Differential actin isoform reorganization in the contracting A7r5 cell

2006 ◽  
Vol 84 (8-9) ◽  
pp. 867-875 ◽  
Author(s):  
D. Brown ◽  
A. Dykes ◽  
J. Black ◽  
S. Thatcher ◽  
M.E. Fultz ◽  
...  
Keyword(s):  
Stress Fibers ◽  
Partial Loss ◽  
Cell Content ◽  
Cytoskeletal Structure ◽  
A7r5 Cell ◽  
Kinase C ◽  
Low Concentrations ◽  
Actin Isoform ◽  
High Concentrations ◽  
Actin Stress Fibers

In the present study, we investigated the reorganization of α- and β-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (≤10−7 mol/L), cells showed an ~70% loss in α-actin stress fibers with robust transport of this isoform to podosomes. By comparison, β-actin remained in stress fibers in cells stimulated at low concentrations (≤10−7 mol/L) of PDBu. However, at high concentrations (≥10−6 mol/L) ~50% of cells showed transport of β-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 × 10−8 mol/L) loss in α-actin stress fibers or reversed podosome formation with re-establishment of α-actin stress fibers. By comparison, these inhibitors caused partial loss of β-actin stress fibers. The results support our earlier conclusion of independent remodeling of α- and β-actin cytoskeletal structure and suggest that the regulation of these structures is different.

scholarly journals Volume-sensitive K influx in human red cell ghosts.

1988 ◽  
Vol 92 (5) ◽  
pp. 685-711 ◽  
Author(s):  
J R Sachs
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Red Cell ◽  
Influx Rate ◽  
Phosphatidylinositol Kinase ◽  
Kinase C ◽  
Low Concentrations ◽  
Atp Analogues ◽  
High Concentrations ◽  
Activate Protein Kinase

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.

scholarly journals 1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells

1990 ◽  
Vol 267 (1) ◽  
pp. 17-22 ◽  
Author(s):  
R N Kolesnick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Pituitary Cells ◽  
Dibutyryl Cyclic Amp ◽  
Kinase C ◽  
Low Concentrations ◽  
High Concentrations ◽  
Phosphatidylcholine Synthesis

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.

scholarly journals Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

1999 ◽  
Vol 144 (3) ◽  
pp. 413-425 ◽  
Author(s):  
Florian Überall ◽  
Karina Hellbert ◽  
Sonja Kampfer ◽  
Karl Maly ◽  
Andreas Villunger ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Atypical Protein Kinase C ◽  
Nih3t3 Cells ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Actin Stress Fibers

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

The Action of an Aniline Derivative (AN 162) on Blood Coagulation and on Platelets

1971 ◽  
Vol 26 (01) ◽  
pp. 145-166
Author(s):  
E Deutsch ◽  
K Lechner ◽  
K Moser ◽  
L Stockinger
Keyword(s):  
Blood Coagulation ◽  
Citric Acid Cycle ◽  
Second Phase ◽  
Aniline Derivative ◽  
Acid Cycle ◽  
Enhancing Effect ◽  
Low Concentrations ◽  
Dual Action ◽  
High Concentrations ◽  
Induced Aggregation

Summary1. The aniline derivative AN 162, Donau Pharmazie, Linz, Austria, has a dual action on the blood coagulation: an anticoagulant and an coagulation enhancing effect.2. The anticoagulant action may only be demonstrated with high concentrations (over 1 X 10”3 M related to plasma) preferentially in PPP. It is partially caused by an inhibition of the endogenous way of generation of the prothrombin converting principle. In addition it is suggested that it interferes with the fibrinogen-fibrin reaction in a manner not yet understood.3. The coagulant action is caused by a greater availability of platelet constituents at low concentrations of AN 162 (over 1 × 10-4 M) and by the induction of a release reaction at higher concentrations. The platelet factors 3 and 4, serotonin, adenine, and acid phosphatase are released.4. AN 162 inhibits platelet aggregation. This inhibition can be demonstrated by the PAT of Breddin and in the stirred aggregation test of Born. It is more effective to inhibit the collagen-induced and the second phase of the adrenaline-induced aggregation than the ADP induced one. The platelet retention (test of Hellem) is also reduced.5. The action of AN 162 on the platelets is caused by a damage of the platelet membrane which becomes permeabel for both, soluble platelet constitutents and granula.6. AN 162 interferes with the energy metabolism of the platelets. It causes a loss of ATP, and inhibits the key-enzymes of glycolysis, citric acid cycle, fatty acid oxydation and glutathione reduction.7. AN 162 inhibits the growth of fibroblasts without influence on mitosis.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

Biological Mechanisms of H2S Formation in Sewer Pipes

1992 ◽  
Vol 26 (3-4) ◽  
pp. 907-914 ◽  
Author(s):  
A. Attal ◽  
M. Brigodiot ◽  
P. Camacho ◽  
J. Manem
Keyword(s):  
Suspended Solid ◽  
Urban Wastewater ◽  
Biological Mechanisms ◽  
Sewer Pipes ◽  
Sulfate Reducing ◽  
Biological Phenomena ◽  
Low Concentrations ◽  
Production Of Hydrogen ◽  
High Concentrations ◽  
Reducing Bacteria

The purpose of this study is to gain a better understanding of the biological phenomena involved in the production of hydrogen sulfide in urban wastewater (UWW) systems. It is found that the UWW itself naturally possesses the biomass needed to consume the sulfates. These heterotrophic sulfate-reducing bacteria populations, though immediately active in strict anaerobic conditions, are present only in very low concentrations in the UWW. A concentration of them was studied within the pressure pipes, in the form of deposits, and this justifies the high concentrations of sulfides measured in certain wastewater networks. There are two reasons why the ferrous sulfate used as a treatment in any wastewater networks should not cause the production of additional sulfides. Firstly, the sulfate consumption kinetics are always too slow, relative to the residence time of the water in the pipe, for all of the sulfates to be consumed anyway. Secondly, the amount of assimilable carbon, soluble carbon, and carbon from suspended solid (SS) hydrolysis is insufficient.

Cultivation of New Emerging Agro-Nutritional Crop of Quinoa at Madinat al-Hikmah Karachi, Sindh, Pakistan

2017 ◽  
Vol 10 (1) ◽  
pp. 70-81 ◽  
Author(s):  
Muhammad Afzal Rizvi ◽  
Syed Abid Ali ◽  
Iqra Munir ◽  
Kousar Yasmeen ◽  
Rubina Abid ◽  
...  
Keyword(s):  
Arid Zone ◽  
Good Health ◽  
Morphological Characters ◽  
Morphological Data ◽  
Low Concentrations ◽  
Sindh Province ◽  
High Concentrations ◽  
Elemental Accumulation ◽  
Semi Arid ◽  
Good Agreement

Aim: Quinoa is a popular source of protein, minerals and alternative to traditional grains. The objective of this study is to introduce the Quinoa in the semi-arid zone of Sindh province of Pakistan. Method: A variety of NARC-9 from the agricultural Punjab province was cultivated and subjected to analyze the growth, morphological characters of the varieties obtained, saponin, protein and the elemental composition viz. Cd, Cu, Fe, K, Na, Pb, and Zn. Result: The result demonstrated the optimum growth and no disease were found in the experimental area. At least three major varieties of quinoa were obtained. Seed morphological data of these three quinoa cultivars were collected. The average saponin levels were quite reasonable. Overall proteins band pattern revealed very high polymorphism in quinoa cultivars and the results were also in good agreement with earlier studies. Conclusion: All quinoa cultivars of Madinat al-Hikmah showed high concentrations of albumin than globulin concentrations (i.e. 48-52% and 24-27%, respectively) as compared to control seeds from market that had similar concentrations of the two fractions i.e. 35.58% and 37.68%, respectively. Likewise, low concentrations of prolamin 14-16% and glutelin 11-12% compared to control seeds 13% rank our crop much better quality than the imported one in the market. The trend of elemental accumulation was followed as K >Na >Fe >Zn >Cu >Pb >Cd, while for comparison it was Na >K >Zn >Fe >Cu >Pb >Cd >Pb for wheat grown under similar conditions. Traditional grains together make a major contribution to the total nutritional element intake of the average Pakistani citizen through diet, not only because of large amounts consumed, but also in part by suitable levels of their proteins and elemental up take for good health. Thus the successful cultivation of quinoa in the semi-arid zone of Sindh will certainly prove beneficial.

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