Depletion of lysophosphatidic acid triggers a loss of oriented detyrosinated microtubules in motile fibroblasts

1996 ◽  
Vol 109 (10) ◽  
pp. 2461-2469 ◽  
Author(s):  
T. Nagasaki ◽  
G.G. Gundersen
Keyword(s):  
Lysophosphatidic Acid ◽  
Enzymatic Degradation ◽  
Plasma Membranes ◽  
Contact Inhibition ◽  
Signalling Pathways ◽  
Serum Factor ◽  
Motile Cells ◽  
Inhibition Response ◽  
High Concentrations

We reported earlier that isolated plasma membranes trigger a number of responses comprising contact inhibition of motility, including loss of oriented detyrosinated microtubules (Glu MTs) from the lamella of motile fibroblasts. In this study, we show that the membranes trigger this loss of Glu MTs, not by binding to cells, but by removing an essential component from the medium necessary to maintain oriented Glu MTs. Preincubation of membranes with medium containing serum followed by removal of the membranes by sedimentation rendered the membrane-treated medium capable of triggering the loss of oriented Glu MTs. Membrane activity was inhibited by high concentrations of serum and removal of serum from medium triggered the loss of oriented Glu MTs similar to that triggered by membranes. These results suggest that the membranes trigger the loss of Glu MTs by inactivating factors in serum that are required for the maintenance of oriented Glu MTs. By fractionating serum, we have identified lysophosphatidic acid (LPA) as the principal serum factor that is responsible for supporting oriented Glu MTs. The activity of LPA to maintain oriented Glu MTs upon serum withdrawal was half maximal at 100 nM and no activity was observed with structurally related phospholipids. Serum LPA levels were sufficient to account for the ability of serum to support oriented Glu MTs. Enzymatic degradation of serum LPA strongly reduced the ability of serum to support oriented Glu MTs. That membranes degrade LPA was shown by the ability of membranes to block LPA's ability to maintain oriented Glu MTs, and by direct measurement of the loss of radiolabeled LPA after incubation with membranes in vitro. These results show that isolated plasma membranes trigger the loss of Glu MTs from the lamella of motile cells by degrading serum LPA. Coupled with earlier results showing that membranes trigger a number of contact inhibition responses, our data suggest a new model for contact inhibition of motility in which local degradation of LPA and/or interference with LPA-stimulated signalling pathways initiates a contact inhibition response in colliding cells.

Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules

2002 ◽  
Vol 115 (10) ◽  
pp. 2053-2065
Author(s):  
Tummala Hemachand ◽  
Bagavathi Gopalakrishnan ◽  
Dinakar M. Salunke ◽  
Satish M. Totey ◽  
Chandrima Shaha
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Capra Hircus ◽  
Bifunctional Molecules ◽  
Sperm Surface ◽  
Sperm Plasma Membrane ◽  
Glutathione S Transferases ◽  
High Concentrations

Glutathione S-transferases (GSTs) are enzymes that detoxify electrophilic compounds. Earlier studies from our laboratory showed that anti-GST antibodies interfered with the fertilising ability of spermatozoa from Capra hircus (goat) in vitro, suggesting that GSTs are localised at the cell surface. In this study, we provide evidence for the presence of GSTs of 24 kDa on the sperm plasma membrane attached by non-covalent interactions. The GST activity associated with the spermatozoal plasma membrane was significantly higher than the activity present in the plasma membranes of brain cells,hepatocytes, spleenocytes and ventriculocytes. Analysis of GST isoforms demonstrates the presence of GST Pi and Mu on the sperm plasma membranes. Both isoforms were able to bind to solubilised as well as intact zona pellucida(ZP) through their N-terminal regions but failed to bind to ZP once the oocytes were fertilised. Solubilised goat ZP separates into three components,one of which, the ZP3-like component, bound to sperm GSTs. High concentrations of anti-GST antibodies or solubilised ZP led to aggregation of sperm GSTs,resulting in the release of acrosin. In contrast, inhibition of sperm GST binding to ZP, by saturation of binding sites for sperm GSTs on the solubilised ZP using peptides designed from the N-terminii of GST Pi or Mu or blocking of binding sites for ZP on sperm GSTs with antibodies raised against the N-terminal GST peptides, inhibited essential prefertilisation changes in sperm. These data therefore demonstrate the strategic location of catalytically active defensive enzymes on the sperm surface that also act as zona-binding proteins. Therefore, sperm-surface GSTs serve as bifunctional molecules in a transcriptionally inactive cell whose requirement for cellular defense and economy of molecules that it can carry is greater than that of any somatic cell type.

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.

Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

1997 ◽  
Vol 78 (04) ◽  
pp. 1173-1177 ◽  
Author(s):  
Jacek Musiał ◽  
Jakub Swadźba ◽  
Miłosz Jankowski ◽  
Marek Grzywacz ◽  
Stanisława Bazan-Socha ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Skin Incision ◽  
Anticardiolipin Antibodies ◽  
Small Blood Vessel ◽  
Anionic Phospholipids ◽  
Increased Risk ◽  
High Concentrations

SummaryAntiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

1982 ◽  
Vol 47 (02) ◽  
pp. 150-153 ◽  
Author(s):  
P Han ◽  
C Boatwright ◽  
N G Ardlie
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Antiarrhythmic Drugs ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Second Phase ◽  
Ionophore A23187 ◽  
High Concentrations ◽  
Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

Mouse Trophoblast Cells Are Constitutive Producers of Thromboplastin (Factor III) In Vitro

1985 ◽  
Vol 54 (02) ◽  
pp. 438-441 ◽  
Author(s):  
K Dalaker ◽  
E Haug ◽  
H Prydz
Keyword(s):  
Complement System ◽  
Serum Factor ◽  
Trophoblast Cells ◽  
Thromboplastin Factor

SummaryTrophoblasts from murine placenta synthesize thromboplastin in the absence of inducing agents and a functional complement system, nor is the rate or level of synthesis enhanced by inducers. A serum factor which is destroyed/removed by addition of oxalate and subsequent dialysis appears to enhance the ability of trophoblasts to synthesize thromboplastin.

Needleless electrospinning of PVP/PGS fibrous scaffolds for skin tissue engineering applications

2018 ◽  
Author(s):  
Antonios Keirouz ◽  
Giuseppino Fortunato ◽  
Anthony Callanan ◽  
Norbert Radacsi
Keyword(s):  
Tissue Engineering ◽  
Tensile Modulus ◽  
Dermal Fibroblasts ◽  
Skin Tissue ◽  
Skin Substitute ◽  
Electrospinning Technique ◽  
Skin Tissue Engineering ◽  
Needleless Electrospinning ◽  
High Concentrations

Scaffolds and implants used for tissue engineering need to be adapted for their mechanical properties with respect to their environment within the human body. Therefore, a novel composite for skin tissue engineering is presented by use of blends of Poly(vinylpyrrolidone) (PVP) and Poly(glycerol sebacate) (PGS) were fabricated via the needleless electrospinning technique. The formed PGS/PVP blends were morphologically, thermochemically and mechanically characterized. The morphology of the developed fibers related to the concentration of PGS, with high concentrations of PGS merging the fibers together plasticizing the scaffold. The tensile modulus appeared to be affected by the concentration of PGS within the blends, with an apparent decrease in the elastic modulus of the electrospun mats and an exponential increase of the elongation at break. Ultraviolet (UV) crosslinking of PGS/PVP significantly decreased and stabilized the wettability of the formed fiber mats, as indicated by contact angle measurements. In vitro examination showed good viability and proliferation of human dermal fibroblasts over the period of a week. The present findings provide important insights for tuning the elastic properties of electrospun material by incorporating this unique elastomer, as a promising future candidate for skin substitute constructs.

Methods of experimental polyploidization and their use in apple breeding

2020 ◽  
Vol 62 ◽  
pp. 85-90
Author(s):  
L. V. Tashmatova ◽  
O. V. Matsneva ◽  
T. M. Khromova ◽  
V. V. Shakhov
Keyword(s):  
Exposure Time ◽  
Cell Walls ◽  
Prolonged Exposure ◽  
Ornamental Plants ◽  
Colchicine Treatment ◽  
Apple Trees ◽  
Open Ground ◽  
High Concentrations ◽  
Diploid Gametes

The article presents methods of experimental polyploidy of fruit, berry and ornamental plants. The purpose of this review is to highlight the problems and prospects of polyploidization of plants in the open ground and in vitro culture and the possibility of their application for apple trees. For the purpose of obtaining apple tetraploids as donors of diploid gametes, seed seedlings were treated with a solution of colchicine in concentrations of 0.1-0.4 % for 24 and 48 hours. Colchicine concentrations of 0.3 % and 0.4 % at 48 hours of treatment had a detrimental eff ect on their development. As a result, tetraploids and chimeras were obtained from seeds from free pollination of the varieties Orlik, Svezhest, Kandil Orlovsky, as well as from seeds obtained from crossing the varieties Svezhest×Bolotovskoe, Moskovskoe Оzherel’e×Imrus, Girlyanda×Venyaminovskoe. The optimal concentration of colchicine was 0.1 %. Methods of colchicine treatment have been studied: 1) adding to the nutrient medium, colchicine concentration: 0.01%, 0.02%, exposure time 24h-19 days; 2) applying amitotic solution to the growth point, colchicine concentration: 0.1 %, 0.2 %, exposure time 24h-7 days. To increase the penetration of colchicine through the cell walls, a 0.1 % dimexide solution was used. Studies have shown that high concentrations and prolonged exposure to colchicine reduce the viability of explants.

The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

2020 ◽  
Vol 65 (9-10) ◽  
pp. 3-7
Author(s):  
V. V. Gostev ◽  
Yu. V. Sopova ◽  
O. S. Kalinogorskaya ◽  
M. E. Velizhanina ◽  
I. V. Lazareva ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
High Concentration ◽  
Short Term ◽  
High Concentrations ◽  
Selection Of ◽  
Selection Of Resistance ◽  
Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

INDUCTIVE AND INHIBITORY ACTIONS OF A LOW MOLECULAR WEIGHT SERUM FACTOR ON IN VITRO MATURATION OF OOCYTES OF THE MEDAKA

1987 ◽  
Vol 8 (5) ◽  
pp. 313-322 ◽  
Author(s):  
TAKASHI IWAMATSU ◽  
SUSUMU Y. TAKAHASHI ◽  
NORIYOSHI SAKAI ◽  
KAORI ASAI
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight ◽  
Serum Factor
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