Association of AP1 adaptor complexes with GLUT4 vesicles

1999 ◽  
Vol 112 (24) ◽  
pp. 4793-4800 ◽  
Author(s):  
A.K. Gillingham ◽  
F. Koumanov ◽  
P.R. Pryor ◽  
B.J. Reaves ◽  
G.D. Holman
Keyword(s):  
Temperature Dependence ◽  
Western Blotting ◽  
Brefeldin A ◽  
Intracellular Sorting ◽  
In Vitro Effects ◽  
Gamma S ◽  
Isolated Cell

Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes.

scholarly journals Biochemical dissection of AP-1 recruitment onto Golgi membranes.

1993 ◽  
Vol 123 (3) ◽  
pp. 561-573 ◽  
Author(s):  
L M Traub ◽  
J A Ostrom ◽  
S Kornfeld
Keyword(s):  
Brefeldin A ◽  
In Vitro Assay ◽  
Coated Vesicle ◽  
Temperature Dependent ◽  
Fungal Metabolite ◽  
Vitro Assay ◽  
Golgi Membranes ◽  
Lag Period ◽  
Gamma S

Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.

The Association of the Adaptor Complexes AP1 and AP3 with GLUT4 Vesicles: Implications for GLUT4 Compartmentalisation

1999 ◽  
Vol 27 (3) ◽  
pp. A100-A100
Author(s):  
Alison K. Gillingham ◽  
Françoise Koumanov ◽  
Paul R. Pryor ◽  
Barbara J. Reaves ◽  
Geoffrey D. Holman
Keyword(s):  
Plasma Membrane ◽  
Western Blotting ◽  
Subcellular Localisation ◽  
Density Gradients ◽  
Transferrin Receptors ◽  
Rat Adipocytes ◽  
In Vitro Effects ◽  
Adaptor Recruitment

By using glycerol density gradients of post-plasma membrane supernatants from rat adipocytes, we have been able to separate the compartment containing GLUT4 from that containing transferrin receptors. The major pool of GLUT4 (≈ 80%) is localised to dense fractions, which also contain VAMP2, while the remainder is in lighter fractions, some of which also contain transferrin receptors. AP1 and AP3 complexes are detected in two fractions, the heavier of which co-sediments with the major pool of GLUT4. Nycodenz gradients have been used to examine the in vitro effects of GTP-γ-S on adaptor recruitment. On addition of GTPγS, GLUT4 vesicles fractionate as a heavier population of vesicles, which we suggest is due to a coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with API, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of AP1 and AP3 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased 3-fold by the addition of GTP-γ-S and an ATP regenerating system to the post-plasma membrane lysate. and 4-fold by carrying out the incubation at 37°C compared with 0°C. These data demonstrate that GLUT4 subcellular localisation can be studied in isolated fractions and that GLUT4 compartments are associated with at least two distinct adaptor complexes.

scholarly journals Targeting and mistargeting of plasma membrane adaptors in vitro.

1993 ◽  
Vol 123 (5) ◽  
pp. 1093-1105 ◽  
Author(s):  
M N Seaman ◽  
C L Ball ◽  
M S Robinson
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Coated Vesicle ◽  
Double Labeling ◽  
In Vitro System ◽  
Specific Antibodies ◽  
Excess Calcium ◽  
Gamma S ◽  
Intracellular Sequestration

Targeting and recruitment of the plasma membrane (PM) clathrin-coated vesicle adaptor complexes has been studied using an in vitro system based on permeabilized acceptor cells and donor cytosol. Through the use of species- and/or tissue-specific antibodies, only newly recruited exogenous PM adaptors are visualized. Targeting of PM adaptors can be switched from the plasma membrane to a perinuclear compartment by GTP gamma S or excess calcium. Prior treatment with brefeldin A prevents GTP gamma S-induced mistargeting. Double-labeling immunofluorescence and immunogold EM indicate that the perinuclear PM adaptor binding compartment is late endosomal. We propose that receptors for PM adaptors cycle between the plasma membrane and an endosomal storage compartment. Normally the receptors would be switched on only at the plasma membrane, but both GTP gamma S and calcium are capable of reversing this switch. Intracellular sequestration of PM adaptor receptors may provide the cell with a mechanism for up-regulating endocytosis following a burst of exocytosis.

Effect of Clofibric Acid on Desmin and Vimentin Contents in Rat Myocardiocytes

2006 ◽  
Vol 25 (5) ◽  
pp. 403-408 ◽  
Author(s):  
Adriana Sampayo-Reyes ◽  
Antonio Narro-Juárez ◽  
Salvador Saíd-Fernández ◽  
Héctor G. Lozano-Garza ◽  
Javier Vargas-Villarreal ◽  
...  
Keyword(s):  
Experimental Study ◽  
Adverse Effects ◽  
Western Blotting ◽  
Intermediate Filaments ◽  
Cytoskeletal Proteins ◽  
Clofibric Acid ◽  
Cellular Organization ◽  
In Vitro Effects ◽  
In Cells

The aim of this experimental study was to analyze in vitro effects of clofibric acid on vimentin and desmin contents in rat myocardiocytes, which was carried out in primary myocardiocyte cells that were treated only with clofibric acid at 0.1 mM. The measurement of vimentin and desmin were done by Western blotting and densitometry. This study showed that myocardiocytes exposed to clofibric acid exhibit a 26.3% decrease in vimentin and a 42.1% decrease in desmin. Considering the role that these intermediate filaments play in the anchorage and cellular organization of myocardiocytes, the decrease of desmin and vimentin observed in cells treated with clofibric acid may be partially responsible for the adverse effects observed in patients. In conclusion, the alteration of cytoskeletal proteins may be a cause of cardiopathy in patients treated with these compounds.

scholarly journals IN VITRO EFFECTS OF POLYPHENOLS EXTRACTED FROM THE AROEIRA PLANT (SCHINUS TEREBINTHIFOLIUS RADDI) ON THE GROWTH OF PROSTATE CANCER CELLS (LNCaP, PC-3 AND DU145)

2013 ◽  
Vol 1 (1) ◽  
Author(s):  
Luis Carlos Queires ◽  
Michel Crépin ◽  
Francis Vacherot ◽  
Alex De la Taille ◽  
Luiz Erlon Rodrigues
Keyword(s):  
Prostate Cancer ◽  
Cellular Growth ◽  
Cell Multiplication ◽  
Schinus Terebinthifolius ◽  
Cell Lineages ◽  
Humid Environment ◽  
In Vitro Effects ◽  
Natural Drugs ◽  
Isolated Cell

Despite the existence of various types of antineoplastic treatments of prostate cancer, most of them induce apoptosis in the tumor cells by means of synthetic or natural drugs. The aim of this work was to study the antiproliferative action of methanolic and aqueous extracts from the leaves of the aroeira plant (Schinus terebinthifolius) a plant from the Northeast from Brazil over isolated prostate cancer cell cultures. The extracts were tested in isolated cell lineages from non hormone dependent cancers (DU145 e PC-3), as well as in hormone-dependent ones (LNCaP). The cells were cultivated in a RPMI medium and were incubated at 37oC in a 5% CO2, humid environment. The activities of the extracts were evaluated within 3 and 6 days of cellular growth. The aqueous extract was more effective than the methanolic one in the inhibition of cell multiplication in all the lineages tested

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

1990 ◽  
Vol 64 (03) ◽  
pp. 402-406 ◽  
Author(s):  
M D Oethinger ◽  
E Seifried
Keyword(s):  
In Vitro Study ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Control Value ◽  
Dose Time ◽  
In Vitro Effects ◽  
Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

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.

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

Observations on the in vitro Effects of Chylomicra, Low-Density Lipoproteins and Phospholipids on Human Plasma Euglobulin Lysis

1963 ◽  
Vol 09 (01) ◽  
pp. 164-174 ◽  
Author(s):  
Albert R Pappenhagen ◽  
J. L Koppel ◽  
John H Olwin
Keyword(s):  
Human Plasma ◽  
Phosphatidyl Ethanolamine ◽  
Phosphatidyl Inositol ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Inhibitory Effects ◽  
Soybean Phospholipids ◽  
In Vitro Effects ◽  
Complete Precipitation

SummaryData have been presented on the in vitro effects of human chylomicra, low-density human plasma lipoproteins, and partially purified preparations of various phospholipids on human plasma euglobulin lysis. Euglobulin lysis was found to be accelerated by preparations of mixed soybean phospholipids (aso-lectin), cephalin, phosphatidyl inositol, phophatidyl serine and phosphatidyl ethanolamine. In contrast, it was found to be inhibited by preparations of human chylomicra, low-density human plasma liproproteins and lecithin. Inhibition of euglobulin lysis produced by any of these three agents could be diminished or completely overcome by the simultaneous presence of suitable levels of any one of the accelerating agents. In all cases studied, both inhibitory and accelerating effects were observed to be concentration-dependent. Evidence has been obtained to suggest that in the case of the accelerating agents the observed increased rate of euglobulin lysis is not a direct effect on lysis itself, but rather is due to more complete precipitation of plasminogen in the presence of these substances. On the other hand, it appears that the inhibitory effects observed are not related to the extent of plasminogen precipitation, but are actually true inhibitions of euglobulin lysis. The possible clinical significance of some of these observations has been briefly discussed.

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