Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.
AuthorsMontero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R
JournalEMBO J
PubMed ID8521803
'Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera ... More
Differential gel electrophoresis and transgenic mitochondrial calcium reporters demonstrate spatiotemporal filtering in calcium control of mitochondria.
AuthorsTerhzaz S, Southall TD, Lilley KS, Kean L, Allan AK, Davies SA, Dow JA
JournalJ Biol Chem
PubMed ID16670086
'Mitochondria must adjust both their intracellular location and their metabolism in order to balance their output to the needs of the cell. Here we show by the proteomic technique of time series difference gel electrophoresis that a major result of neuroendocrine stimulation of the Drosophila renal tubule is an extensive ... More
Crystal structure of a Ca2+-discharged photoprotein: implications for mechanisms of the calcium trigger and bioluminescence.
AuthorsDeng L, Markova SV, Vysotski ES, Liu ZJ, Lee J, Rose J, Wang BC
JournalJ Biol Chem
PubMed ID15155735
'Ca2+-regulated photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have postulated mechanisms for ... More
Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.
AuthorsBerestovskaya NG, Shaloiko LA, Gorokhovatsky AY, Bondar VS, Vysotski ES, Maximov JE, von Doehren H, Alakhov YB
JournalAnal Biochem
PubMed ID10036164
'Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in ... More
Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector.
AuthorsRembold CM, Kendall JM, Campbell AK
JournalCell Calcium
PubMed ID9056079
'The physiologic relevance of Ca2+ release from the sarcoplasmic reticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmic reticulum free [Ca2+] (i.e. [Ca2+]sr) in the intact rat tail artery. We exploited a novel technique to measure [Ca2+]sr with genetically targeted apoaequorin acting as ... More
Differential regulation of nuclear and cytosolic Ca2+ in HeLa cells.
AuthorsBadminton MN, Campbell AK, Rembold CM
JournalJ Biol Chem
PubMed ID8940122
'The results reported in this study address the controversial issue that nuclear free Ca2+ ([Ca2+]n) may be regulated independently of cytosolic free Ca2+ ([Ca2+]c). We have measured [Ca2+]n and [Ca2+]c with recombinant aequorin targeted to the nucleus and cytosol in HeLa cells. We found that histamine, ATP, and ionomycin increased ... More
Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils.
AuthorsLucas M, Solano F
JournalAnal Biochem
PubMed ID1332545
'The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in ... More
Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.
AuthorsWang Y, Yu YA, Shabahang S, Wang G, Szalay AA
JournalMol Genet Genomics
PubMed ID12395190
'Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the ... More
Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase.
'In this report we show that ubiquinone cytochrome c reductase (complex III) from isolated rat heart mitochondria when inhibited with antimycin A, produces a large amount of superoxide as measured by the chemiluminescent probe coelenterazine. When mitochondria are inhibited with myxothiazol or stigmatellin, there is no detectable formation of superoxide. ... More
Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.
AuthorsCasadei J, Powell MJ, Kenten JH
JournalProc Natl Acad Sci U S A
PubMed ID2315301
'A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in ... More
Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin.
AuthorsWatanabe M, Kurihara S, Inouye S, Ohno T
JournalFEBS Lett
PubMed ID2468529
'Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. ... More
Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling.
AuthorsChiesa A, Rapizzi E, Tosello V, Pinton P, de Virgilio M, Fogarty KE, Rizzuto R
JournalBiochem J
PubMed ID11256942
'Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene ... More
Chemiluminescent detection of oxidants in vascular tissue. Lucigenin but not coelenterazine enhances superoxide formation.
AuthorsTarpey MM, White CR, Suarez E, Richardson G, Radi R, Freeman BA
JournalCirc Res
PubMed ID10347095
'Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to ... More
Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium.
AuthorsKnight MR, Campbell AK, Smith SM, Trewavas AJ
JournalNature
PubMed ID1865907
'Methods for measuring plant cytoplasmic calcium using microelectrodes or microinjected fluorescent dyes are associated with extensive technical problems, so measurements have been limited to single or small groups of cells in tissue strips or protoplasts. Aequorin is a calcium-sensitive luminescent protein from the coelenterate Aequorea victoria (A. forskalea) which is ... More
Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.
AuthorsPadua RA, Baron KT, Thyagarajan B, Campbell C, Thayer SA
JournalAm J Physiol
PubMed ID9530092
'Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization ... More
Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin.
AuthorsXiong ZH, Ruben L
JournalMol Biochem Parasitol
PubMed ID9010842
'The following study was undertaken to determine if calcium ions move from the plasma membrane to the nucleus of Trypanosoma brucei. Nuclear and cytosolic calcium flux was measured with the calcium sensitive photoprotein, aequorin which was targeted to various locations in stably transformed procyclic cells. Immunoblots revealed that the recombinant ... More
Changes in free calcium in the endoplasmic reticulum of living cells detected using targeted aequorin.
AuthorsKendall JM, Badminton MN, Dormer RL, Campbell AK
JournalAnal Biochem
PubMed ID7985790
'The Ca(2+)-activated photoprotein aequorin has been engineered with the endoplasmic reticulum (ER)-targeting sequence from calreticulin at the N-terminus and the KDEL sequence at the C-terminus so that it locates in the ER of living cells. Targeting of apoaequorin to the ER of COS7 cells was demonstrated by immunolocalization. Selective permeabilization ... More
Genetically engineered obelin as a bioluminescent label in an assay for a peptide.
AuthorsMatveev SV, Lewis JC, Daunert S
JournalAnal Biochem
PubMed ID10328766
'The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation ... More
Violet bioluminescence and fast kinetics from W92F obelin: structure-based proposals for the bioluminescence triggering and the identification of the emitting species.
AuthorsVysotski ES, Liu ZJ, Markova SV, Blinks JR, Deng L, Frank LA, Herko M, Malikova NP, Rose JP, Wang BC, Lee J
JournalBiochemistry
PubMed ID12755603
'Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation ... More
Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin.
AuthorsWatkins NJ, Knight MR, Trewavas AJ, Campbell AK
JournalBiochem J
PubMed ID7702585
'Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli. However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established. In order to monitor internal free Ca2+ in E. coli during challenge with chemotactic agents, the ... More
The origins of marine bioluminescence: turning oxygen defence mechanisms into deep-sea communication tools.
AuthorsRees JF, de Wergifosse B, Noiset O, Dubuisson M, Janssens B, Thompson EM
JournalJ Exp Biol
PubMed ID9510532
'Bioluminescence, the emission of ecologically functional light by living organisms, emerged independently on several occasions, yet the evolutionary origins of most bioluminescent systems remain obscure. We propose that the luminescent substrates of the luminous reactions (luciferins) are the evolutionary core of most systems, while luciferases, the enzymes catalysing the photogenic ... More
Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme.
AuthorsMarkova SV, Golz S, Frank LA, Kalthof B, Vysotski ES
JournalJ Biol Chem
PubMed ID14583604
'Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned ... More
Bioluminescence in the sea: photoprotein systems.
AuthorsShimomura O
JournalSymp Soc Exp Biol
PubMed ID2871634
'Photoproteins are the primary reactants of the light-emitting reactions of various bioluminescent organisms. A photoprotein emits light in proportion to its amount, like a luciferin, but its light-emitting reaction does not require a luciferase. There are about two dozen types of bioluminescent organisms for which substantial biochemical knowledge is presently ... More
Chemiluminescence microscopy as a tool in biomedical research.
AuthorsCréton R, Jaffe LF
JournalBiotechniques
PubMed ID11730016
'Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress ... More
Targeting aequorin and green fluorescent protein to intracellular organelles.
AuthorsDe Giorgi F, Brini M, Bastianutto C, Marsault R, Montero M, Pizzo P, Rossi R, Rizzuto R
JournalGene
PubMed ID8707049
'Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca(2+)-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of ... More
Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins.
AuthorsKnight MR, Read ND, Campbell AK, Trewavas AJ
JournalJ Cell Biol
PubMed ID8458875
'The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, ... More
Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.
AuthorsWatkins NJ, Campbell AK
JournalBiochem J
PubMed ID8101077
'cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal ... More
Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments.
AuthorsMartin PE, George CH, Castro C, Kendall JM, Capel J, Campbell AK, Revilla A, Barrio LC, Evans WH
JournalJ Biol Chem
PubMed ID9430718
'Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of ... More
Mn(2+)-activated luminescence of the photoprotein obelin.
AuthorsVysotski ES, Trofimov CP, Bondar VS, Frank LA, Markova SV, Illarionov BA
JournalArch Biochem Biophys
PubMed ID7840683
'The light emission of obelin may be initiated by Mn2+ under alkaline conditions. The luminescence takes place in a pH range from 7 to 12 with a sharp optimum at 11.75. The first-order rate constant for Mn(2+)-activated luminescence decay is more than 9 s-1, while that for Ca(2+)-activated luminescence decay ... More
A noncommercial dual luciferase enzyme assay system for reporter gene analysis.
AuthorsDyer BW, Ferrer FA, Klinedinst DK, Rodriguez R
JournalAnal Biochem
PubMed ID10860516
Red-shifted Renilla reniformis luciferase variants for imaging in living subjects.
AuthorsLoening AM, Wu AM, Gambhir SS
JournalNat Methods
PubMed ID17618292
'The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm ... More
Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase(1).
AuthorsInouye S, Watanabe K, Nakamura H, Shimomura O
JournalFEBS Lett
PubMed ID10984608
'The deep-sea shrimp Oplophorus gracilirostris secretes a luciferase that catalyzes the oxidation of coelenterazine to emit blue light. The luciferase (M(r) approx. 106000) was found to be a complex composed of 35 kDa and 19 kDa proteins, and the cDNAs encoding these two proteins were cloned. The expression of the ... More
Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin.
AuthorsCubitt AB, Firtel RA, Fischer G, Jaffe LF, Miller AL
JournalDevelopment
PubMed ID7671796
'To examine the patterns of high free cytosolic calcium or [Ca2+]i during Dictyostelium''s development, we expressed apoaequorin in D. discoideum, reconstituted aequorin and observed the resultant patterns of calcium-dependent luminescence. Specific, high calcium zones are seen throughout normal multicellular development and are roughly coincident with those regions that later differentiate ... More
Endothelin-1 induces NAD(P)H oxidase in human endothelial cells.
AuthorsDuerrschmidt N, Wippich N, Goettsch W, Broemme HJ, Morawietz H
JournalBiochem Biophys Res Commun
PubMed ID10720482
'Superoxide anions (O(*-)(2)) induce oxidative stress and reduce endothelial NO availability by peroxynitrite formation. In human endothelial cells gp91(phox) was identified as the limiting subunit of the forming NAD(P)H oxidase. Because endothelin-1 (ET-1) is considered as a pro-atherosclerotic stimulus, we analyzed the effect of ET-1 on gp91(phox) expression and O(*-)(2) ... More
Light-emitters involved in the luminescence of coelenterazine.
AuthorsShimomura O, Teranishi K
JournalLuminescence
PubMed ID10660666
'Coelenterazine emits light by chemi-and bioluminescence reactions, decomposing into coelenteramide and CO(2). To ascertain the light emitters involved, the fluorescence of coelenteramide and five analogues were studies in four kinds of solvent. The results showed that coelenteramides can form five kinds of light emitters, ie unionized (lambda(max) 386-423 nm), phenolate ... More
Salicylic acid induces a cytosolic Ca2+ elevation in yeast.
AuthorsMori IC, Iida H, Tsuji FI, Isobe M, Uozumi N, Muto S
JournalBiosci Biotechnol Biochem
PubMed ID9648231
'Cytosolic free calcium ion concentration ([Ca2+]cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca(2+)-sensitive luminescent protein, aequorin, when combined with coelenterazine. SA induced a transient [Ca2+]cyt elevation that was dependent on the concentration of SA and pH of ... More
The Golgi apparatus plays a significant role in the maintenance of Ca2+ homeostasis in the vps33Delta vacuolar biogenesis mutant of Saccharomyces cerevisiae.
AuthorsMiseta A, Fu L, Kellermayer R, Buckley J, Bedwell DM
JournalJ Biol Chem
PubMed ID10026219
'The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range. In this study, we examined how cellular Ca2+ homeostasis is maintained in a vps33Delta vacuolar biogenesis mutant. We found that growth of the vps33Delta strain was ... More
Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals.
AuthorsZhao H, Doyle TC, Wong RJ, Cao Y, Stevenson DK, Piwnica-Worms D, Contag CH
JournalMol Imaging
PubMed ID15142411
'In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which ... More
Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley.
AuthorsBlume B, Nürnberger T, Nass N, Scheel D
JournalPlant Cell
PubMed ID10948260
'Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined ... More
Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells.
AuthorsRutter GA, White MR, Tavaré JM
JournalCurr Biol
PubMed ID7583147
'BACKGROUND: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis ... More
Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.
AuthorsDaiber A, August M, Baldus S, Wendt M, Oelze M, Sydow K, Kleschyov AL, Munzel T
JournalFree Radic Biol Med
PubMed ID14732294
'In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, ... More
Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity.
AuthorsSouza HP, Souza LC, Anastacio VM, Pereira AC, Junqueira ML, Krieger JE, da Luz PL, Augusto O, Laurindo FR
JournalFree Radic Biol Med
PubMed ID10889453
'Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that ... More
Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification.
AuthorsKurose K, Inouye S, Sakaki Y, Tsuji FI
JournalProc Natl Acad Sci U S A
PubMed ID2643108
'Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the ... More
Locating a protein-protein interaction in living cells via split Renilla luciferase complementation.
AuthorsKaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y,
JournalAnal Chem
PubMed ID14632132
'For spatial and quantitative kinetic analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence using a cell membrane permeable substrate, coelenterazine. This split Renilla luciferase complementation readout was shown to work for locating ... More
Isolation and expression of a cDNA encoding Renilla reniformis luciferase.
AuthorsLorenz WW, McCann RO, Longiaru M, Cormier MJ
JournalProc Natl Acad Sci U S A
PubMed ID1674607
'Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which ... More
Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.
AuthorsRizzuto R, Brini M, Murgia M, Pozzan T
JournalScience
PubMed ID8235595
'Microdomains of high intracellular calcium ion concentration, [Ca2+]i, have been hypothesized to occur in living cells exposed to stimuli that generate inositol 1,4,5-trisphosphate (IP3). Mitochondrially targeted recombinant aequorin was used to show that IP3-induced Ca2+ mobilization from intracellular stores caused increases of mitochondrial Ca2+ concentration, [Ca2+]m, the speed and amplitude ... More
The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.
AuthorsShimomura O, Inouye S
JournalProtein Expr Purif
PubMed ID10336865
'Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in ... More
Low-pH-mediated elevations in cytosolic calcium are inhibited by aluminium: a potential mechanism for aluminium toxicity.
'Aluminium, the most abundant metal in the earth''s crust, is highly toxic to most plant species. One of the prevailing dogmas is that aluminium exerts this effect by disrupting cellular calcium homeostasis. However, recent research gives strongly conflicting results: aluminium was shown to provoke either an increase or a decrease ... More
Receptor for motilin identified in the human gastrointestinal system.
AuthorsFeighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, MacNeil D, Cascieri MA, Nargund R, Bakshi R, Abramovitz M, Stocco R, Kargman S, O'Neill G, Van Der Ploeg LH, Evans J, Patchett AA, Smith RG, Howard AD
JournalScience
PubMed ID10381885
'Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was ... More
Activity screening of bacteria containing Renilla luciferase plasmids.
AuthorsJubin R, Murray MG
JournalBiotechniques
PubMed ID9494710
Photoproteins as indicators of intracellular free Ca2+.
AuthorsCampbell AK, Patel A, Houston WA, Scolding NJ, Frith S, Morgan BP, Compston DA
JournalJ Biolumin Chemilumin
PubMed ID2801233
Recombinant aequorin methods for intracellular calcium measurement in plants.
AuthorsKnight H, Knight MR
JournalMethods Cell Biol
PubMed ID8531755
Chemiluminescent and bioluminescent reporter gene assays.
AuthorsBronstein I, Fortin J, Stanley PE, Stewart GS, Kricka LJ
JournalAnal Biochem
PubMed ID8080073
Shining the light: the mechanism of the bioluminescence reaction of calcium-binding photoproteins.
AuthorsOhmiya Y, Hirano T
JournalChem Biol
PubMed ID8807862
The Ca2+-binding photoproteins from jellyfish have the unique ability to emit blue light in the presence of calcium ions but without molecular oxygen or any other cofactor. Although there is no crystallographic data on the structure of the photoprotein complex, structure-activity studies have elucidated many features of the complex and ... More
Quenching of biotinylated aequorin bioluminescence by dye-labeled avidin conjugates: application to homogeneous bioluminescence resonance energy transfer assays.
AuthorsAdamczyk M, Moore JA, Shreder K
JournalOrg Lett
PubMed ID11405714
[see reaction]. Avidin conjugates containing the covalently attached dyes QSY-7 and dabcyl were prepared and shown to quench the bioluminescence of biotinylated aequorin. Quenching efficiency was shown to be dependent on both the label-to-avidin ratio and the concentration of the avidin conjugate. These properties were exploited to develop a homogeneous ... More
Glowing jellyfish, luminescence and a molecule called coelenterazine.
AuthorsJones K, Hibbert F, Keenan M
JournalTrends Biotechnol
PubMed ID10557160
Luminescence has assumed an important role in analytical biochemistry and molecular biology as an extremely sensitive method for determining the concentration of specific ions and molecules. The luminescent system of the jellyfish Aequorea victoria consists of the photoprotein aequorin, which contains the molecule coelenterazine as a prosthetic group and shows ... More
Protective effect of imidazolopyrazinone antioxidants on ischemia/reperfusion injury.
AuthorsArrault A, Dubuisson M, Gharbi S, Marchand C, Verbeuren T, Rupin A, Cordi A, Bouskela E, Rees JF, Marchand-Brynaert J
JournalBioorg Med Chem Lett
PubMed ID12639551
A series of 2-substituted 3,7-dihydroimidazolo[1,2-a]pyrazine-3-ones has been synthesized and evaluated for their antioxidant activity. Compounds 1-8 are inhibitors of AAPH-induced lipid peroxidation (in vitro) and excellent protectors against microvascular damages in ischemia/reperfusion (in vivo). Hence, the bicyclic structure typical of coelenterazine (luciferin) could be considered as a useful lead in ... More
Targeting aequorin to the endoplasmic reticulum of living cells.
AuthorsKendall JM, Dormer RL, Campbell AK
JournalBiochem Biophys Res Commun
PubMed ID1472014
The photoprotein aequorin has been engineered with an ER targeting sequence at the N-terminus, with and without KDEL at the C-terminus, so that it locates in the ER-secretory pathway. For the first time the free Ca2+ has been quantified inside the ER and shown to be 5-20 times that in ... More
Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli.
AuthorsKnight MR, Campbell AK, Smith SM, Trewavas AJ
JournalFEBS Lett
PubMed ID2037058
We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ([Ca2+]i) using recombinant aequorin reconstituted within live bacterial cells. Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the [Ca2+]i of Escherichia coli. In principle this method should ... More
Targeting of aequorin for calcium monitoring in intracellular compartments.
AuthorsBrini M, Pasti L, Bastianutto C, Murgia M, Pozzan T, Rizzuto R
JournalJ Biolumin Chemilumin
PubMed ID7942123
We have recently developed a new method for monitoring Ca2+ concentrations in defined cell compartments. The cDNA encoding the Ca(2+)-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 ... More
Preparation and handling of aequorin solutions for the measurement of cellular Ca2+.
AuthorsShimomura O
JournalCell Calcium
PubMed ID1959127
Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing ... More
Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells.
AuthorsKendall JM, Badminton MN, Sala-Newby GB, Campbell AK, Rembold CM
JournalBiochem J
PubMed ID8809023
We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1-100 microM. In intact HeLa cells, addition of ionomycin increased ... More
Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations.
AuthorsTarpey MM, Wink DA, Grisham MB
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID14761864
Facile detection of reactive oxygen and nitrogen species in biologic systems is often problematic. This is a result of the numerous cellular mechanisms, both enzymatic and nonenzymatic involved in their catabolism/decomposition, the complex and overlapping nature of their reactivities, as well as the often limited intracellular access of detector systems. ... More
Engineering the CA(2+)-activated photoprotein aequorin with reduced affinity for calcium.
AuthorsKendall JM, Sala-Newby G, Ghalaut V, Dormer RL, Campbell AK
JournalBiochem Biophys Res Commun
PubMed ID1530606
Two stage PCR has been used to introduce single amino acid substitutions into the EF hand structures of the Ca(2+)-activated photoprotein aequorin. Transcription of PCR products, followed by cell free translation of the mRNA, allowed characterisation of recombinant proteins in vitro. Substitution of D to A at position 119 produced ... More
Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin.
AuthorsMasuda H, Takenaka Y, Shikamoto Y, Kagawa M, Mizuno H, Tsuji FI
JournalProtein Expr Purif
PubMed ID14550635
Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, ... More
Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein.
AuthorsPrasher D, McCann RO, Cormier MJ
JournalBiochem Biophys Res Commun
PubMed ID2579647
Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen. Aequorin produces blue light upon binding Ca2+. We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic oligonucleotide ... More
Differences in stability of recombinant apoaequorin within subcellular compartments.
AuthorsBadminton MN, Sala-Newby GB, Kendall JM, Campbell AK
JournalBiochem Biophys Res Commun
PubMed ID8554620
Recombinant acquorin is widely used as an intracellular Ca2+ indicator within live cells. Our data shows that recombinant apoacquorin was unstable within the cytosol, with a half life of approximately 20 minutes. Targeting of the protein to subcellular organelles resulted in an increase in stability which may be due to ... More
The moss, Physcomitrella patens, transformed with apoaequorin cDNA responds to cold shock, mechanical perturbation and pH with transient increases in cytoplasmic calcium.
The gene for apoaequorin has been used previously to indicate cytosolic calcium changes in higher plants. Here we report the transformation of the moss Physcomitrella patens with the cDNA for apoaequorin. Stable transformants were obtained in the wild type which reconstitute the calcium-sensitive luminescent protein aequorin in vivo after incubation ... More
Cloning and sequence analysis of cDNA for the luminescent protein aequorin.
AuthorsInouye S, Noguchi M, Sakaki Y, Takagi Y, Miyata T, Iwanaga S, Miyata T, Tsuji FI
JournalProc Natl Acad Sci U S A
PubMed ID3858813
The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic ... More
Aequorea victoria bioluminescence moves into an exciting new era.
AuthorsKendall JM, Badminton MN
JournalTrends Biotechnol
PubMed ID9621461
Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellular signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins involved in bioluminescence of the jellyfish Aequorea victoria--aequorin and green fluorescent protein. Engineered recombinant aequorin has led to a novel ... More
Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.
AuthorsShimomura O, Musicki B, Kishi Y, Inouye S
JournalCell Calcium
PubMed ID8519061
15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa ... More
Simultaneous indirect activity measurements of GH and PRL genes in the same, living mammosomatotrope.
AuthorsWillard ST, Amstutz MD, Abraham EJ, Castaño JP, Leaumont DC, Faught WJ, Frawley LS
JournalAm J Physiol
PubMed ID10600806
Dynamic intracellular processes in endocrine cells are usually controlled by the coordinated modulation of two or more functionally related genes. Attempts to gain a more complete understanding of these processes would be facilitated greatly by a method enabling activity measurements of two genes at the same time. Here we describe ... More
Membrane permeability of coelenterazine analogues measured with fish eggs.
AuthorsShimomura O
JournalBiochem J
PubMed ID9291095
To determine the suitability of various coelenterazine analogues for the regeneration of aequorin in living cells, the membrane permeabilities of 11 analogues were measured using the eggs of the killifish Fundulus grandis by soaking the eggs in solutions containing the analogues. The results indicated that e-coelenterazine, which has an exceptionally ... More
Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.
Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. ... More
Characterization of a chimeric aequorin molecule expressed in myeloma cells.
AuthorsCasadei J, Powell MJ, Kenten JH
JournalJ Biolumin Chemilumin
PubMed ID2801222
We have constructed a chimeric aequorin consisting of a fragment of the anti-NP immunoglobulin gene fused to the aequorin gene. Expression in a myeloma cell line has produced a Fab'-like molecule that has the ability to bind NIP specifically and generate bioluminescent activity. It takes approximately 8 h at 4 ... More
Can coelenterates make coelenterazine? Dietary requirement for luciferin in cnidarian bioluminescence.
AuthorsHaddock SH, Rivers TJ, Robison BH
JournalProc Natl Acad Sci U S A
PubMed ID11572972
In the calcium-activated photoprotein aequorin, light is produced by the oxidation of coelenterazine, the luciferin used by at least seven marine phyla. However, despite extensive research on photoproteins, there has been no evidence to indicate the origin of coelenterazine within the phylum Cnidaria. Here we report that the hydromedusa Aequorea ... More
Yeast respond to hypotonic shock with a calcium pulse.
AuthorsBatiza AF, Schulz T, Masson PH
JournalJ Biol Chem
PubMed ID8798538
We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in ... More
Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes.
AuthorsKervinen M, Pätsi J, Finel M, Hassinen IE
JournalAnal Biochem
PubMed ID14654044
The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex ... More
Preparation and X-ray crystallographic analysis of the Ca2+-discharged photoprotein obelin.
AuthorsDeng L, Markova SV, Vysotski ES, Liu ZJ, Lee J, Rose J, Wang BC
JournalActa Crystallogr D Biol Crystallogr
PubMed ID14993677
Ca(2+)-regulated photoproteins belong to the EF-hand Ca(2+)-binding protein family. The addition of calcium ions initiates bright blue bioluminescence of the photoproteins, a result of the oxidative breakdown of coelenterazine peroxide to coelenteramide. Crystals of the Ca(2+)-discharged W92F mutant of obelin from Obelia longissima have been grown, representing the first crystallization ... More
Obelin mRNA--a new tool for studies of translation in cell-free systems.
Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system. Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific ... More
Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin.
AuthorsSanchez-Bueno A, Yoshida R, Tsuji FI
JournalInt J Biochem Cell Biol
PubMed ID8930127
Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary ... More
NADPH oxidase contributes to coronary endothelial dysfunction in the failing heart.
AuthorsZhang P, Hou M, Li Y, Xu X, Barsoum M, Chen Y, Bache RJ,
JournalAm J Physiol Heart Circ Physiol
PubMed ID19168727
Increased reactive oxygen species (ROS) produced by the failing heart can react with nitric oxide (NO), thereby decreasing NO bioavailability. This study tested the hypothesis that increased ROS generation contributes to coronary endothelial dysfunction in the failing heart. Congestive heart failure (CHF) was produced in six dogs by ventricular pacing ... More
Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA.
AuthorsSaran S, Nakao H, Tasaka M, Iida H, Tsuji FI, Nanjundiah V, Takeuchi I
JournalFEBS Lett
PubMed ID8276111
A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] ... More
Galactose-dependent expression of the recombinant Ca2(+)-binding photoprotein aequorin in yeast.
AuthorsNakajima-Shimada J, Iida H, Tsuji FI, Anraku Y
JournalBiochem Biophys Res Commun
PubMed ID1989594
Aequorin is a Ca2(+)-binding protein that emits light upon reacting with Ca2+ and has been used as a probe for monitoring changes in the intracellular free Ca2+ concentration, [Ca2+]i. The protein consists of three components: apoaequorin (apoprotein), molecular oxygen and a chromophore. The present study was designed to conditionally express ... More
Atomic resolution structure of obelin: soaking with calcium enhances electron density of the second oxygen atom substituted at the C2-position of coelenterazine.
AuthorsLiu ZJ, Vysotski ES, Deng L, Lee J, Rose J, Wang BC
JournalBiochem Biophys Res Commun
PubMed ID14592432
The spatial structure of the Ca(2+)-regulated photoprotein obelin has been solved to resolution of 1.1A. Two oxygen atoms are revealed substituted at the C2-position of the coelenterazine in contrast to the obelin structure at 1.73A resolution where one oxygen atom only was disclosed. The electron density of the second oxygen ... More
Mechanism of photoinactivation and re-activation in the bioluminescence system of the ctenophore Mnemiopsis.
AuthorsAnctil M, Shimomura O
JournalBiochem J
PubMed ID6147135
The bioluminescence of the ctenophore Mnemiopsis takes place when the photoprotein mnemiopsin in the photocytes reacts with Ca2+. The luminescence is inhibited in sunlight and this photoinhibition is reversible by keeping the live specimens in the dark. The extracts of mnemiopsin are similarly photoinhibited, but the photoinhibition cannot be reversed ... More
Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.
AuthorsInouye S, Tsuji FI
JournalAnal Biochem
PubMed ID1621948
A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by ... More
Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
AuthorsKotarsky K, Antonsson L, Owman C, Olde B
JournalAnal Biochem
PubMed ID12711342
Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently ... More
The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues.
AuthorsShimomura O, Kishi Y, Inouye S
JournalBiochem J
PubMed ID8280050
The regeneration of an active semi-synthetic aequorin, from apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic aequorins from apoaequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate ... More
Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells.
AuthorsPinton P, Ferrari D, Magalhães P, Schulze-Osthoff K, Di Virgilio F, Pozzan T, Rizzuto R
JournalJ Cell Biol
PubMed ID10704437
The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression ... More
Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells.
AuthorsSheu YA, Kricka LJ, Pritchett DB
JournalAnal Biochem
PubMed ID8470808
Changes in intracellular free calcium concentration ([Ca2+]i) are involved in many important physiological responses. Detecting changes in [Ca2+]i is crucial to understanding the physiologic roles of intracellular free calcium. We have characterized changes of [Ca2+]i in human cells transfected with apoaequorin cDNA. When reconstituted in vivo by incubating transfected cells ... More
Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.
AuthorsShimomura O, Musicki B, Kishi Y
JournalBiochem J
PubMed ID2803250
Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities ... More
Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.
AuthorsShimomura O, Inouye S, Musicki B, Kishi Y
JournalBiochem J
PubMed ID2400391
Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin ... More
Light production by the arm tips of the deep-sea cephalopod Vampyroteuthis infernalis.
AuthorsRobison BH, Reisenbichler KR, Hunt JC, Haddock SH
JournalBiol Bull
PubMed ID14583508
The archaic, deep-sea cephalopod Vampyroteuthis infernalis occurs in dark, oxygen-poor waters below 600 m off Monterey Bay, California. Living specimens, collected gently with a remotely operated vehicle (ROV) and quickly transported to a laboratory ashore, have revealed two hitherto undescribed means of bioluminescent expression for the species. In the first, ... More
Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.
AuthorsCampbell AK, Patel AK, Razavi ZS, McCapra F
JournalBiochem J
PubMed ID3421897
1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of ... More
Nucleoplasmin-targeted aequorin provides evidence for a nuclear calcium barrier.
AuthorsBadminton MN, Kendall JM, Sala-Newby G, Campbell AK
JournalExp Cell Res
PubMed ID7813626
The Ca(2+)-activated photoprotein aequorin has been targeted to the nucleus of live cells by engineering nucleoplasmin, a nuclear structural protein from Xenopus laevis, onto the amino terminus. Successful targeting of the apoprotein was demonstrated by immunolocalization and selective permeabilization of the plasma membrane. Functional protein was reconstituted in live cells ... More
A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway.
We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular ... More
Nuclear targeting of aequorin. A new approach for measuring nuclear Ca2+ concentration in intact cells.
AuthorsBrini M, Marsault R, Bastianutto C, Pozzan T, Rizzuto R
JournalCell Calcium
PubMed ID7820845
We here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca(2+)-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but ... More
Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae.
AuthorsForster C, Kane PM
JournalJ Biol Chem
PubMed ID10991947
The vacuole is the major site of intracellular Ca(2+) storage in yeast and functions to maintain cytosolic Ca(2+) levels within a narrow physiological range via a Ca(2+) pump (Pmc1p) and a H(+)/Ca(2+) antiporter (Vcx1p) driven by the vacuolar H(+)-ATPase (V-ATPase). We examined the function of the V-ATPase in cytosolic Ca(2+) ... More
Coelenterazine: a two-stage antioxidant in lipid micelles.
Authorsde Wergifosse B, Dubuisson M, Marchand-Brynaert J, Trouet A, Rees JF
JournalFree Radic Biol Med
PubMed ID15036347
Coelenterazine is a luciferin found in many marine bioluminescent organisms. This luciferin also possesses high antioxidant properties and an exceptional ability to protect cells exposed to oxidative stress. It has been suggested that coelenterazine's antioxidative mechanisms include the formation of an oxidation product, coelenteramine, also endowed with chain-breaking properties. In ... More