scholarly journals Erythrocyte deformation in shear flow: influences of internal viscosity, membrane stiffness, and hematocrit

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 727-734 ◽  
Author(s):  
K Kon ◽  
N Maeda ◽  
T Shiga
Keyword(s):  
Viscoelastic Properties ◽  
Shear Force ◽  
Flow Lines ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Suspension Viscosity ◽  
Applied Shear Stress ◽  
Erythrocyte Deformation ◽  
Membrane Stiffness ◽  
Internal Viscosity

The effect of shear force (depending on shear rate and viscosity of extracellular medium) and hematocrit of RBC suspension on RBC deformation was studied quantitatively using a cone-plate rheoscope with various kinds of cells, ie, partially hemolyzed (PH) cells, density-fractionated intact cells, and diamide-treated cells. The deformation index (DI) of ellipsoidally deformed cells was shown to be a function of beta gamma eta ex(eta ex/eta in)alpha, where gamma eta ex is applied shear stress, eta ex and eta in are external and internal viscosities, respectively, and alpha and beta are adjustable parameters related to the membrane viscoelastic properties. The increase of suspension viscosity at higher hematocrits (Hts) generally enhanced the ellipsoidal deformation of cells, in the same manner as increasing the suspending medium viscosity of a diluted cell suspension. The suppressing effect on cell deformation appeared above a certain Ht. When intact cells were mixed with glutaraldehyde-treated, hardened cells, the ellipsoidal deformation of intact cells was disturbed. The suppression of deformation probably occurred through disturbance of laminar flow-lines around intact cells.

scholarly journals Erythrocyte deformation in shear flow: influences of internal viscosity, membrane stiffness, and hematocrit

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 727-734 ◽  
Author(s):  
K Kon ◽  
N Maeda ◽  
T Shiga
Keyword(s):  
Viscoelastic Properties ◽  
Shear Force ◽  
Flow Lines ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Suspension Viscosity ◽  
Applied Shear Stress ◽  
Erythrocyte Deformation ◽  
Membrane Stiffness ◽  
Internal Viscosity

Abstract The effect of shear force (depending on shear rate and viscosity of extracellular medium) and hematocrit of RBC suspension on RBC deformation was studied quantitatively using a cone-plate rheoscope with various kinds of cells, ie, partially hemolyzed (PH) cells, density-fractionated intact cells, and diamide-treated cells. The deformation index (DI) of ellipsoidally deformed cells was shown to be a function of beta gamma eta ex(eta ex/eta in)alpha, where gamma eta ex is applied shear stress, eta ex and eta in are external and internal viscosities, respectively, and alpha and beta are adjustable parameters related to the membrane viscoelastic properties. The increase of suspension viscosity at higher hematocrits (Hts) generally enhanced the ellipsoidal deformation of cells, in the same manner as increasing the suspending medium viscosity of a diluted cell suspension. The suppressing effect on cell deformation appeared above a certain Ht. When intact cells were mixed with glutaraldehyde-treated, hardened cells, the ellipsoidal deformation of intact cells was disturbed. The suppression of deformation probably occurred through disturbance of laminar flow-lines around intact cells.

Stimulus-Secretion Coupling: A Perspective Highlighting the Contributions of Peter Baker

1988 ◽  
Vol 139 (1) ◽  
pp. 1-30
Author(s):  
T. J. RINK ◽  
D. E. KNIGHT
Keyword(s):  
Fluorescent Dyes ◽  
Motor Nerve ◽  
Giant Axon ◽  
Model Systems ◽  
Motor Nerve Terminal ◽  
Coupling Mechanism ◽  
Extracellular Medium ◽  
Adrenal Chromaffin Cell ◽  
Intact Cells ◽  
Stimulus Secretion Coupling

Many investigators are using numerous preparations for contributing to our present understanding of stimulus-secretion coupling, by which we mean stimulus-dependent exocytosis, sometimes known as the regulated pathway. However, a few model systems have been particularly illuminating and several of these were exploited by Peter Baker and his close associates: namely, the motor nerve terminal, the adrenal chromaffin cell, the sea urchin egg and the blood platelet. In fact, Peter's first real contribution in this area came from his seminal studies on calcium transport in his favourite preparation, the squid giant axon, where he investigated Ca2+/Na+ exchange, Ca2+ distribution and voltage-gated Ca2+ entry. More direct investigations into stimulus-secretion coupling came from work on neurone transmitter release in collaboration with Andrew Crawford, and on catecholamine secretion from the adrenal medulla in collaboration (with TJR). His most important generic contribution to this field was in the development (with DEK), of the electropermeabilized cell, which allows control of the low molecular weight components of the cytosol while leaving the exocytotic apparatus and process intact. In the initial experiments on the cells it was finally proved that Ca2+-dependent secretion of catecholamines is indeed from the granules and not from the cytosol. The quantification of the Ca2+ requirement of secretory exocytosis was an important step, as was the investigation of many factors purported to be important in the coupling mechanism or in the exocytotic process itself. Work with the human platelet, using this technique, has proved to be especially valuable in unravelling the complex interactions between different second messengers and has been neatly complemented by work in intact cells containing Ca2+-indicator fluorescent dyes. Peter was also intrigued by postsecretory events both in the early seventies, and at the end of his career when he embarked on analysis of the membrane retrieval process and the associated uptake of extracellular medium.

scholarly journals High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells

Lab on a Chip ◽  
2019 ◽  
Vol 19 (21) ◽  
pp. 3652-3663 ◽  
Author(s):  
Patricia M. Davidson ◽  
Gregory R. Fedorchak ◽  
Solenne Mondésert-Deveraux ◽  
Emily S. Bell ◽  
Philipp Isermann ◽  
...  
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Time Scale ◽  
Viscoelastic Properties ◽  
Micropipette Aspiration ◽  
Automated Image Analysis ◽  
Cell Nuclei ◽  
Intact Cells ◽  
Nuclear Mechanics ◽  
Analysis Platform

We report the development, validation, and application of an easy-to-use microfluidic micropipette aspiration device and automated image analysis platform that enables high-throughput measurements of the viscoelastic properties of cell nuclei.

Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa

1987 ◽  
Vol 65 (7) ◽  
pp. 602-609 ◽  
Author(s):  
Madhabi Barua ◽  
Gopal C. Majumder
Keyword(s):  
Specific Activity ◽  
Enzymic Activity ◽  
Significant Inhibition ◽  
Appreciable Effect ◽  
Flagellar Motility ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Phosphoprotein Phosphatase ◽  
Nitrophenyl Phosphate ◽  
Enzyme Markers

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, β-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.

Temperature Dependence of Viscoelasticity of PFPE Lubricant Confined in Nanometer-Sized Gaps

2008 ◽  
Author(s):  
Shintaro Itoh ◽  
Kenji Fukuzawa ◽  
Yuya Hamamoto ◽  
Hedong Zhang
Keyword(s):  
Temperature Dependence ◽  
Molecular Mobility ◽  
Viscoelastic Properties ◽  
Shear Force ◽  
Measuring Method ◽  
Measured Temperature ◽  
Highly Sensitive ◽  
Pfpe Lubricant ◽  
Gap Width ◽  
Reduced Temperature

In this study, we measured temperature dependence of viscoelastic properties of PFPE lubricant that was confined in nanometer-sized gap widths. In the viscoelastic measurement, we used the fiber wobbling method, which is the highly sensitive shear force measuring method that we have originally developed. Our experimental results showed that the temperature dependence of the lubricant’s viscosity was declined at the gap width of less than 5 nm. Since the elasticity also appeared at the gap width of 5 nm, the reduced temperature dependence is considered to be caused by the repression of molecular mobility due to the solidification of the confined lubricant.

scholarly journals Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action

1995 ◽  
Vol 305 (2) ◽  
pp. 529-537 ◽  
Author(s):  
M Xie ◽  
M G Low
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase D ◽  
Bovine Serum ◽  
Binding Agent ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Streptolysin O ◽  
Binding Agents ◽  
Triton X ◽  
Cholesterol Binding

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.

scholarly journals Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts

1988 ◽  
Vol 250 (3) ◽  
pp. 653-658 ◽  
Author(s):  
J P Slotte ◽  
E L Bierman
Keyword(s):  
Plasma Membrane ◽  
Cholesteryl Ester ◽  
Time Course ◽  
Plasma Membranes ◽  
Mass Movement ◽  
Extracellular Medium ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Cultured Fibroblasts ◽  
Time Course Study

This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.

scholarly journals Cell fatty acid composition affects free radical formation during lipid peroxidation

1994 ◽  
Vol 267 (1) ◽  
pp. C177-C188 ◽  
Author(s):  
J. A. North ◽  
A. A. Spector ◽  
G. R. Buettner
Keyword(s):  
Oxidative Stress ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Acid Composition ◽  
Spectral Characteristics ◽  
Radical Formation ◽  
Spin Adduct ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Lipid Radical

Lipid-derived free radicals generated from intact human U937 monocytes exposed to iron-induced oxidative stress were detected by electron paramagnetic resonance (EPR) with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Lipid radical formation was enhanced when the cells were enriched with n-3 or n-6 polyunsaturated fatty acids. Computer simulation indicated that at least two POBN spin adducts were formed, having spectral characteristics consistent with carbon-centered radicals (aN = 15.9 G and aH = 2.6 G; aN = 15.1 G and aH = 2.8 G). These alkyl radicals are probably formed by beta-scission of alkoxyl radicals. POBN spin adduct formation correlated with ethane generation. Addition of ascorbate to the assay medium greatly increased the radical signal intensity. Although radical generation was cell dependent and POBN spin adducts were observed in cell homogenates, the adducts formed by the intact cells were detected only in the extracellular medium. These findings indicate that the extent of lipid radical formation in response to oxidative stress can be influenced by changes in the polyunsaturated fatty acid composition of the cell lipids and suggest the possibility that carbon-centered lipi radicals may interact with extracellular structures.

scholarly journals Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

1975 ◽  
Vol 64 (1) ◽  
pp. 254-260 ◽  
Author(s):  
RT Briggs ◽  
ML Karnovsky ◽  
MJ Karnovsky
Keyword(s):  
Hydrogen Peroxide ◽  
Polymorphonuclear Leukocytes ◽  
Halide Ions ◽  
Reaction Products ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Cytochemical Demonstration ◽  
Halide System ◽  
Primary Granules ◽  
Micro Organisms

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.

Membrane Changes in Polymorphonuclear Leukocytes During Ionophore (A23187) - Induced Lysosomal Release

1977 ◽  
Vol 35 ◽  
pp. 482-483
Author(s):  
P.L. Moore ◽  
P.L. Sannes ◽  
H.L. Bank ◽  
S.S. Spicer
Keyword(s):  
Structural Changes ◽  
Calcium Release ◽  
Clear Understanding ◽  
Ionophore A23187 ◽  
Enzyme Release ◽  
Extracellular Medium ◽  
Pmn Leukocytes ◽  
Intracellular Ion Concentrations ◽  
Membrane Changes ◽  
Pmn Leukocyte

It is thought that calcium and/or magnesium may play important roles in polymorphonuclear (PMN) leukocyte functions such as chemotaxis, adhesion and phagocytosis. Yet, a clear understanding of the biological roles of these ions has awaited the development of techniques which permit a selective alteration of intracellular ion concentrations. Recently, treatment of cells with the ionophore A23187 has been used to alter intracellular divalent cation concentrations. This ionophore is a lipid soluble antibiotic produced by Streptomyces chartreusensis that complexes with both calcium and magnesium (3) and is believed to carry these ions across biological membranes (4). Biochemical investigations of human PMN leukocytes demonstrate that cells treated with A23187 and extracellular calcium release their lysosomal enzymes into the extracellular medium without rupturing and releasing their soluble cytoplasmic enzymes (5,6). The aim of the present study and and a companion report (7) was to investigate the structural changes that occur in leukocytes during ionophore-induced lysosomal enzyme release.

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