Role of a new bioassay for thyroid-stimulating antibodies (aequorin TSAb) in Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is characterized by an autoimmune reaction against thyrotropin (TSH) receptors and is diagnosed by TSH receptor antibody (TRAb). A novel assay for thyroid-stimulating antibody (TSAb) was recently introduced using a frozen Chinese hamster ovary cell line expressing TSH receptors, cyclic adenosine monophosphate (cAMP)-gated calcium channel, and aequorin (aequorin TSAb). The aim of this study was to evaluate the role of aequorin TSAb in GO. We studied 136 Japanese patients with GO (22 euthyroid and 8 hypothyroid GO patients) at our hospital. TRAbs were estimated by first generation TRAb (TRAb 1st), second generation TRAb (hTRAb 2nd), conventional porcine TSAb, and the new aequorin TSAb assays. Aequorin TSAb, porcine TSAb, TRAb 1st, and hTRAb 2nd were positive in 125/136 (92%), 110/136 (81%), 81/130 (62%), and 93/114 (82%) patients, respectively. In patients with hyperthyroid GO, they were positive in 98/106 (98%), 96/106 (91%), 78/101 (77%), and 84/93 (90%) patients, respectively. In patients with euthyroid GO, they were positive in 19/22 (86%), 9/22 (41%), 1/21 (5%), and 6/17 (35%) patients, respectively. Aequorin TSAb levels were significantly related to TRAb 1st (r = 0.4172, p < 0.0001), hTRAb 2nd (r = 0.2592, p < 0.0001), and porcine TSAb (r = 0.4665, p < 0.0001). Clinical activity score (CAS) was significantly greater in patients with high titers of aequorin TSAb than in those with low titers. Aequorin TSAb levels were significantly related to the signal intensity ratio of the enlarged eye muscle and proptosis evaluated by MRI before steroid pulse therapy. Aequorin TSAb assay was more sensitive than the conventional assays, especially in euthyroid GO.

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca(2+) Dynamics in High-Ca(2+) Organelles.

Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease.

Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots.

It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H2O2 induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H2O2. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H2O2 are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H2O2-induced gene expressions respectively, and present a Ca(2+)- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.

Spying on organelle Ca²âº in living cells: the mitochondrial point of view.

Over the past years, the use of genetically encoded Ca(2+) indicators (GECIs), derived from aequorin and green fluorescent protein, has profoundly transformed the study of Ca(2+) homeostasis in living cells leading to novel insights into functional aspects of Ca(2+) signalling. Particularly relevant for a deeper understanding of these key aspects of cell pathophysiology has been the possibility of imaging changes in Ca(2+) concentration not only in the cytoplasm, but also inside organelles. In this review, we will provide an overview of the ongoing developments in the use of GECIs, with particular focus on mitochondrially targeted probes. Indeed, due to recent advances in organelle Ca(2+) imaging with GECIs, mitochondria are now at the centre of renewed interest: they play key roles both in the physiology of the cell and in multiple pathological conditions relevant to human health.

Neuronal network imaging in acute slices using Ca2+ sensitive bioluminescent reporter.

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen]. / Melanin koncentráló hormon receptor-1 (MCHR1) antagonista fókuszált könyvtár kiválasztása és in vitro biológiai szurése AequosCreen esszével.

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.

Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3ß4α5 nicotinic acetylcholine receptors.

The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3ß4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3ß4* receptor did not affect total [(125)I]-epibatidine binding or surface expression of the receptor. However, addition of α5(D398) into α3ß4* receptor decreased the maximal response to agonist without significantly affecting EC(50) in aequorin intracellular calcium assay. α3ß4α5(N398) nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3ß4α5 nAChRs led to significantly greater intracellular calcium release from IP(3) stores relative to α3ß4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC(50) for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3ß4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of α5 into an α3ß4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling.

Identification of endomorphin-1 and endomorphin-2 binding sites in human mu-opioid receptor by antisense oligonucleotide strategy.

The effects of phosphorothioate antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 of the human mu-opioid receptor were studied in the CHO-mu-opioid receptor cells using aequorin luminescence-based calcium assay. All four antisense oligodeoxynucleotides significantly decreased the level of mu-opioid receptor mRNA in comparison with the non-treated cells, used as control. However, no statistically significant differences between antisense oligodeoxynucleotides were observed. antisense oligodeoxynucleotides against exon-2 attenuated endomorphin-1-induced intracellular calcium response in a concentration-dependent manner. antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 inhibited endomorphin-2-induced intracellular calcium response in a concentration-dependent manner and the effect of antisense oligodeoxynucleotides against exons-3 and -4 was most pronounced. The mismatch oligodeoxynucleotides against respective exons failed to exert any effect. The selective actions of antisense probes directed against different exons of the human mu-opioid receptor gene, that resulted, at the protein level, in attenuation of calcium responses induced by endomorphin-1 and endomorphin-2, suggest that the binding sites for endomorphins are structurally and functionally different. The presence of functionally distinct binding sites might play a crucial role in the modulation of pain and may be important clinically.

Comparison of luminescent immunoassays using biotinylated proteins of aequorin, alkaline phosphatase and horseradish peroxidase as reporters.

We compare aequorin, alkaline phosphatase and horseradish peroxidase as reporters for luminescent immunoassays using alpha-fetoprotein (AFP) as a model analyte. Biotinylated aequorin was prepared from mutated aequorin containing a reactive cysteine residue by chemical conjugation. The measurable range of AFP using this biotinylated aequorin was 0.02-200 ng/ml, with a lower background level than the other biotinylated enzymes tested.

Recombinant aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody.

The mutated recombinant aequorin with a reactive cysteine residue (Cys-aequorin) was highly purified and then conjugated with a maleimide-activated antibody without significant loss of luminescence activity. The conjugate ratio of Cys-aequorin to heavy chain of immunoglobulin G (IgG) was estimated to be 1:1. To test the bioluminescent immunoassay with aequorin-labeled antibody, alpha-fetoprotein (AFP), a serological marker of liver cancer, was used as a model analyte. The measurable range of AFP was 0.02 to 200 ng/ml with the coefficient of variation between 2.1 and 4.5%.

Development of a chemical screening system using aqueorin-fused protein.

We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.

Characterization of intracellular Ca2+ transient by the hybrid logistic function in aequorin-injected rabbit and mouse papillary muscles.

Myocardial intracellular calcium (Ca2+) transients (CaTs) regulate tension generation and relaxation. Isometric tension curves are often analyzed using exponential equations; however, we previously demonstrated that hybrid logistic (HL) functions, which describe the difference between two S-shaped logistic functions, provide more accurate representations. In the present study, we investigated the potential application of HL functions for analyzing CaTs directly. CaTs were measured using the calcium-sensitive bioluminescent protein, aequorin, in 7 isolated rabbit right ventricular and 15 isolated mouse left ventricular papillary muscles. CaT data were fit by the least-squares method using HL and polynomial exponential (PE) function equations. The mean correlation coefficient (r) values of HL and PE fits were 0.9934 vs. 0.9523 in rabbit and 0.9980 vs. 0.9407 in mouse, respectively. The Z transformation of r value and the adjusted coefficient of determination (r squares) were higher, and the residual mean squares and Akaike information criterion values, which estimate goodness of fit between functions with different numbers of parameters, were lower for the HL curves than for the PE curves in both rabbit and mouse. There were significant correlations between the calculated values from the best-fit HL function curve and the primary CaT data. Thus the HL function curves more accurately described the amplitudes and time courses of CaTs in both rabbit and mouse papillary muscles. We speculate that the first logistic component curve reflects the concentration and time course of Ca2+ inflow into the cytoplasmic space, and that the second logistic component curve reflects the concentrations and time courses of Ca2+ removal from the cytoplasmic space as well as Ca2+ binding to troponin. This approach might provide a more robust model for studying CaTs and cardiac cycle regulation.

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples.

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.

Mu-opioid receptor ligands lack receptor subtype selectivity in the aequorin luminescence-based calcium assay.

The aim of the present study was to characterize the binding selectivity of the mu-opioid receptor ligands, endomorphin-1, endomorphin-2, and DAMGO, in the in vitro functional assay, based on the changes in intracellular calcium levels. For the experiments Chinese hamster ovary cells, stably expressing human mu-receptor, were used. The mu-agonist-induced calcium responses were significantly inhibited by naloxone, an opioid antagonist with high preference for the mu-opioid receptors. Naloxonazine, a mu1-non-peptide antagonist, inhibited the effect of all tested mu-agonists. However, there was no significant difference in the antagonist effect of naloxonazine on the calcium response induced by mu1- (endomorphin-2) and mu2-agonists (endomorphin-1, DAMGO). [D-Pro2]endomorphin-1 and [D-Pro2]endomorphin-2, putative peptide mu2- and mu1-antagonists, respectively, which had been shown in vivo to inhibit the antinociception induced by mu-agonists, produced no inhibitory effect in our in vitro experiments. Our results demonstrated that there is only one population of the mu-opioid receptors expressed in the Chinese hamster ovary cells. We suggest that the mu-opioid receptors form a homogenous population in the in vitro systems. However, the existence of mu-receptor subtypes in vivo is still pharmacologically possible.

Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin.

A bioluminescence DNA hybridization assay for the detection of Plasmodium falciparum, the most deadly species of malaria, using the photoprotein aequorin as a bioluminescent label has been developed. The current gold standard for the detection of malaria is light microscopy, which can detect down to approximately 50 parasites/microL of blood, but has low-throughput, high costs, and requires high skill, which limit the applicability of the method, especially in the developing regions where malaria detection is mostly needed. The utilization of aequorin as a bioluminescence label offers the advantages of high signal-to-noise ratio and reliable detection down to attomole levels, allowing for the development of highly sensitive and miniaturized high-throughput bioluminescence assays. Herein, we developed a DNA hybridization assay for the detection of P. falciparum based on the competition between the target DNA and the signal generating DNA streptavidin-aequorin for hybridization with the probe DNA. This bioluminescence hybridization assay demonstrated a detection limit of 3 pg/microL and was employed for the detection of target DNA in standard and spiked human serum samples. The DNA hybridization assay was developed in a microplate format without the need for sample PCR amplification, showing the potential suitability of this method in the parallel analysis of samples by low-trained personnel, such as that typically encountered in developing regions.

Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening.

The present work describes the engineering and characterization of a new Ca(2+)-activated photoprotein (Photina) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein-coupled receptors (GPCRs), targeting Photina to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina. The mitochondrial-targeted Photina also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR(3), FLIPR(TETRA), CyBi-Lumax flash HT, Lumilux, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS.

Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2 signaling.

Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.

[D-1-Nal4]endomorphin-2 is a potent micro-opioid receptor antagonist in the aequorin luminescence-based calcium assay.

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels, was used to examine relative potency and efficacy of the micro-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) analogues containing D-3-(1-naphthyl)-alanine (D-1-Nal) or D-3-(2-naphthyl)-alanine (D-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their micro-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the micro-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with D-1-Nal or D-2-Nal substitutions in position 4 of endomorphin-2 are strong micro-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [D-1-Nal(4)]endomorphin-2. The pA(2) value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [D-1-Nal(4)]endomorphin-2 was also the most potent analogue of the series.