bluuebunny/crossref_metadata_2025_split huggingface dataset:
Luciferase cDNA from Japanese firefly, Luciola cruciata: Cloning, structure and expression in Escherichia coli
Authors: Tatsumi Hiroki , Masuda Tsutomu , Kajiyama Naoki , Nakano Eiichi 
Published: May 2005

AbstractWe have cloned the cDNA for luciferase from lantern poly(A)+ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.
Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra
Authors: Morse D. , Fritz L. , Pappenheimer A. M. , Hastings J. W. 
Published: May 2005

AbstractA luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti‐LBP and immunofluorescence microscopy. The amount of LBP is ten‐fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.
Chemiluminescent immunoassay (CLIA) for urinary albumin
Authors: Bagazgoitia F. Javier , Garcia J. Luis , Ibarra J. M. , Patiño R. , Diéquez Carlos , Weeks Ian , Woodhead J. Stuart 
Published: May 2005

AbstractA simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester‐labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol‐assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1‐5 mg/l, and the inter‐assay CVs are ≤ 15%. Using 10− and 50‐fold sample dilutions in assay buffer, a wide working range (1‐250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 ± 2.7 μg/min (x ± SD), range 1‐15.9 μg/min in 36 healthy subjects (17♂, 19♀, ages 4‐56 years), with day‐to‐day variations of 28.5 ± 20% (x ± SD), range 3.3‐76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf‐life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.
Comparison of the lux systems in Vibrio harveyi and Vibrio fischeri
Authors: Miyamoto C. , Boylan M. , Cragg L. , Meighen E. 
Published: May 2005

AbstractComparison of the nucleotide sequences and gene organization of the lux systems from Vibrio harveyi and Vibrio fischeri has demonstrated that the location and order of the lux structural genes are highly conserved whereas considerable divergence has arisen in the location and/or presence of lux regulatory genes in the two marine bacteria. The order of the lux structural genes (luxCDABE) are identical in the two bacteria with the three fatty acid reductase genes (luxCDE) required for aldehyde biosynthesis flanking the luciferase genes (luxAB). Complementation in trans of the upstream V. fischeri DNA containing the luxC and luxD structural genes with the downstream luxA, B and E genes of V. harveyi resulted in luminescent Escherichia coli that did not require any exogenous aldehyde. These results indicate that the light‐emitting systems are very similar in the two bacteria.However, the lux regulatory systems in these two bacteria appear to have clearly diverged. In V. harveyi, an open reading frame of >40 codons does not exist within 600 bp of the start of luxC in contrast to the luxl regulatory gene present in the V. fischeri lux system. An open reading frame of 615 bp is present farther upstream of luxC with the same direction of transcription and approximate location as the luxR regulatory gene of V. fischeri; however, apparent homologies do not exist between the two genes. The similarities in organization of the lux structural genes and the differences in the existence and/or location of lux regulatory genes in the two Vibrio species raises the question of how these marine bacteria can have a similar growth‐dependent regulation of luminescence expression.
Chemistry, clones, and circadian control of the dinoflagellate bioluminescent system. The Marlene DeLuca memorial lecture
Authors: Hastings J. Woodland 
Published: May 2005

AbstractBioluminescence in the dinoflagellate Gonyaulax polyedra occurs as brief bright flashes, originating from many (∼400) small (∼0.5 μm) cytoplasmic organelles which protrude into the acidic vacuole, and are thus surrounded by the tonoplast. Biochemically, the substrate is unusual; it is an open chain tetrapyrrole, highly unstable to air but protected in the cell at pH̃ 8 by virtue of a luciferin binding protein (LBP). This molecule is a dimer of 72 kDa subunits which, upon a decrease in pH, releases luciferin to react with oxygen in the luciferase (∼140 kDa) catalysed luminescent reaction. cDNAs for both luciferase and LBP have been isolated and cloned, and the identity of LBP was confirmed by hybrid selection and in vitro translation of the message. The tenfold circadian (day to night) change in the amount of LBP, which parallels the in vivo rhythm of luminescence, is due to de novo synthesis and subsequent degradation of the protein each day. The LBP mRNA levels, as determined by in vitro translations and by Northern hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated at the level of translation.
Bioluminescent click beetles revisited
Authors: Wood Keith V. , Lam Y. Amy , McElroy William D. , Seliger Howard H. 
Published: May 2005

AbstractIn studying beetle bioluminescence in the early 1960s, Dr McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light. They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases. We have recently cloned cDNAs from this beetle species which code for at least four different luciferases. The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli. The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few. Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.
High specific activity chemiluminescent and fluorescent markers: Their potential application to high sensitivity and ‘multi‐analyte’ immunoassays
Authors: Ekins Roger , Chu Frederick , Micallef Jacob 
Published: May 2005

AbstractThe sensitivities of immunoassays relying on conventional radioisotopic labels (i.e. radioimmunoassay (RIA) and immunoradiometric assay (IRMA)) permit the measurement of analyte concentrations above ca 107 molecules/ml. This limitation primarily derives, in the case of ‘competitive’ or ‘limited reagent’ assays, from the manipulation errors arising in the system combined with the physicochemical characteristics of the particular antibody used; however, in the case of ‘non‐competitive’ systems, the specific activity of the label may play a more important constraining role. It is theoretically demonstrable that the development of assay techniques yielding detection limits significantly lower than 107 molecules/ml depends on:

the adoption of ‘non‐competitive’ assays designs;
the use of labels of higher specific activity than radioisotopes;
highly efficient discrimination between the products of the immunological reactions involved.
Chemiluminescent and fluorescent substances are capable of yielding higher specific activities than commonly used radioisotopes when used as direct reagent labels in this context, and both thus provide a basis for the development of ‘ultra‐sensitive’, non‐competitive, immunoassay methodologies. Enzymes catalysing chemiluminescent reactions or yielding fluorescent reaction products can likewise be used as labels yielding high effective specific activities and hence enhanced assay sensitivities.A particular advantage of fluorescent labels (albeit one not necessarily confined to them) lies in the possibility they offer of revealing immunological reactions localized in ‘microspots’ distributed on an inert solid support. This opens the way to the development of an entirely new generatio of ‘ambient analyte’ microspot immunoassays perrnitting the simultaneous measurement of tens or even hundreds of different analytes in the same small sample, using (for example) laser scanning techniques. Early experience suggests that microspot assays with sensitivities surpassing that of isotopically based methodologies can readily be developed.
1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays
Authors: Bronstein Irena , Edwards Brooks , Voyta John C. 
Published: May 2005

AbstractWe have synthesized and studied two 1,2‐dioxetane‐based chemiluminescent enzyme substrates: 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(3″‐phosphoryloxy)phenyl‐1,2‐dioxetane (AMPPD), and, 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐ (3″‐β‐D′‐galactopyrano‐yloxy)phenyl‐1,2‐dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD‐galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which ‘stabilizes’ the dephosphorylated AMPPD emitter.Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer.AMPPD and AMPGD offer alternatives to colorimetric and fluorescent subsrates for alkaline phosphatase and β‐D‐galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β‐hCG, LH, TSH and others).
The development of non‐separation time‐resolved fluoroimmunoassays for the measurement of urinary metabolites
Authors: Barnard Geoff , Kohen Fortune , Mikola Heikki , Lovgren Timo 
Published: May 2005

AbstractWe describe the principles of a new generation of sequential or simultaneous time‐resolved fluoroimmunoassays, namely, simple, rapid, liquid‐phase non‐separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone‐3‐glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone‐3‐glucuronide. The antibody‐binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone‐3‐glucuronyl‐6‐BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody‐free label is measured in a time‐resolved fluorometer and is proportional to the concentration of estrone‐3‐glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.
A new luminescence immunoassay for thyrotropin using coated tubes: Evaluation and comparison with immunoradiometric assay
Authors: Pilo A. , Zucchelli G. C. , Ferdeghini M. , Masini S. , Calvo S. 
Published: May 2005

AbstractA recently available chemiluminescence immunoassay (LIA‐MAT) for TSH, set up by Byk‐Sangtec Diagnostica, has been evaluated and compared with IRMA methods. The system LIA‐MAT uses two monoclonal antibodies: one coated on tubes and the other labelled with isoluminol. To compute the within‐assay precision profile, we estimated the response error relationship from all the duplicates (420) of eleven experiments. The CV of the response was 4–5% from the maximal response until 10,000 RLU (corresponding to about 1 μlU/ml); in the lower response range the CV worsened up to 8%. The sensitivity, derived from the precision profile, was 0.052 μlU/ml similar to that found in IRMA‐MAT (0.044 μlU/ml); the working range extended from 0.33 to 100 μlU/ml. Results from LIA‐MAT in the concentration range 1–30 μlU/ml were compared with the consensus mean produced by users of IRMA methods (IRMA‐MAT, IRMA‐Behring, Maiaclone Serono) participating in an inter‐laboratory survey; a good correlation with IRMA techniques was found. The distribution of TSH determinations produced by LIA‐MAT on 62 low concentration sample (<0.3 μlU/ml) from patients unresponsive to TRH test, was found similar to that observed for the kit IRMA Boots‐Celltech assumed as reference.
Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation
Authors: Cadenas Enrique 
Published: May 2005

AbstractThe formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the OO bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H2O2by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl‐hydroxo complexes, as that formed during the oxidation of metmyoglobin by H2O2, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.
Formation of electronically excited states during the interaction ofp‐benzoquinone with hydrogen peroxide
Authors: Brunmark Anders 
Published: May 2005

AbstractThe reaction betweenp‐benzoquinone and H2O2in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3‐epoxy‐p‐benzoquinone, (b) 2‐hydroxy‐p‐benzoquinone and (c)p‐benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2‐hydroxy‐p‐benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C2C3double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in thep‐benzoquinone molecule leading to a decrease in the half‐wave potential, as compared to the parentp‐benzoquinone.The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2‐hydroxy‐p‐benzosemiquinone, H2O2and hydroxyl radical. The process involves the primary oxidation of 2‐hydroxy‐p‐benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10−8photons/2‐hydroxy‐p‐benzoquinone molecule formed.Photoemission arising from thep‐benzoquinone/H2O2reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2‐glutathionyl‐p‐benzohydroquinone adduct, involving thioether α‐hydrogen atom‐transfer to the triplet ketone.
Low‐level chemiluminescence during lipid peroxidations and enzymatic reactions
Authors: Nakano Minoru 
Published: May 2005

AbstractLow‐level chemiluminescence during lipid peroxidation and enzymatic reaction have been analysed by a filter type spectrometer. Tyrosine and tryprophan residues in proteins were found to be emitters in the visible region during their enzymatic oxidation. The natural chemiluminescence from fertilization of sea urchin eggs was found to have originated from tyrosine – cation radical mediated reaction in ovo‐peroxidase – membrane protein – H2O2 system.
The lipoxygenase pathway and chemiluminescence in horse eosinophilic leukocytes
Authors: Müller T. , Chavaillaz P. A. , Jörg A. , Grob M. , Peterhans E. 
Published: May 2005

AbstractIt was shown in several cell types that the dual lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid but not the cyclooxygenase inhibitor acetylsalicylic acid suppressed luminol‐dependent chemiluminescence. Since lipoxygenase is known to generate chemiluminescence in vitro, these observations were interpreted as evidence for a direct contribution of the lipoxygenase pathway to light emission in intact cells.We have investigated a possible contribution of the lipoxygenase to the chemiluminescence of horse eosinophils by directly comparing the formation of the byproduct chemiluminescence with the formation of stable end‐products of the lipoxygenase pathway, leukotrienes and HETEs. Azide as well as eicosatetraynoic acid almost completely inhibited chemiluminescence stimulated by the calcium ionophore A23187 but had less effect on the formation of leukotrienes. The tumour‐promoting ester, phorbol myristate acetate, stimulated chemiluminescence in an azide‐ and eicosatetraynoic acid‐sensitive manner and failed to evoke the production of leukotrienes. Azide, but also eicosatetraynoic acid inhibited the luminol‐dependent chemiluminescence generated by isolated eosinophil peroxidase in the presence of H2O2.Our results argue against a direct role of the lipoxygenase pathway in the generation of light in horse eosinophilic leukocytes but do not exclude that product(s) of this pathway may be involved in stimulus‐response coupling.
Introduction to beetle luciferases and their applications
Authors: Wood Keith V. , Lam Y. Amy , McElroy William D. 
Published: May 2005

AbstractAll beetle luciferases have evolved from a common ancestor: they all use ATP, O2, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.
The regulatory control of the bacterial luminescence system—A new view
Authors: Ulitzur S. 
Published: May 2005

AbstractWe have recently shown that the transcription of the PR lux operon for Vibrio fischeri luminescence is positively controlled by the htpR (σ32) protein. It was suggested that the LexA protein might negatively control the lux genes. This paper extends these findings. It was found that Escherichia coli cells that contain the entire lux operon (pChv1) in RecA or LexA mutants which are unable to remove the LexA protein are considerable dimmer than the wild‐type strain. Mutants that do not make LexA or from a weakly bound LexA are very bright. The role of σ32 protein was studied on luxR‐luxl genes that are fused to β‐galactosidase. The addition of V. fischeri inducer brings about the formation of β‐galactosidase activity in htpR+ but not in htpR− strains of E. coli/pMJ3. Similar to the effect of starvation on the induction of luminescence in marine bacteria and in E. coli/pChv1 cells, β‐galactrosidase activity in such constructs is preferentially induced by low nutrient concentrations. A new model for the regulatory control of the V. fischeri luminescence system is discussed.
Mutants of luminous bacteria selected for bioluminescent toxicity tests
Authors: Wagner A. , Winkler U. , Lümmen P. 
Published: May 2005

AbstractMutants of the luminescent bacterial strain NRRL B‐11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS‐PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs‐mutants was altered. QSAR analysis showed that the inhibitory effect of chloro‐substituted phenols on bioluminescence of the ahs‐mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs‐mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.