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Quintana-Bustamante O.,CIEMAT | Grueso E.,CIEMAT | Grueso E.,Paul Ehrlich Institute | Garcia-Escudero R.,CIEMAT | And 7 more authors.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β 1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. © 2012 Quintana-Bustamante et al. Source

Aguilar L.F.,Pontifical Catholic University of Valparaiso | Pino J.A.,Pontifical Catholic University of Valparaiso | Soto-Arriaza M.A.,University of Santiago de Chile | Cuevas F.J.,Vina del Mar University | And 3 more authors.

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo). © 2012 Aguilar et al. Source

Pellinen T.,University of Helsinki | Sanchez S.,Microscopy and Dynamic Imaging Unit | Sanchez S.,University of Concepcion | Wang Y.,University of Helsinki | And 2 more authors.
Developmental Cell

Nuclear membrane microdomains are proposed to act as platforms for regulation of nuclear function, but little is known about the mechanisms controlling their formation. Organization of the plasma membrane is regulated by actin polymerization, and the existence of an actin pool in the nucleus suggests that a similar mechanism might operate here. Weshow that nuclear membrane organization and morphology are regulated by the nuclear level of active Rac1 through actin polymerization-dependent mechanisms. Rac1 nuclear export is mediated by two internal nuclear export signals and through its interaction with nucleophosmin-1 (B23), which acts as a Rac1 chaperone inside the nucleus. Rac1 nuclear accumulation alters the balance between cytosolic Rac1 and Rho, increasing RhoA signaling in the cytoplasm and promoting a highly invasive phenotype. Nuclear Rac1 shuttling is a finely tuned mechanism for controlling nuclear shape and organization and cell invasiveness. © 2015 Elsevier Inc. Source

Caracciolo G.,University of Rome La Sapienza | Pozzi D.,University of Rome La Sapienza | Capriotti A.L.,University of Rome La Sapienza | Marianecci C.,University of Rome La Sapienza | And 10 more authors.
Journal of Medicinal Chemistry

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical-chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery. © 2011 American Chemical Society. Source

Sanchez S.A.,University of California at Irvine | Sanchez S.A.,Microscopy and Dynamic Imaging Unit | Gratton E.,University of California at Irvine | Zanocco A.L.,University of Chile | And 2 more authors.

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R h. As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured. © 2011 Sanchez et al. Source

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