Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon

AbstractRegulated exocytosis involves the Ca2+-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca2+-binding proteins [1]. The Ca2+-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow ‘cameleon’ (Ycam2) – comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP – which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin–Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic β cells, apparent resting values of cytosolic [Ca2+] and [Ca2+]gd were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within ∼1 μm of the plasma membrane underwent increases in [Ca2+]gd that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes

Advances in multispectral and hyperspectral imaging for archaeology and art conservation

Multispectral imaging has been applied to the field of art conservation and art history since the early 1990s. It is attractive as a noninvasive imaging technique because it is fast and hence capable of imaging large areas of an object giving both spatial and spectral information. This paper gives an overview of the different instrumental designs, image processing techniques and various applications of multispectral and hyperspectral imaging to art conservation, art history and archaeology. Recent advances in the development of remote and versatile multispectral and hyperspectral imaging as well as techniques in pigment identification will be presented. Future prospects including combination of spectral imaging with other noninvasive imaging and analytical techniques will be discussed

The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

<p>Abstract</p> <p>Background</p> <p>Cigarette smoke (CS) is the main cause in the development of chronic obstructive pulmonary disease (COPD), the pathogenesis of which is related to an extended inflammatory response. In this study, we investigated the effect of low and high doses of gas phase cigarette smoke (GPS) on cultured lymphocyte progenitor cells, using techniques to assess cell viability and to elucidate whether cells die of apoptosis or necrosis upon exposure to different doses of GPS.</p> <p>Methods</p> <p>In our approach we utilised a newly-established system of exposure of cells to GPS that is highly controlled, accurately reproducible and simulates CS dosage and kinetics that take place in the smokers' lung. This system was used to study the mode of cell death upon exposure to GPS in conjunction with a range of techniques widely used for cell death studies such as Annexin V staining, activation of caspase -3, cytoplasmic release of cytochrome C, loss of mitochondrial membrane potential and DNA fragmentation.</p> <p>Results</p> <p>Low doses of GPS induced specific apoptotic indexes in CCRF-CEM cells. Specifically, cytochrome C release and cleaved caspase-3 were detected by immunofluorescence, upon treatment with 1-3 puffs GPS. At 4 h post-exposure, caspase-3 activation was observed in western blot analysis, showing a decreasing pattern as GPS doses increased. Concomitant with this behaviour, a dose-dependent change in Δψ_mdepolarization was monitored by flow cytometry 2 h post-exposure, while at 4 h Δψ_mcollapse was observed at the higher doses, indicative of a shift to a necrotic demise. A reduction in DNA fragmentation events produced by 5 puffs GPS as compared to those provoked by 3 puffs GPS, also pointed towards a necrotic response at the higher dose of GPS.</p> <p>Conclusion</p> <p>Collectively, our results support that at low doses gas phase cigarette smoke induces apoptosis in cultured T-lymphocytes, whereas at high doses GPS leads to necrotic death, by-passing the characteristic stage of caspase-3 activation and, thus, the apoptotic route.</p

Laser cleaning studies on stonework and polychromed surfaces

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN034535 / BLDSC - British Library Document Supply CentreGBUnited Kingdo