May 2005

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane‐chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H2O2 and substrates, the pH value of the micelle polar phase and the H2O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15‐fold higher than for the ABI‐COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.
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 2: Photographs
Authors: Stanley Philip E.
Published: May 2005

Part 1 of this survey comprised a description of the luminometers (J. Biolumin. Chemilumin. 7, 77‐108 (1992)) and 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. This part includes photographs of around 40 of these instruments. 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.
In vitro effect of ascorbic acid on neutrophil–endothelial cell interaction
Authors: Jonas Eleonore , Dwenger Alexander , Hager Anke
Published: May 2005

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil–endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen‐derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil‐mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil‐mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant‐mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).
Interaction between myeloperoxidase and antibodies against myeloperoxidase measured by chemiluminescence
Authors: Nässberger Lennart
Published: May 2005

We investigated interaction between antibodies directed against myeloperoxidase (anti‐MPO) and myeloperoxidase (MPO) with chemiluminescence. Whole serum diluted 1/10 containing circulating anti‐MPO antibodies as well as serum from normal blood donors inhibited myeloperoxidase (MPO) enzyme activity when incubated with 1.42 m̈g MPO. When further diluted the inhibitory effect was abolished. Incubation with purified human lgG fraction of anti‐MPO, did not cause any inhibition when diluted 1/10 and incubated with 1.42 m̈g MPO. When adding MPO to normal sera a rapid increase of the enzyme activity was seen above 5 m̈g, and the inhibitory effect was completely abolished when 10 m̈g was added. Both sera from healthy individuals, as well as sera from patients with circulating anti‐MPO inhibited MPO activity. The inability of pure lgG fractions from anti‐MPO sera to inhibit MPO enzyme activity, clearly indicates the presence of an inhibitory factor, unspecific or specific, in serum.
Application of in vivo bioluminescence to the study of lonophoretic action
Authors: Simpson W. J.
Published: May 2005

Ionophores (carbonyl cyanide m‐chlorophenylhydrazone; valinomycin; and the hopderived compounds colupulone, trans‐isohumulone and trans‐humulinic acid) reduced the rate of dodecanal‐dependent light emission from luxA/B‐transformed cells of Lactococcus lactis subsp. diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose. This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non‐destructive technique in real time.
GroESL proteins facilitate binding of externally added inducer by luxR protein‐containing E. coli cells
Authors: Adar Y. Y. , Ulitzur S.
Published: May 2005

htpR− (rpoH, σ32 minus) strain of E. coli harbouring the whole lux system of Vibrio fischeri is very dim. We have recently shown that GroESL proteins fully recover the expression of the lux system in this strain. This work has been undertaken to study our assumption that the GroESL proteins stabilize the LuxR protein, thus enhancing the formation of LuxR–Inducer complex. E. coli htpR− cells harbouring the luxR gene were unable to bind extracellularly added inducer, while late logarithmically growing htpR+ strain bound small quantities of the inducer. Reduction in the nutrient content of the growth medium resulted in a large increase in the capability of these cells to bind the inducer. htpR+ or htpR− E. coli strains harbouring both the luxR and the groESL genes bound large quantities of the inducer. The molecular and ecological significance of these results is discussed.
Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite
Authors: Arnhold J. , Mueller S. , Arnold K. , Sonntag K.
Published: May 2005

Two different mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite were found. Most substances investigated in these experiments acted by scavenging NaOCI. This mechanism was independent of the concentration of hydrogen peroxide and the incubation time between luminol and inhibitors. The most potent inhibitors were substances containing SH groups. Compounds with amino groups as a target for HOCI/OCI− to yield chloramines were much less effective inhibitors. Another mechanism of inhibition was found for catalase. It depended on the presence of hydrogen peroxide in the incubation medium and the incubation time between luminol and catalase. The enzyme inhibited the luminescence by removing H2O2 at molar concentrations much smaller than those found for all other inhibitors. Our results confirm the present models of the mechanism of generation of luminescence in luminol oxidation.
Improvements to enhanced horseradish peroxidase detection sensitivity
Authors: Motsenbocker M. A. , Kondo K.
Published: May 2005

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13‐fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.
Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring
Authors: Nyrén Pål
Published: May 2005

A novel technique that is useful for the control of the luminescence output in a bioluminometric assay has been developed. The method relies on the immobilization on paramagnetic beads of an enzyme that is capable of catalysing the hydrolysis or oxidation of the substrate for the luminescence reaction. This technique has been used for control of the level of ATP in the luciferin‐luciferase system. Biotinylated apyrase was bound to streptavidin coated paramagnetic beads and added to the assay mixture. The level of ATP in the reaction mixture was then conveniently controlled using an external magnet. The possible use of this approach in other systems, such as the bacterial luciferase–oxidoreductase is discussed.
Commercially available luminometers and imaging devices for low‐light level measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update 2
Authors: Stanley Philip E.
Published: May 2005

This survey was completed in December 1993 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). Technical details are provided together with company address and contact information.
Photon emission from chemically perturbed yeast cells
Authors: Rajfur Zenon
Published: May 2005

Photon emission (PE) from yeast cells Saccharomyces cerevisiae strain SP‐4 in normal conditions and in conditions perturbed by the addition of formaldehyde was investigated using single‐photon counting equipment. PE from yeast cells, growing in a standard nutrient medium (YPG) then centrifuged and resuspended in a phosphate buffer (pH = 6.5), was measured in the presence of oxygen or argon. The solution of formaldehyde (2%) was injected into the sample. The intensity of PE increased and reached a maximum, then slowly decreased to a level which was higher than the PE level without the perturbing factor. The kinetics of PE was found to be strongly dependent upon the presence of oxygen. The model of formation and recombination of free radicals was tested. The results indicate that PE can arise during the recombination reactions of free radicals like R⋅ + R⋅, RO⋅ + RO⋅, RO⋅2 + RO which are formed in the enzymatic oxidative reactions.
Imaging of chemiluminescent reactions in mesoscale silicon‐glass microstructures
Authors: Kricka Larry J. , Ji Xiaoying , Nozaki Osamu , Wilding Peter
Published: May 2005

Chemiluminescent reactions in mesoscale analytical structures (chips) containing micrometer‐sized interconnecting channels and chambers (pL‐nL total volume) were imaged. The chips were fabricated by bonding Pyrex glass to etched pieces of silicon using a high‐temperature diffusive bonding technique. In initial experiments light emission from an enhanced chemiluminescent horseradish peroxidase reaction and from a peroxyoxalate reaction contained in straight channels (300 μm wide × 20μ deep; volume 70.2 nL) and open chambers (812 μm wide, 400 μm deep, 5.2 mm long) linked by channels (100μm wide, 20 μm deep) to an exit and entry port were studied using a specially modified microplate holder and an Amerlite microplate luminometer. Light emission from more complex structures (two chambers interconnected by a branching channel 100 μm wide, 20 μm deep) filled with a solution containing alkaline phosphatase, Emerald, and CSPDTM was imaged using a Photometrics Star 1 CCD camera. Detailed investigation of the detection and spatial resolution of the signal was performed on a Berthold Luminograph LB 980 using both the enhanced chemiluminescent horseradish peroxidase reaction and a peroxyoxalate reaction. We successfully resolved light emission from silicon structures with dimensions 100 μm wide and 20 μm deep. These simple silicon structures served as models for more complex designs that will be used for simultaneous multi‐analyte assays in which an imaging system resolves and quantitates light emission from different locations on a silicon‐glass analytical device.
Imaging reactive oxygen species in asthma
Authors: Vachier Isabelle , Doucen Christian Le , Loubatière Jacques , Damon Marcelle , Térouanne Béatrice , Nicolas Jean‐Claude , Chanez Pascal , Godard Philippe
Published: May 2005

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA‐stimulation. Secondly a video‐imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti‐IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.
Targeting of aequorin for calcium monitoring in intracellular compartments
Authors: Brini Marisa , Pasti Lucia , Bastianutto Carlo , Murgia Marta , Pozzan Tullio , Rizzuto Rosario
Published: May 2005

We have recently developed a new method for monitoring Ca2+ concentrations in defined cell compartments. The cDNA encoding the Ca2+‐sensitive photoprotein aequorin has been modified in order to include specific targeting sequences and expressed in eukaryotic cells; the recombinant protein, specifically located inside the cells, has allowed the direct study of mitochondrial and nuclear Ca2+ concentrations in living cells. The principles, and the application, of this new methodology are discussed in this article.
Chimeric receptors as a tool for luminescent measurement of biological activities of steroid hormones
Authors: Jausons‐Loffreda N. , Balaguer P. , Roux S. , Fuentes M. , Pons M. , Nicolas J.‐C. , Gelmini S. , Pazzagli M.
Published: May 2005

Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17‐mer DNA sequence, specific for Gal‐4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub‐cloning was allowed with the help of an imaging system using a single‐photon‐counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector‐concentration‐dependent and their biological activities will be compared to those of native receptors.
Measurement of endogenous and TNFα‐mediated H2O2 production in supernatants of SK‐N‐SH neuroblastoma cells with an enhanced chemiluminescence assay
Authors: Carmine T. C. , Bruchelt G. , Hahn T. , Niethammer D.
Published: May 2005

A sensitive peroxidase‐dependent luminol‐enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H2O2) generation by tumour cells was established. This test system allows determination of H2O2 in concentrations as low as 25 pmol (50nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin flurorescence assay. After 3h incubation time 104SK‐N‐SH neuroblastoma cells released 60° 5 pmol H2O2 in the supernatant and this level was significantly (p<0.025) increased by about 70% in the presence of 5pmol (100 ng/mL) recombinant tumour necrosis factor α (TNFα). In contrast, H2O2 production was slightly reduced by TNFα at a very low concentration of 0.5 fmol (0.01 ng/mL).
Comparison of MTT and ATP‐based assays for the measurement of viable cell number
Authors: Petty Russell D. , Sutherland Lesley A. , Hunter Elizabeth M. , Cree Ian A.
Published: May 2005

Cell viability assays are widely used to assess the effect of chemotherapeutic drugs and other agents on cell lines and have shown promise for the prediction of tumour chemosensitivity. In this study we have compared two viability assays using Daudi and CCRF‐CEM cell lines over a range of 1500–100,000 cells/well of a microplate. The ATP assay was able to detect the lower limit of 1563 cells/well with luminescence values at least 100× background readings, while the MTT assay could not detect less than 25,000 cells/well above background readings. The ATP assay also showed better reproducibility and sensitivity when cells were grown in microtitre plates over several days, and is particularly useful for the measurement of viability with low cell numbers.
Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme
Authors: Rauch Pavel , Poplstein Martin , Hochel Igor , Fukal Ladislav , Ferri Elida , Abagnato Carlo Alberto , Girotti Stefano , Roda Aldo
Published: May 2005

A sensitive chemiluminescent sandwich‐type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol‐enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o‐phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.
4‐phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol
Authors: Kricka Larry J. , Ji Xiaoying
Published: May 2005

4‐Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.
Luminol‐enhanced chemiluminescence after reaction of hydroperoxides with opsonized zymosan
Authors: Heinle Helmut , El Dessouki Jasmin
Published: May 2005

Luminol‐enhanced chemiluminescence (LEC) is very sensitive in detecting free radicals but relatively insensitive for hydroperoxides (hydrogen peroxide, tert‐butyl hydroperoxide). However, in the presence of opsonized zymosan (often used for stimulation of phagocytic cells) hydroperoxides also induce LEC, suggesting that free radicals are produced under these conditions. Therefore careful interpretation with respect to the nature of the reactive species is necessary when LEC is used for characterization of zymosan‐induced phagocytosis. We studied the properties of zymosan‐induced LEC under different test conditions and with various inhibitors. Typical radical scavengers, e.g. nordihydroguaiaretic acid and superoxide dismutase, are strong inhibitors, indicating the importance of the superoxide anion. This system is useful for drug testing with respect to antioxidative or radical scavenging activity.
The roles of the two highly unstable components F and P involved in the bioluminescence of euphausiid shrimps
Authors: Shimomura Osamu
Published: May 2005

Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50‐h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow‐working type, contrary to early report.