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Principles Of Fluorescence Spectroscopy

: The unpolarized absorption and circular dichroism spectra of the fundamental vibrational transitions of the chiral molecule, 4-methyl-2-oxetanone, are calculated ab initio. Harmonic force fields are obtained using Density Functional Theory (DFT), MP2, and SCF methodologies and a 5S4P2D/3S2P (TZ2P) basis set. DFT calculations use the Local Spin Density Approximation (LSDA), BLYP, and Becke3LYP (B3LYP) density functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with experiment. The MP2 force field yields spectra in slightly worse agreement with experiment than the B3LYP force field. The SCF force field yields spectra in poor agreement with experiment.The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreements with experiment. jg

Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields

Lipid Rafts: Elusive or Illusive?

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

The mystery of membrane organization: composition, regulation and roles of lipid rafts

Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargo that enters cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in such diverse processes as cytokinesis, cell adhesion and morphogenesis, cell fusion, and learning and memory.

Pathways and mechanisms of endocytic recycling.

https://findresearcher.sdu.dk:8443/ws/files/141851016/Niemann_Pick_C2_protein_regulates_sterol_transport_between_plasma_membrane_and_late_endosomes_in_human_fibroblasts.pdf

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

Transport of cholesterol derived from hydrolysis of lipoprotein associated cholesteryl esters out of late endosomes depends critically on the function of the Niemann Pick C1 (NPC1) and C2 (NPC2) proteins. Both proteins bind cholesterol but also various other sterols and both with strongly varying affinity. The molecular mechanisms underlying this multiligand specificity are not known. On the basis of the crystal structure of NPC2, we have here investigated structural details of NPC2–sterol interactions using molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) calculations. We found that an aliphatic side chain in the sterol ligand results in strong binding to NPC2, while side-chain oxidized sterols gave weaker binding. Estradiol and the hydrophobic amine U18666A had the lowest affinity of all tested ligands and at the same time showed the highest flexibility within the NPC2 binding pocket. The binding affinity of all ligands correlated highly with their calculated partitioning coefficient (logP) ...

Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein

Intrinsically fluorescent sterols, like dehydroergosterol (DHE), mimic cholesterol closely and are therefore suitable to determine cholesterol transport by fluorescence microscopy. Disadvantages of DHE are its low quantum yield, rapid bleaching, and the fact that its excitation and emission is in the UV region of the spectrum. Thus, one has to deal with chromatic aberration and low signal-to-noise ratio. We developed a method to correct for chromatic aberration between the UV channel and the red/green channel in multicolor imaging of DHE compared with the lipid droplet marker Nile Red in living macrophage foam cells and in adipocytes. We used deconvolution microscopy and developed image segmentation techniques to assess the DHE content of lipid droplets in both cell types in an automated manner. Pulse-chase studies and colocalization analysis were performed to monitor the redistribution of DHE upon adipocyte differentiation. DHE is targeted to transferrin-positive recycling endosomes in preadipocytes but associates with droplets in mature adipocytes. Only in adipocytes but not in foam cells fluorescent sterol was confined to the droplet-limiting membrane. We developed an approach to visualize and quantify sterol content of lipid droplets in living cells with potential for automated high content screening of cellular sterol transport.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.20593

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma

http://www.ibpc.fr/UMR7099/Publis/pdf/Tannert03.pdf

Daniel Wüstner

Papers

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma

Aminophospholipids Have No Access to the Luminal Side of the Biliary Canaliculus IMPLICATIONS FOR THE SPECIFIC LIPID COMPOSITION OF THE BILE FLUID