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  • The ecto-enzyme CD38 is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2+ mobilization from lysosomes in pancreatic acinar cells.

    19 March 2018

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca(2+) signals by discharging acidic Ca(2+) stores and leading to digestive enzyme secretion. From cells derived from CD38(-/-) mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca(2+) release from lysosomal stores in pancreatic acinar cells.

  • Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate.

    6 March 2018

    The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.

  • Three-dimensional imaging of direct-written photonic structures.

    4 April 2018

    Third-harmonic generation microscopy has been used to analyze the morphology of photonic structures created using the femtosecond laser direct-write technique. Three-dimensional waveguide arrays and waveguide-Bragg gratings written in fused-silica and doped phosphate glass were investigated. A sensorless adaptive-optical system was used to correct the optical aberrations occurring in the sample and microscope system, which had a lateral resolution of less than 500 nm. This nondestructive testing method creates volume reconstructions of photonic devices and reveals details invisible to other linear microscopy and index profilometry techniques.

  • Adaptive optics for structured illumination microscopy.

    4 April 2018

    We implement wave front sensor-less adaptive optics in a structured illumination microscope. We investigate how the image formation process in this type of microscope is affected by aberrations. It is found that aberrations can be classified into two groups, those that affect imaging of the illumination pattern and those that have no influence on this pattern. We derive a set of aberration modes ideally suited to this application and use these modes as the basis for an efficient aberration correction scheme. Each mode is corrected independently through the sequential optimisation of an image quality metric. Aberration corrected imaging is demonstrated using fixed fluorescent specimens. Images are further improved using differential aberration imaging for reduction of background fluorescence.

  • Addressable microlens array for parallel laser microfabrication.

    3 April 2018

    Parallel processing in femtosecond-laser-based microfabrication is demonstrated using a microlens array in conjunction with a liquid-crystal spatial light modulator (SLM). A portion of the SLM is mapped onto each individual lenslet in the array and can be used to effectively switch foci on and off for fabrication. In addition, the technique allows for homogenizing the intensity of the array of foci and translating spots relative to their natural focus. The technique demonstrates the potential for high efficiency processing of aperiodic structures.

  • Effects of catecholamine stress on diastolic function and myocardial energetics in obesity

    6 March 2018

    Background-Obesity is characterized by impaired cardiac energetics, which may play a role in the development of diastolic dysfunction and inappropriate shortness of breath. We assessed whether, in obesity, derangement of energetics and diastolic function is further altered during acute cardiac stress. Methods and Results-Normal-weight (body mass index, 22±2 kg/m 2 ; n=9-17) and obese (body mass index, 39±7 kg/m 2 ; n=17-46) subjects underwent assessment of diastolic left ventricular function (cine magnetic resonance imaging volume-time curve analysis) and cardiac energetics (phosphocreatine/ATP ratio; P-magnetic resonance spectroscopy) at rest and during dobutamine stress (heart rate increase, 65±22% and 69±14%, respectively; P=0.61). At rest, obesity was associated with a 22% lower peak filling rate (P < 0.001) and a 15% lower phosphocreatine/ATP ratio (1.73±0.40 versus 2.03±0.28; P=0.048). Peak filling rate correlated with fat mass, left ventricular mass, leptin, waist-to-hip ratio, and phosphocreatine/ATP ratio. On multivariable analysis, phosphocreatine/ATP was the only independent predictor of peak filling rate (β=0.50; P=0.03). During stress, a further reduction in phosphocreatine/ATP occurred in obese (from 1.73±0.40 to 1.53±0.50; P=0.03) but not in normal-weight (from 1.98±0.24 to 2.04±0.34; P=0.50) subject. For similar levels of inotropic stress, there were smaller increases in peak filling rate in obesity (38% versus 70%; P=0.01). Conclusions-In obesity, cardiac energetics are further deranged during inotropic stress, in association with continued diastolic dysfunction. Myocardial energetics may play a key role in the impairment of diastolic function in obesity. © 2012 American Heart Association, Inc.

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    14 February 2018