635 Vesicular transport simulation based on large deformation of shell-like plasma membrane vesicle.

2004 ◽  
Vol 2004 (0) ◽  
pp. _635-1_-_635-6_
Author(s):  
Tadashi KOSAWADA ◽  
Takeshi OHKUSHI ◽  
Tatsu KOMATSUDA
Keyword(s):  
Plasma Membrane ◽  
Large Deformation ◽  
Membrane Vesicle ◽  
Vesicular Transport ◽  
Plasma Membrane Vesicle ◽  
Transport Simulation ◽  
Simulation Based

The iodide channel of the thyroid: a plasma membrane vesicle study

1992 ◽  
Vol 263 (3) ◽  
pp. C590-C597 ◽  
Author(s):  
P. Golstein ◽  
M. Abramow ◽  
J. E. Dumont ◽  
R. Beauwens
Keyword(s):  
Plasma Membrane ◽  
Chloride Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Anion Channel ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Bovine Thyroid ◽  
Set Up

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.

scholarly journals Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

2002 ◽  
Vol 277 (51) ◽  
pp. 49767-49775 ◽  
Author(s):  
Hanbang Zhang ◽  
Ellen M. Howard ◽  
Paul D. Roepe
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Chloroquine Resistance ◽  
Malarial Parasite ◽  
Plasma Membrane Vesicle ◽  
Wild Type ◽  
Stem Loop

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

scholarly journals ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

1969 ◽  
Vol 41 (2) ◽  
pp. 378-392 ◽  
Author(s):  
Charles W. Boone ◽  
Lincoln E. Ford ◽  
Howard E. Bond ◽  
Donald C. Stuart ◽  
Dianne Lorenz
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicle ◽  
Whole Cells ◽  
Continuous Sucrose Gradient

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

Stimulation of P2 receptors causes release of IL-1β–loaded microvesicles from human dendritic cells

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 3856-3864 ◽  
Author(s):  
Cinzia Pizzirani ◽  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Elena Adinolfi ◽  
Dorianna Sandonà ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Ionic Strength ◽  
Membrane Vesicle ◽  
P2 Receptors ◽  
Membrane Receptors ◽  
Extracellular Nucleotides ◽  
Receptor Subtype ◽  
Plasma Membrane Vesicle ◽  
Stimulation Of

Abstract Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X7 receptor subtype (P2X7R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1β. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X7R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X7R by extracellular ATP causes IL-1β release from the vesicle lumen. Previous studies demonstrated that high extracellular K+ inhibits IL-1β processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1β loading.

scholarly journals Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release

1999 ◽  
Vol 354 (1381) ◽  
pp. 307-318 ◽  
Author(s):  
Martin Oheim ◽  
Dinah Loerke ◽  
Robert H. Chow ◽  
Walter Stühmer
Keyword(s):  
Plasma Membrane ◽  
Evanescent Wave ◽  
Membrane Vesicle ◽  
Three Dimensional ◽  
Plasma Membrane Vesicle ◽  
Wave Excitation ◽  
Time Resolved ◽  
Before And After ◽  
Adrenal Chromaffin ◽  
Evanescent Wave Excitation

Evanescent–wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass–water interface on top of which the cells are grown. Whereas epi–illumination images appeared blurred and structureless, evanescent–wave excitation highlighted acridine orange–labelled vesicles as individual pinpoints. Three–dimensional (3D) trajectories of individual vesicles were obtained from time–resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D (3) . Based on the single–vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [ K + ]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.

The oxalate/sulfate antiporter in lobster hepatopancreas: internal and external binding constants

2000 ◽  
Vol 203 (9) ◽  
pp. 1497-1502 ◽  
Author(s):  
G.A. Gerencser ◽  
C. Burgin ◽  
F. Robbins ◽  
G.A. Ahearn
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
Binding Constants ◽  
Binding Specificity ◽  
Organic Anions ◽  
Transport Rate ◽  
Disulfonic Acid ◽  
Plasma Membrane Vesicle ◽  
Binding Characteristics ◽  
Basolateral Plasma Membrane

Utilizing a purified basolateral plasma membrane vesicle (BLMV) preparation containing a sulfate/oxalate antiporter, it was demonstrated that sulfate exhibited similar binding characteristics to the transporter whether bound internally or externally. It was also demonstrated that oxalate had similar binding characteristics to the antiporter whether it was bound internally or externally. Oxalate had a greater affinity to the transporter than did sulfate. Several organic anions affected binding and, therefore, overall transport by the antiporter. Most notably, sulfate was the only anion that stimulated oxalate uptake into BLMVs, which suggests a conservative binding specificity for the antiporter. 4-Acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and/or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited the transport rate, confirming the existence of oxalate/sulfate exchange by the transporter. These results suggest that oxalate, not sulfate, regulates the transport rate because of its greater affinity to the transporter.

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