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Hypoxia, Free Radicals, and Reperfusion Injury Following Cold Storage and Reperfusion of Livers for Transplantation
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Ronald G. Thurman, Wenshi Gao, Henry D. Connor, Sigrid Bachmann, Robert T. Currin, Ronald P. Mason, John J. Lemasters
In hepatocytes loaded with aequorin, a Ca2+-indicating photoprotein, a biphasic response of free Ca2+ to anoxia in isolated hepatocytes was reported.64 Ca2+ increased initially and then returned toward baseline. Subsequently, as lactate dehydrogenase was released, free Ca2+ increased again. In these experiments, hepatocytes were centrifuged in Ca2+-free, aequorin-containing medium to “gravity load” aequorin. In this work, cytosolic lactate dehydrogenase, but not aequorin, was reportedly released from hepatocytes during anoxic injury. If this is true, aequorin cannot be loaded exclusively into the cytosol by gravity. Therefore, all or part of the Ca2+ changes measured by aequorin may result from changes of Ca2+ in endosomes or other subcellular compartments that aequorin enters while hepatocytes were exposed to high gravitational fields.
Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
Aequorin-based experiments are ideal for identifying Ca2+ signals generated by intact zebrafish embryos over the long periods of time that are required for developmental events; this method is non-disturbing and allows for continual visualization of Ca2+ signalling events from fertilization to >30 hpf (Webb and Miller, 2003, 2007; Webb et al., 2010). One disadvantage of this method, however, is that it only generates two-dimensional and relatively low-resolution Ca2+ information (Cheung et al., 2006, 2011; Yuen et al., 2013). Thus, after using the aequorin-based approach to establish the location and time that Ca2+ transients arose in the developing skeletal musculature, the signals were then examined at higher spatial resolution by loading embryos with the fluorescent Ca2+ reporter, calcium green-1 dextran and using high-resolution laser scanning confocal microscopy. This method allowed the subcellular identification of Ca2+ signals in the SMCs of normally developing embryos between ~17 hpf and ~24 hpf (Cheung et al., 2011). Furthermore, this imaging technique would be very useful for investigating at high resolution the effect of TPC2 knock down, knock out and inhibition on the SMC-generated Ca2+ signals.
Introduction
Published in Shoogo Ueno, Bioimaging, 2020
Aequorin and green fluorescent protein (GFP) were discovered by Osamu Shimomura (1928–2018) in Japan in 1962 (Shimomura et al., 1962). Using the jelly fish Aequorea victoria, Shimomura’s target was a luminescent substance, aequorin. GFP was isolated as a by-product of aequorin owing to its bright conspicuous fluorescence. Both are unusual proteins but they had no particular importance when Shimomura and his co-authors first reported them; 40 years after their discovery, they are well known and widely used, aequorin as a calcium probe and GFP as a marker protein (Shimomura, 2005).
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
Bioluminescence resonance energy transfer (BRET) is a process where energy transfer occurs from a bioluminescent donor to a fluorescent acceptor. Interestingly, the green fluorescent protein (GFP), isolated by Shimomura and colleagues in early 1960s from the Aequorea jellyfish, emits green light in those jellyfishes by absorbing the excited state energy of the luciferase aequorin, which itself catalyzes the oxidation of the substrate molecule coelenterazine triggering the chemiluminescence process [54].
Modulation of neuromuscular synapses and contraction in Drosophila 3rd instar larvae
Published in Journal of Neurogenetics, 2018
Kiel G. Ormerod, JaeHwan Jung, A. Joffre Mercier
Hewes, Snowdeal, Saitoe, and Taghert (1998) performed a detailed analysis of effects of the peptides encoded in dFMRFa on nerve-evoked contractions of larval body wall muscles. SAPQDFVRSamide had no effect, but the other seven peptides increased contraction amplitude, with thresholds of ∼10 nM for DPKQDFMRFamide, SPKQDFMRFamide, SDNFMRFamide, PDNFMRFamide and SVQDNFMHRamide, and 10-fold lower for TPAEDFMRFamide and MDSNFIRFamide. EC50 values were ∼40 nM for most of the peptides, and their effects were equivalent whether applied separately or in combination, suggesting that the peptides are functionally redundant (Hewes et al., 1998). This view was supported by subsequent work identifying one GPCR that, when expressed in CHO cells and assayed for bioluminescence of co-transfected aequorin, fails to respond to SAPQDFVRSamide but responds to the other peptides encoded in dFMRFa with similar EC50 values, ranging as low as 0.9–2 nM (Cazzamali & Grimmelikhuijzen, 2002; Meeusen et al., 2002). This GPCR also responded to Drosophila myosuppressin, sulfakinin-1 and short neuropeptide F, but at concentrations too high to be considered physiologically relevant (EC50 values 38–110 nM), so it was designated the Drosophila FMRFamide receptor (FR). Genes for two receptors for Drosophila myosuppressin (DmsR-1 and DmsR-2) were subsequently identified, and when expressed in CHO cells and examined with a bioluminescence assay, they failed to respond to FMRFamide, Drosophila short neuropeptide F-1 and perisulfakinin (Egerod et al., 2003a). DmsR-2 was also expressed in HEK cells and assayed for translocation of [beta]-arrestin2-green fluorescent protein ([beta]ARR2-GFP), a protein involved in desensitization of nearly all GPCRs (Kohout & Lefkowitz, 2003). These cells responded to both Drosophila myosuppressin and DPKQDFMRFamide if a G-protein coupled receptor kinase was co-expressed to accelerate [beta]ARR2-GFP translocation (Johnson, Bohn, et al., 2003). HEK cells expressing either DmsR-1 or DmsR-2 also showed decreased cAMP levels in response to both myosuppressin and DPKQDFMRFamide but were ∼10-fold less sensitive to the latter peptide. Thus, many people have favoured the view that there is only one receptor for the peptides encoded in dFMRFa, at least at physiologically relevant peptide concentrations. Receptor and ligand modeling indicates that the five Drosophila peptides containing the sequence ‘FMRFamide’ exhibit subtle differences in docking and linking with the FMRFamide receptor, but they all elicit very similar responses in cardiac bioassays (Maynard et al., 2013).