Search

Abstract: We have observed single DNA strands labeled with a small fluorescent molecule, BODIPY-FL, using objective based total internal fluorescence microscopy. In spite of its photobleaching, this small fluorophore is potentially useful for monitoring biochemical processes like protein synthesis because it can be efficiently incorporated into proteins by the ribosome through a charged epsilon-labeled fluorescent lysine tRNA. Using evanescent wave laser excitation at 488nm, we have been able to observe single Bodipy molecules using integration times as low as 20ms with a good signal to noise ratio, and for several images before photobleaching. We have measured the fluorescence decay due to photobleaching and have been able to lengthen it by a factor of 5 using oxygen scavenger systems. [Copyright &y& Elsevier]

Photon emission from polycrystalline silver induced by scanning tunneling microscopy is studied for three different tip materials (Au, PtIr and W). Photon emission intensity curves as a function of the tip voltage are observed to be almost identical for platinum–iridium alloy and gold tips (and more than 10 times enhanced as compared with the tungsten tip). An evolution in topography and photon map for different applied voltages is investigated along with the study of the spatial distribution of photon emission in dependence upon the surface local differential height. It turns out that no clear correlation between a local curvature and enhancement of light emission can be found. Simultaneous measurements of tunneling current and photon intensity as a function of vertical tip displacement confirm the earlier observation, namely, that similar apparent barrier heights exist for both elastic and inelastic tunneling channels. The role of the tip material as well as its shape is discussed. [Copyright &y& Elsevier]

Levels of cholesterol are elevated in the brains of Alzheimer’s disease (AD) patients [1,2]. While the precise pathophysiological consequences remain unclear, an increase in cholesterol at the plasma membrane promotes endocytosis of the amyloid precursor protein (APP) thereby leading to increased Aβ production [3]. An interaction between the Aβ fragment C99 (resulting from cleavage of APP by β-secretase), and cholesterol was recently proposed [4]. In the present work, we studied the nature of this putative cholesterol binding site (CBS) on APP by analyzing APP mutants within the CBS (located between 691-710 of APP770). We measured the influence of mutants on production of amyloid peptides, cleavage by secretases and the subcellular localization of APP mutants in HEK293T cells. A variety of mutants of the CBS of APP751-mCherry (APP isoform which contain 751 residues fused to the fluorescent protein mCherry) were constructed using site-directed mutagenesis: E693K, S697A, K699A, K699E, G700A, G704A, V710A, S697A/K699A and G700A/G704A (numbering according to APP770 sequence). The amounts of secreted Aβ40 and Aβ42 peptides were measured by ELISA (Meso Scale Discovery) and short peptides (28 to 42aa) by ELISA from IBL (Immuno-Biological Laboratories) in the culture medium of HEK293T cells transfected with various mutants. We studied the activity of secretases by analyzing the levels of C99 and AICD (APP Intracellular Domain) using western blot. In addition, we analyzed the subcellular localization of mutant APP-mCherry by confocal microscopy, using immunocytochemistry with anti-Early Endosome Antigen 1 antibodies. Levels of secreted Aβ40 and Aβ42 were significantly decreased in HEK293T cells transfected with all mutants (except for S697A) as compared to HEK293T cells transfected with APPwt (p<0.05 to p<0.0001, 3 independent experiments with 6