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Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism.
May JM
(1989) Biochem J 263: 875-81
MeSH Terms: Biological Transport, Cell Membrane Permeability, Cysteine, Erythrocyte Membrane, Humans, In Vitro Techniques, Maleimides, Monosaccharide Transport Proteins, Peptide Mapping, Structure-Activity Relationship, Sulfhydryl Compounds, Sulfhydryl Reagents, Trypsin
Show Abstract · Added December 10, 2013
S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.
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13 MeSH Terms
Permeability characteristics of cultured endothelial cell monolayers.
Albelda SM, Sampson PM, Haselton FR, McNiff JM, Mueller SN, Williams SK, Fishman AP, Levine EM
(1988) J Appl Physiol (1985) 64: 308-22
MeSH Terms: Animals, Aorta, Cattle, Cell Membrane Permeability, Cells, Cultured, Dextrans, Endothelium, Vascular, Fetus, Membrane Potentials
Show Abstract · Added May 27, 2014
The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.
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9 MeSH Terms
NMR measurements of intracellular sodium in the rabbit proximal tubule.
Gullans SR, Avison MJ, Ogino T, Giebisch G, Shulman RG
(1985) Am J Physiol 249: F160-8
MeSH Terms: Animals, Biological Transport, Active, Cell Membrane Permeability, Detergents, Dysprosium, Ion Channels, Kidney Tubules, Proximal, Magnetic Resonance Spectroscopy, Nystatin, Octoxynol, Ouabain, Polyethylene Glycols, Polyphosphates, Rabbits, Sodium, Sodium Isotopes, Spectrum Analysis
Show Abstract · Added December 10, 2013
The present study evaluated the use of nuclear magnetic resonance (NMR) spectroscopy to monitor directly and continuously intracellular sodium levels in rabbit renal cortical tubule suspensions. When the paramagnetic shift reagent dysprosium tripolyphosphate was added to the extracellular medium it was possible to resolve signals from intracellular and extracellular sodium without adversely affecting cellular viability. An efflux of intracellular sodium against a significant concentration gradient was observed when sodium-loaded cells were warmed from 4 to 37 degrees C. At 37 degrees C in steady state, inhibition of Na+-K+-ATPase activity by ouabain increased intracellular sodium content in a dose-dependent and time-dependent manner. A biphasic time course of increased intracellular sodium following ouabain (10(-3) M) suggested that the sodium permeability of the plasma membrane may decrease following pump inhibition, thus limiting sodium influx. Nystatin, an agent known to facilitate sodium entry across cell membranes, increased intracellular sodium fivefold. In another series of experiments several maneuvers were performed to ascertain the fraction of intracellular sodium that was NMR visible. Quantitative assessment of either an efflux or influx of sodium indicated that the NMR visibility of the transported sodium was 100%. Furthermore, disruption of the cell membranes with Triton X-100 showed that the entire pool of intracellular sodium was 100% NMR visible.(ABSTRACT TRUNCATED AT 250 WORDS)
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17 MeSH Terms
Heterologous expression of mammalian P450 in COS cells.
Clark BJ, Waterman MR
(1991) Methods Enzymol 206: 100-8
MeSH Terms: Animals, Cell Fractionation, Cell Line, Cell Membrane Permeability, Cytochrome P-450 Enzyme System, DNA, Electric Stimulation, Indicators and Reagents, Mammals, Recombinant Proteins, Transfection, Ultracentrifugation
Added February 12, 2015
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12 MeSH Terms
Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape.
Sims JR, Karp S, Ingber DE
(1992) J Cell Sci 103 ( Pt 4): 1215-22
MeSH Terms: Actomyosin, Adenosine Triphosphate, Amino Acid Sequence, Animals, Cattle, Cell Membrane Permeability, Cell Nucleus, Cells, Cells, Cultured, Cytoskeleton, Endothelium, Vascular, Fibronectins, Integrins, Molecular Sequence Data, Morphogenesis, Peptide Fragments, Stress, Mechanical
Show Abstract · Added May 22, 2014
Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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17 MeSH Terms
Shear stress-facilitated calcium ion transport across lipid bilayers.
Chakravarthy SR, Giorgio TD
(1992) Biochim Biophys Acta 1112: 197-204
MeSH Terms: Biological Transport, Calcium, Cations, Divalent, Cell Membrane Permeability, Fluorescent Dyes, Indoles, Lipid Bilayers, Liposomes, Particle Size, Stress, Mechanical, Surface Properties
Show Abstract · Added March 20, 2014
Small unilamellar liposomes were used in this study of shear stress effects on the trans-bilayer flux of calcium ions (Ca2+). Liposome suspensions were prepared from 99% egg phosphatidylcholine by a microporous filter extrusion technique. The inner aqueous phase of the unilamellar liposomes contained indo-1(5-), a fluorescent indicator of free Ca2+. The external aqueous phase was composed of Hepes-buffered saline containing normal physiological levels of common ionic species. Calcium ion levels were set at 100 nM and 1 mM in the inner and outer aqueous phases, respectively. Liposome suspensions were exposed to graded levels of uniform shear stress in an optically modified rotational viscometer. Intraliposome Ca2+ concentration was estimated from continuous measurement of indo-1(5-) fluorescence. Electronically measured particle size distribution was used to determine liposome surface area for estimation of trans-bilayer Ca2+ flux. Trans-bilayer Ca2+ flux increased linearly with applied shear rate from 27 s-1 to 2700 s-1. Diffusional resistance of the lipid bilayer, not the convective resistance of the surrounding fluid, was the limiting step in the transport of Ca2+. Liposome permeability to Ca2+ increased by nearly two orders of magnitude over the physiologically relevant shear rate range studied. Solute transport in injectable liposome preparations may be dramatically influenced by cardiovascular fluid stress. Solute delivery rates determined in liposomes exposed to static conditions may not accurately predict in vivo, cardiovascular solute transport.
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11 MeSH Terms
The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.
de Caestecker MP, Telfer BA, Hutchinson IV, Ballardie FW
(1992) J Immunol Methods 154: 11-20
MeSH Terms: Antibodies, Monoclonal, Cell Membrane Permeability, Dose-Response Relationship, Drug, Dose-Response Relationship, Immunologic, Flow Cytometry, Fluorescein-5-isothiocyanate, Fluorescent Antibody Technique, Formaldehyde, Humans, Interleukin-1, Lipopolysaccharides, Microscopy, Fluorescence, Monocytes, Phycoerythrin, Polymers, Reproducibility of Results, Time Factors, Tissue Fixation, Tumor Necrosis Factor-alpha
Show Abstract · Added January 5, 2011
Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.
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19 MeSH Terms