scholarly journals Snake venom cardiotoxin can rapidly induce actin polymerization in intact platelets

1993 ◽  
Vol 290 (2) ◽  
pp. 591-594 ◽  
Author(s):  
R F Liou ◽  
W C Chang ◽  
S T Chu ◽  
Y H Chen
Keyword(s):  
Actin Polymerization ◽  
Cytochalasin B ◽  
Cell Lysis ◽  
Toxin Concentration ◽  
Intact Cells ◽  
Lactate Dehydrogenase Activity ◽  
Rabbit Platelets ◽  
Cytoskeletal Change ◽  
Naja Atra ◽  
Rapid Incorporation

The action of Taiwan cobra (Naja naja atra) venom cardiotoxin on rabbit platelets at 37 degrees C was characterized by observing cytoskeletal alterations and cell lysis. At a concentration of 21.4 microM the toxin produced no cell lysis within 30 s, and less than 5% of the total lactate dehydrogenase activity of intact cells was detected in the suspending medium after the interaction had proceeded for 3 min. The extent of cell lysis was proportional to toxin concentration and interaction time. Before cell lysis, the toxin caused rapid incorporation of actin monomers into cross-linked actin filaments. The actin incorporation could be inhibited by either the presence of cytochalasin B or increased CaCl2 concentration in the suspending medium. However, addition of indomethacin did not influence the toxin-induced cytoskeletal change.

scholarly journals Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan

1992 ◽  
Vol 282 (2) ◽  
pp. 393-397 ◽  
Author(s):  
J Norgauer ◽  
M Eberle ◽  
H D Lemke ◽  
K Aktories
Keyword(s):  
Pertussis Toxin ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Superoxide Radical ◽  
Human Neutrophils ◽  
Superoxide Anion Production ◽  
Radical Production ◽  
Rapid Formation ◽  
Primary Granules ◽  
Type 3

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

scholarly journals A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle

2010 ◽  
Vol 299 (5) ◽  
pp. C988-C993 ◽  
Author(s):  
Hak Rim Kim ◽  
Paul C. Leavis ◽  
Philip Graceffa ◽  
Cynthia Gallant ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Direct Detection ◽  
New Method ◽  
Actin Dynamics ◽  
Tat Peptide ◽  
Isolated Cells ◽  
Intact Cells ◽  
Permeabilized Cells

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.

Sulfhydryl substituents of the human erythrocyte hexose transport mechanism

1986 ◽  
Vol 250 (6) ◽  
pp. C853-C860 ◽  
Author(s):  
R. E. Abbott ◽  
D. Schachter ◽  
E. R. Batt ◽  
M. Flamm
Keyword(s):  
Human Erythrocyte ◽  
Transport Mechanism ◽  
Cytochalasin B ◽  
Erythrocyte Membranes ◽  
Type I ◽  
Type Ii ◽  
Hexose Transport ◽  
Intact Cells ◽  
Type Iii ◽  
Human Erythrocyte Membranes

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.

scholarly journals Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation.

1980 ◽  
Vol 84 (2) ◽  
pp. 455-460 ◽  
Author(s):  
D C Lin ◽  
K D Tobin ◽  
M Grumet ◽  
S Lin
Keyword(s):  
Binding Sites ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Molecular Size ◽  
Muscle Actin ◽  
Competitive Displacement ◽  
Low Concentrations ◽  
Relative Affinity ◽  
Cytochalasin E ◽  
Low Ionic Strength

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'-phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.

Interaction of Mycotoxins with Copper-Folin Reagent

1978 ◽  
Vol 41 (5) ◽  
pp. 370-372
Author(s):  
MOHAMED Y. SIRAJ ◽  
TIMOTHY D. PHILLIPS ◽  
A. WALLACE HAYES
Keyword(s):  
Linear Relationship ◽  
Aflatoxin B1 ◽  
Analytical Method ◽  
Cytochalasin B ◽  
High Sensitivity ◽  
Blue Color ◽  
Penicillic Acid ◽  
Toxin Concentration ◽  
Color Development ◽  
Positive Materials

To study the interference by mycotoxins in protein measurements, solutions of various concentrations of aflatoxin B1, citrinin, cytochalasin B, ochratoxin A, patulin, penicillic acid, rubratoxins A and B, T-2 toxin and zearalenone were tested for a positive copper-Folin reaction. Except for T-2 toxin, all mycotoxins tested developed a blue color, characteristic of proteins, in the copper-Folin solution. The Lowry reaction for the mycotoxins was linear over the concentration range of 20–100 μg/ml for most of these toxins. Aflatoxin B1 and citrinin at 2 μg/ml and zearalenone at 0.5 mg/ml developed a blue color in the copper-Folin solution. The linear relationship between color development and toxin concentration plus high sensitivity makes the copper-Folin reaction a potential analytical method for these toxins but only in the absence of protein or other copper-Folin positive materials.

Discrepancy between glucose transport and transporters in human femoral adipocytes

1989 ◽  
Vol 256 (1) ◽  
pp. E179-E185 ◽  
Author(s):  
E. Karnieli ◽  
R. Moscona ◽  
R. Rafaeloff ◽  
Y. G. Illouz ◽  
M. Armoni
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Intact Cell ◽  
Transport Activity ◽  
Intact Cells ◽  
Insulin Resistant ◽  
Large Size ◽  
Microsomal Membranes ◽  
Subcutaneous Adipose

Obesity is known to be associated with insulin resistance in human and rat adipocytes. However, it is not known what are the perturbations in insulin action that contribute to disproportional femoral obesity. Thus femoral subcutaneous adipose tissue was obtained from lean women with various degrees of disproportional obesity, by liposuction. 3-O-methylglucose (3-O-methyl-D-glucopyranose) transport was measured in intact cells, and glucose transporter levels in plasma and low-density microsomal membranes were assessed using the cytochalasin B binding assay. A sixfold cellular enlargement was associated with increase in both basal and insulin-stimulated glucose transport activity in the intact cell, and a 300-600% increase in insulin stimulating effect per se. However, when glucose transporter levels were assessed, this cellular enlargement was accompanied by a 40-70% transporter depletion (in largest cells compared with smallest ones) in both subcellular fractions examined, from either basal or insulin-stimulated cells. This discrepancy, between increasing cellular glucose transport rates and relative depletion of transporter levels, suggests that these cells are not insulin resistant, as could be expected from their large size. A role for other factor(s), additional to glucose transporter levels, in the regulation of cellular glucose uptake rate is thus suggested.

scholarly journals Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.

1981 ◽  
Vol 88 (3) ◽  
pp. 654-659 ◽  
Author(s):  
T D Pollard ◽  
M S Mooseker
Keyword(s):  
Electron Microscopy ◽  
Growth Rates ◽  
Rate Constants ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Monomer Concentration ◽  
Dissociation Rate ◽  
Filament Bundles ◽  
Dissociation Rate Constants ◽  
Pointed End

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

2007 ◽  
Vol 292 (4) ◽  
pp. C1562-C1566 ◽  
Author(s):  
Christopher J. Guerriero ◽  
Ora A. Weisz
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Cell Function ◽  
Actin Dynamics ◽  
Cellular Functions ◽  
Intact Cells ◽  
Atp Levels ◽  
Dependent Inhibition

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

Complex Influence of Cytochalasin B on Actin Polymerization

1979 ◽  
Vol 34 (7-8) ◽  
pp. 555-557 ◽  
Author(s):  
P. Dancker ◽  
I. Low
Keyword(s):  
Light Scattering ◽  
Actin Polymerization ◽  
Time Course ◽  
Cytochalasin B ◽  
Degree Of Polymerization ◽  
Viscosity Measurements ◽  
Low Concentrations ◽  
Scattering Measurements ◽  
Final Length ◽  
Complex Influence

Abstract In the presence of very low concentrations (about 2 x 10-7 ᴍ) of cytochalasin B (CB) the time course of actin polymerization is much more sigmoidal when followed by viscosity measurements than when followed by light scattering measurements. This suggests that under these conditions actin polymers do not immediately reach their final length but only via short ”bent“ polymers which can be detected only by light scattering but not by viscosity measurements. At higher CB concentrations (about equimolar to those of actin) CB reduces the average degree of polymerization and favors the nucleation step necessary for polymerization.

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