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Counterilluminating animals produce a ventral light to hide their silhouette in the water column. This midwater camouflage technique requires a fine and dynamic control of the wavelength, angular distribution, and intensity of their luminescence, which needs to continuously match ambient downwelling light. Recently, extraocular opsins have been suggested to play a role in the bioluminescence control of several organisms, such as squids, comb jellies, or brittle stars, providing a way for photogenic structures to perceive their own light output. By analysing a growing embryonic series of the velvet belly lanternshark, Etmopterus spinax, we show that the development of lanternshark luminescence competence is associated with the expression of encephalopsin within epidermal cells and in the light-regulating structure of the photogenic organs. Such an intra-uterine expression of encephalopsin strongly supports this blue-sensitive extraocular opsin to allow bioluminescence perception in lanternshark photophores and suggests a clear physiological interaction between photoemission and photoperception., Author(s): Laurent Duchatelet [sup.1] , Julien M. Claes [sup.1] , Jérôme Mallefet [sup.1] Author Affiliations: (Aff1) 0000 0001 2294 713X, grid.7942.8, Marine Biology Laboratory, Earth and Life Institute, Université Catholique [...]

Light plays a critical role in the functioning of the marine environment. In the dark ocean, bioluminescent organisms are the only visually relevant sources of light. Cameras of different sensitivities were used to compare the density of pelagic bioluminescent sources (BL) of different light intensities at a regional scale: the image-intensified charge-coupled device for deep-sea research (ICDeep), an image-intensified silicon intensifier target (ISIT) camera and a silicon intensifier target (SIT) camera. Pelagic ICDeep values were higher than ISIT measurements by a mean factor of 7.6 in the Mediterranean Sea and 3.5 in the Atlantic Ocean. Atlantic ISIT values were higher than SIT values by a mean factor of 4.5. Standardising bioluminescence measurements to the near-seafloor (0-400 m above bottom) layer, [BL.sub.NSF], a logarithmic decrease with depth was observed from three independent datasets (slopes not significantly different): ISIT (Atlantic, Mediterranean), ICDeep (Mediterranean). Intercepts from ICDeep measurements were higher than ISIT measurements by a factor of 4.4. From these trends, a conversion factor to calculate benthopelagic plankton biomass from near-seafloor [BL.sub.NSF] density was derived. Calibration of the ICDeep enabled calculation of the minimum intensity of source visible to that camera. [BL.sub.NSF] sources of low-light intensity (≥ 1.4 x [10.sup.-7] W [m.sup.-2]) outnumber fourfold sources of greater intensity (>ca. [10.sup.-6] W [m.sup.-2] ([λ.sub.peak] = 470 nm). This reveals a high abundance of low-light bioluminescent sources in the marine environment, with mean pelagic densities of 33.15 sources [m.sup.-3] (Atlantic) and 6.79 sources [m.sup.-3] (Mediterranean) between 500 and 1500 m depth., Introduction Bioluminescence is ubiquitous in the marine environment, with bioluminescent species in most of the major marine phyla (Herring 1987; Widder 2010). Bioluminescence is highly effective for signalling in water; […]

ABSTRACT Fluorescent molecules have revolutionized the field of molecular biology and biotechnology, and could be of benefit to research conducted on economically important haliotid species (abalone) for applications such as [...]

Living organisms produce hydrogen peroxide ([H.sub.2][O.sub.2]) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease, The effects of [H.sub.2][O.sub.2] on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of [H.sub.2][O.sub.2] within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with [H.sub.2][O.sub.2] to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for [H.sub.2][O.sub.2], combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of [H.sub.2][O.sub.2] generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in [H.sub.2][O.sub.2] levels by directly imaging elevations in [H.sub.2][O.sub.2] within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor [H.sub.2][O.sub.2] fluxes in real time in living animals offers opportunities to dissect [H.sub.2][O.sub.2]'s disparate contributions to health, aging, and disease. cancer | molecular imaging | redox biology doi/ 10.1073/pnas.1012864107

Microscale mechanical forces can determine important outcomes ranging from the site of material fracture to stem cell fate. However, local stresses in a vast majority of systems cannot be measured due to the limitations of current techniques. In this work, we present the design and implementation of the CdSe-CdS core-shell tetrapod nanocrystal, a local stress sensor with bright luminescence readout. We calibrate the tetrapod luminescence response to stress and use the luminescence signal to report the spatial distribution of local stresses in single polyester fibers under uniaxial strain. The bright stress-dependent emission of the tetrapod, its nanoscale size, and its colloidal nature provide a unique tool that may be incorporated into a variety of micromechanical systems including materials and biological samples to quantify local stresses with high spatial resolution. nanoscience | semiconductor nanocrystals | fluorescence | luminescent stress gauge doi/ 10.1073/pnas.1016022107

Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and D-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and D-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quanlitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects. 10.1021/ac102692u

We describe a novel multiplex 'amino acid array' for simultaneously quantifying different amino acids based on the rapid growth of amino acid auxotrophic E. coli First, we constructed genetically engineered amino acid auxotrophs of E. coli containing a bioluminescence reporter gene, yielding concomitant luminescence as a response to cell growth, and then immobilized the reporter cells within individual agarose of respective wells in a 96-well plate serving as a mimic of a biochip. Using the amino acid array, we were able to determine quantitatively the concentrations of 16 amino acids in biological fluid by simply measuring bioluminescent signals from the immobilized cells within 4 h without pre- and post-treatment. The clinical utility of this method was verified by quantifying different amino acids in dried blood spot specimens from clinical samples for the diagnosis of metabolic diseases of newborn babies. This method serves as a convenient route to the rapid and simultaneous analysis of multiple amino acids from complex biological fluids and represents a new analytical paradigm that can replace conventional, yet laborious methods currently in use. 10.1021/ac100087r

From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chemical generation of light) for finding food, attracting mates, and evading predators. Disparate biochemical systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecological benefits of bioluminescence, with biochemical and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, I review recent advances in understanding bioluminescence in the ocean and highlight future research efforts that will unite molecular details with ecological and evolutionary relationships. 10.1126/science.1174269

Conventional anticancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter antitumor drug activity. To address this limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (for example, myeloma, leukemia and solid tumors) stably expressing luciferase are cultured with nonmalignant accessory cells (for example, stromal cells) for selective quantification of tumor cell viability, in presence versus absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib resistance in leukemic cells. A stroma-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-κB, HIF-1α, myc, hTERT and IRF4; for biological aggressiveness; and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, shows more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anticancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stroma interactions., Tumor-stroma interactions are increasingly recognized as critical components of tumor biology (1), including invasion and metastatic potential (2). The interactions of bone marrow stromal cells (BMSCs) with tumor cells are [...]

We aimed to develop a far-red luminescence imaging technology for visualization of disease specific antigens on cell surfaces in a living body. First, we conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase. This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in the far-red region as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting and imaging capabilities of tumors, we then linked this conjugate to an anti-human DIk-1 monoclonal antibody via the biotin-avidin interaction. This far-red luminescent probe enabled us to obtain high-resolution microscopic images of live, DIk-1-expressing Huh-7 cells without an external light source, and to monitor the accumulation of this probe in tumor-bearing mice. Thus this far-red luminescent probe is a convenient analytical tool for the evaluations of monoclonal antibody localization in a living body. Cypridina luciferase | far-red luminescent probe | luciferin | tumor

The goal of the present study is to develop a small luminescence template, in which any protein may be incorporated, for illuminating the dynamics of protein signaling. We first determined optimal fragmentation sites of Gaussia princeps-derived luciferase (GLuc). The utility of the template was demonstrated with calmodulin (CAM) and a peptide-linked CAM, which were sandwiched between the fragments of split GLuc dissected at Q105. Living mammalian cells with the probe quickly emitted luminescence in response to endo- and exogenous [Ca.sup.2+] levels. The applicability of the template was expanded to the visualization of the phosphorylation of the estrogen receptor and interaction of the androgen receptor with a peptide via an intramolecular complementation between GLuc fragments. It also provides the smallest bioluminescence template ever synthesized. Considering that conformational changes of proteins generally occur for signal transductions, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins.

We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-[alpha] and angiotensin II, two important cardiovascular molecules.

The development of a prototype dual-modality optical and PET (OPET) small animal imaging tomograph is underway in the Crump Institute for Molecular Imaging at the University of California Los Angeles. OPET consists of a single ring of six detector modules with a diameter of 3.5 cm. Each detector has an 8 x 8 array of optically isolated BGO scintillators which are coupled to multichannel photomultiplier tubes and open on the front end. The system operates in either PET or optical mode and reconstructs the data sets as 3D tomograms. The detectors are capable of detecting both annihilation events (511 keV) from PET tracers as well as Single Photon Events (SPEs) (2-3 eV) from bioluminescence. Detector channels are readout using a custom multiplex readout scheme and then filtered in analog circuitry using either a [gamma]-ray or SPE specific filter. Shaped pulses are sent to a Digital Signal Processing (DSP) unit for event processing. The DSP unit has 100 MHz Analog-to-Digital Converters on the front-end which send digitized samples to Field Programmable Gate Arrays which are programmed via user configurable algorithms to process PET coincidence events or bioluminescence SPEs. Information determined using DSP includes: event timing, energy determination-discrimination, position determination-lookup, and coincidence processing. Coincidence or SPE events are recorded to an external disk and minimal post processing is required prior to image reconstruction. Initial imaging results from a phantom filled with [sup.18]FDG solution and an optical pattern placed on the front end of a detector module in the vicinity of a SPE source are shown. Index Terms--Bioluminescence, optical imaging, positron emission tomography, small animal imaging.

We report the detection of luciferase by implementing a bioluminescent assay in microfluidic reactors. The reactors were fabricated in poly(methyl methacrylate) by hot embossing using a mold master with the reactor layouts made by high-precision micromilling. The overall fabrication process was simple to implement and had a quick turnaround time with low cost. Two reactors, one with smooth channels (called reactor I) and the other with staggered herringbone mixers (called reactor II), were studied for the bioluminescent assay. The assay was implemented by introducing a sample and an assay solution into the reactors and then mixing took place to achieve the enzymatic reactions. We found that the mixing efficiency in reactor II was 17.8 times higher than reactor I. Theoretical analysis of the experimental results indicated that the required channel length of mixing was linearly proportional to the flow rate. A calibration curve for luciferase was obtained for both reactors. We found that the detection sensitivity of reactor II was 3 times higher than reactor I. The limit of detection in reactor II was determined to be 0.14 [micro]g/mL luciferase. The device was further exploited to determine the concentration of luciferase samples obtained from in vitro protein expression.

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death, miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker.

There are two goals in this simulation study: (1) to show that the time variation of the bioluminescence source can cause artifacts in the tomographic images such that quantification and localization becomes impossible; and (2) to show that the a priori knowledge of the light kinetics can be used to eliminate these artifacts. These goals are motivated by the fact that the half-life of luciferase has been reported as 30 min to 2 h in vivo. We perform two-dimensional simulations. We consider a 40 mm diameter circular region with an inclusion of 6 mm diameter located 10 mm away from the center. The measurement data is simulated using a finite-element-based forward solver. We model the noncontact measurements such that four-wavelength data is collected from four 90[degrees] apart views. The results show that the ratio of the total imaging time to the half-life of the exponentially decaying bioluminescent source is the deciding factor in the reconstruction of the source. It is also demonstrated that a priori knowledge of the source kinetics is required to perform tomographic bioluminescence imaging of short half-life bioluminescent sources and the use of spatial a priori information alone is not adequate. OCIS codes: 170.3010, 110.3080.

The present study examines pyruvate kinase-conjugated antibodies for potential use in ELISA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.

Accuracy in cellular function has to be achieved despite random fluctuations (noise) in the concentrations of different molecular constituents inside and outside the cell. The circadian oscillator in cyanobacteria is an example of resilience to noise. This resilience could be either the consequence of intercellular communication or the intrinsic property of the built-in biochemical network. Here we investigate the intercellular coupling hypothesis. A short theoretical depiction of interacting noisy phase oscillators, confirmed by numerical simulations, allows us to discriminate the effect of coupling from noise. Experimentally, by studying the phase of concurrent populations of different initial phases, we evaluate a very small upper limit of the intercellular coupling strength. In addition, in situ entrainment experiments confirm our ability to detect a coupling of the circadian oscillator to an external force and to describe explicitly the dynamic change of the mean phase. We demonstrate, therefore, that the cyanobacterial clock stability is a built-in property as the intercellular coupling effect is negligible. biological clock | bioluminescence | dynamics | synechoccocus

Two tests are conducted for explaining the cryptic bioluminescence phenotype through molecular characterization of the lux operon and its bioluminescence gene cluster associated with virulence. It is seen that the cold-water-fish pathogen, Vibrio salmonicide, produces insufficient levels of its aliphatic-aldehyde that are detectably luminous in culture.

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first [...]

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size ([approximately equal to] 100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca. bioluminescence | luciferin-binding protein | gene fusion | gene fission | domain duplication

In various multicellular organisms, circadian clocks are present not only in the central nervous system, but also in peripheral organs and tissues. In mammals peripheral oscillators are not directly responsive to light, but are entrained by the central oscillator in the suprachiasmatic nucleus. These individual oscillators are diverse in their free-running periods and phases. In contrast, cultured peripheral tissues and cell lines from zebrafish are not only rhythmic, but can also be directly entrained by light. Because of the convenience of studying rhythms in cultured cells, however, little has been known about properties of individual oscillators in intact zebrafish. Here, we show the remarkable diversity and consistency of oscillator properties in various peripheral organs and tissues from the period3-luciferase (per3-luc) transgenic zebrafish. Tissue-dependent differences were found in free-running period, phase, response to light, and temperature compensation. Furthermore, cycling amplitudes were reduced at lower temperatures in some, but not all, of the organs tested. Finally, we found that per3-luc rhythms can free run in both constant dark and constant light with remarkably similar amplitudes, phases, and periods, despite the fact that the mRNA of per2 and per1 has been shown not to oscillate in constant light. circadian | period3 | bioluminescence | temperature compensation

Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1[DELTA], which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1[DELTA] apoprotein (apoCph1[DELTA]) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1[DELTA] altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1[DELTA] and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein. biliprotein photoreceptor | phytofluor | single-molecule fluorescence | biophotonics

A combination of bioluminescence (BRET) and fluorescence (FRET) resonance energy transfer and fluorescence microscopy is used to study the molecular mechanism of the oligomerization of the prototypic family B secretin receptor. The results of the analysis demonstrate that the motif-independent interactions of transmembrane segments during the maturation of nascent molecules lead to the oligomerization of the secretin receptor, as they form intracellularly through the core domains.

The crystal structure at 1.93-[Angstrom] resolution is determined for the [Ca.sup.2+]-discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound [Ca.sup.2+] and/or coelenteramide. Among these structures, global conformational changes are small, typical of the class of 'calcium signal modulators' within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins. coelenterazine | photoprotein | EF hand | luciferase | aequorin

Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIF[alpha] subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules. bioluminescence | imaging | von Hippel-Lindau

On many occasions over the centuries, mariners have reported witnessing surreal nocturnal displays where the surface of the sea produces an intense, uniform, and sustained glow that extends to the horizon in all directions. Although such emissions cannot be fully reconciled with the known features of any light-emitting organism, these so-called 'milky seas' are hypothesized to be manifestations of unusually strong bioluminescence produced by colonies of bacteria in association with a microalgal bloom in the surface waters. Because of their ephemeral nature and the paucity of scientific observations, an explanation of milky seas has remained elusive. Here, we report the first satellite observations of the phenomenon. An [approximately equal to] 15,400-[km.sup.2] area of the northwestern Indian Ocean, roughly the size of the state of Connecticut, was observed to glow over 3 consecutive nights, corroborated on the first night by a ship-based account. This unanticipated application of satellite remote-sensing technology provides insights pertaining to the formation and scale of these poorly understood events. bacterial bioluminescence | satellite remote sensing | microbial ecology | quorum sensing | marine biology

The colors of deep-sea species are generally assumed to be cryptic, but it is not known how cryptic they are and under what conditions. This study measured the color of approximately 70 deep-sea species, both pelagic and benthic, and compared the results with two sets of predictions: 1) optimal crypsis under ambient light, 2) optimal crypsis when viewed by bioluminescent 'searchlights.' The reflectances of the pelagic species at the blue-green wavelengths important for deep-sea vision were far lower than the predicted reflectances for crypsis under ambient light and closer to the zero reflectance prediction for crypsis under searchlights. This suggests that bioluminescence is more important than ambient light for the visual detection of pelagic species at mesopelagic depths. The reflectances of the benthic species were highly variable and a relatively poor match to the substrates on which they were found. However, estimates of the contrast sensitivity of deep-sea visual systems suggest that even approximate matches may be sufficient for crypsis in visually complex benthic habitats. Body coloration was generally uniform, but many crabs had striking patterns that may serve to disrupt the outlines of their bodies.

An investigation on the production of N-acylated homoserine lactonens (AHLs) in packed fish is presented. The nature of the AHL found in the fish and the AHL produced by P. phosphoreum is also elucidated, and the AHL and light production kinetics in P phosphoreum are investigated.

Dinoflagellate bioluminescence is believed to serve a defensive function, decreasing grazing at night. Previous characterization of bioluminescence stimulated by fully developed flows might have underestimated the true sensitivity of bioluminescence by not observing the initial response. Also, it has been suggested that bioluminescence may be more sensitive to time-varying flow than to constant flow conditions. We used developing laminar Couette flow to characterize the sensitivity of the initial bioluminescent response of the dinoflagellate Lingulodinium polyedrum in time-varying flow. Both the absolute sensitivity (threshold) and dynamic sensitivity were consistent with that determined previously in fully developed flows, although there were differences between different cultured isolates of the same species and between those isolates and cells harvested from a unialgal bloom of the same species. When the rate of increase of shear was varied while keeping the maximum shear level similar, the threshold was independent of the rate of increase of shear. Surprisingly, the integrated bioluminescence was strongly dependent on the rate of increase of shear. The mechanism behind the preferential response to rapidly increasing shear was determined to be desensitization. Desensitization may influence which naturally occurring flows strongly stimulate bioluminescence either by allowing cells to avoid producing a primary response in certain slowly changing flows or, more generally, to avoid the cost of repeated stimulation when entrained in environmental flows containing above-threshold shears.

Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease [...]

Bioluminescence is the production of visible light by a living organism. The light commonly appears as flashes from point sources (involving one or more cells, usually described as photocytes) or as a glandular secretion. A visible flash usually involves synchronous light emission from a group of cells or, if from a single-celled organism such as a dinoflagellate, from a group of organelles. The number of cells (or organelles) responding synchronously is the main determinant of the flash intensity. Bioluminescence is a common phenomenon in many deep-sea animals and is widespread among the Cnidaria. In this paper, we compare and contrast in situ and laboratory recordings of the bioluminescent responses of specimens of the deep-sea scyphozoans Atolla wyvillei, Atolla vanhoffeni, Atolla parva, Nausithoe rubra, Paraphyllina intermedia, Periphyllopsis braueri and Periphylla periphylla. Displays in all seven species consist of localised flashes and propagated waves of light in the surface epithelium. The first few single waves propagate at rates of up to 60 cm [s.sup.-1] but subsequent ones in any sequence of stimuli gradually decrease in speed. After several single wave responses, a subsequent stimulus may elicit multiple waves that persist for several seconds. Following such a frenzy, the specimen becomes temporarily refractory to further stimuli, but if rested will recover its normal responses and may produce further frenzies. The dome area, situated above the coronal groove, of the genera Paraphyllina, Periphylla, and Nausithoe is covered with luminescent point sources. Such point sources are generally absent from the dome of species of Atolla. Captured specimens of A. parva also produce secretory bioluminescence, corroborating prior in situ observations of this ability. Secretory bioluminescence in P. periphylla takes the form of scintillating particles released from the lappet margins. We did not observe secretory displays in specimens of any other species in the laboratory, but one instance of apparent secretory luminescence was recorded in situ in a specimen of A. wyvillei.

A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen [alpha] receptor (ER[alpha]) homodimerization as a result of estrogen-like compound binding. ER[alpha] monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ER[alpha] dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ER[alpha]-Rluc and ER[alpha]-EYFP) expressed in Eseherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-[beta] estradiol. The 'in vivo' BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-[beta] estradiol. This new 'in vivo' BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.

The results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate is reported. An alternative mechanism of bioluminescence color was developed and the basis of the mechanism is that luciferase modulates emission color by controlling the resonance based charge delocalization of the anionic keto form of the oxyluciferin excited state.

The cDNA for Gaussia luciferase (GLuc), the enzyme responsible for the bioluminescent reaction of the marine copepod Gaussia princeps, has been cloned recently. GLuc (MW = 19 900) catalyzes the oxidative decarboxylation of coelenterazine to produce coelenteramide and light. We report the first quantitative analytical study of GLuc and examine its potential as a new reporter for DNA hybridization. A plasmid encoding both a biotin acceptor peptide--GLuc fusion protein as well as the enzyme biotin protein ligase (BPL) is engineered by using GLuc cDNA as a starting template. BPL catalyzes the covalent attachment of a single biotin to the fusion protein in vivo. Purification of GLuc is then accomplished by affinity chromatography using immobilized monomeric avidin. Moreover, the in vivo biotinylation enables subsequent complexation of GLuc with streptavidin (SA), thereby avoiding chemical conjugation reactions that are known to inactivate luciferases. Purified GLuc can be detected down to 1 amol with a signal-to-background ratio of 2 and a linear range extending over 5 orders of magnitude. The background luminescence of coelenterazine is the main limiting factor for even higher detectability of GLuc. Furthermore, the GLuc--SA complex is used as a detection reagent in a microtiter well-based DNA hybridization assay. The analytical range extends from 1.6 to 800 pmol/L of target DNA. Biotinylated GLuc produced from 1 L of bacterial culture is sufficient for 150 000 hybridization assays.

Bioluminescence resonance energy transfer has developed in recent years as a new technique to study protein--protein interactions. Protein partners of interest are tagged with either luciferase or green fluorescent protein (GFP). Non-radiative energy transfer between the excited luciferase and the GFP permits the study of spatial relationships between the two partners. This technique constitutes an important tool for the study of the functional activity of different types of receptors, and can be used in sensitive, homogenous high-throughput screening assays.