Cell Permeabilization (cell + permeabilization)

Distribution by Scientific Domains
Life Sciences75%

Selected Abstracts

Cell permeabilization by poliovirus 2B viroporin triggers bystander permeabilization in neighbouring cells through a mechanism involving gap junctions

CELLULAR MICROBIOLOGY, Issue 8 2010
Vanesa Madan
Summary Poliovirus 2B protein is a well-known viroporin implicated in plasma membrane permeabilization to ions and low-molecular-weight compounds during infection. Translation in mammalian cells expressing 2B protein is inhibited by hygromycin B (HB) but remains unaffected in mock cells, which are not permeable to the inhibitor. Here we describe a previously unreported bystander effect in which healthy baby hamster kidney (BHK) cells become sensitive to HB when co-cultured with a low proportion of cells expressing poliovirus 2B. Viroporins E from mouse hepatitis virus, 6K from Sindbis virus and NS4A protein from hepatitis C virus were also able to permeabilize neighbouring cells to different extents. Expression of 2B induced permeabilization of neighbouring cell lines other than BHK. We found that gap junctions are responsible mediating the observed bystander permeabilization. Gap junctional communication was confirmed in 2B-expressing co-cultures by fluorescent dye transfer. Moreover, the presence of connexin 43 was confirmed in both mock and 2B-transfected cells. Finally, inhibition of HB entry to neighbouring cells was observed with 18,-glycyrrhethinic acid, an inhibitor of gap junctions. Taken together, these findings support a mechanism involving gap junctional intercellular communication in the bystander permeabilization effect observed in healthy cells co-cultured with poliovirus 2B-expressing cells. [source]

Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells

FEBS JOURNAL, Issue 4 2006
Jorge Alegre-Cebollada
Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source]

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications

THE JOURNAL OF GENE MEDICINE, Issue S1 2004
Carole Bloquel
Abstract In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue. Copyright © 2004 John Wiley & Sons, Ltd. [source]

An Arabidopsis inositol phospholipid kinase strongly expressed in procambial cells: Synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in insect cells by 5-phosphorylation of precursors

THE PLANT JOURNAL, Issue 6 2001
Stephan Elge
Summary We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole-cell membrane fractions of AtP5K1-expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5-phosphory lation of precursors. Analysis of promoter-reporter gene (,-glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single-cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase , polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation. [source]