scholarly journals Relationship between phosphoinositide kinase activities and protein tyrosine phosphorylation in plasma membranes from A431 cells

1990 ◽  
Vol 272 (3) ◽  
pp. 665-670 ◽  
Author(s):  
B Payrastre ◽  
M Plantavid ◽  
M Breton ◽  
E Chambaz ◽  
H Chap
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
A431 Cells ◽  
Phosphoinositide Kinase ◽  
Epidermal Growth ◽  
First Time ◽  
Dose Dependent

Production of PtdIns(4)P and PtdIns(4,5)P2 by plasma-membrane preparations from A431 cells was selectively stimulated in a dose-dependent manner by epidermal growth factor (EGF) in the presence of Na3VO4. Na3VO4 itself mimicked this effect, which was overcome after treatment by a specific phosphotyrosyl phosphatase isolated from A431 cells. PtdIns and PtdIns(4)P kinase activities were present in phosphotyrosyl-proteins isolated from EGF- and/or Na3VO4-stimulated A431 cells by immunoaffinity using an anti-phosphotyrosine antibody. These data suggest for the first time an EGF-dependent regulation of PtdIns 4-kinase and PtdIns(4)P 5-kinase activities by a mechanism involving a tyrosine-phosphorylation process.

EFFECTS OF VALINOMYCIN ON Rb+ FLUXES, ATP CONTENT AND INSULIN RELEASE IN PANCREATIC ISLETS

1978 ◽  
Vol 88 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Lars-Åke Idahl ◽  
Janove Sehlin ◽  
Inge-Bert Täljedal ◽  
Jorge Tamarit-Rodriguez
Keyword(s):  
Plasma Membrane ◽  
Linear Regression ◽  
Pancreatic Islets ◽  
Insulin Release ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Atp Content ◽  
Ion Permeability ◽  
Β Cells ◽  
Dose Dependent

ABSTRACT Valinomycin, 0.5–500 nm, was tested for its effects on pancreatic islets microdissected from non-inbred ob/ob-mice. Valinomycin decreased the islet accumulation of Rb+ and the content of ATP in a dose-dependent manner; efflux of Rb+ from pre-loaded islets was not noticeably changed. Rb+ accumulation and ATP content correlated markedly; on the model of linear regression, less than 10% of the change of Rb+ accumulation in valinomycin-treated islets was statistically attributable to factors other than ATP. Valinomycin did not cause a prompt inhibition of glucose-stimulated insulin release that could reflect hyperpolarization due to increased K+ permeability. The following conclusions are drawn: 1) The plasma membranes of β-cells resemble those of neurons in having such a high ion permeability as to be relatively little influenced by valinomycin; 2) Islet accumulation of Rb+ is due to a vectorial catalyst in the plasma membrane rather than to uptake by mitochondria; 3) Rb+ accumulation in islets is ATP-dependent.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach

1996 ◽  
Vol 109 (3) ◽  
pp. 687-698 ◽  
Author(s):  
T. Pomorski ◽  
P. Muller ◽  
B. Zimmermann ◽  
K. Burger ◽  
P.F. Devaux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Initial Velocity ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Aminophospholipid Translocase ◽  
Order Of Magnitude ◽  
Complicating Factors ◽  
First Time

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

scholarly journals Tyrosine phosphorylation of guanosine triphosphatase activating protein by activation via surface IgG in human B cells

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1535-1539
Author(s):  
H Yagura ◽  
N Oyaizu ◽  
S Pahwa
Keyword(s):  
B Cells ◽  
Tyrosine Phosphorylation ◽  
Signal Transduction Pathway ◽  
Dependent Manner ◽  
Transduction Pathway ◽  
Gtpase Activating Protein ◽  
Human B Cells ◽  
Associated Proteins ◽  
Guanosine Triphosphatase ◽  
Dose Dependent

In this study, we analyzed tyrosine phosphorylation of guanosine triphosphatase (GTPase) activating protein in human B cells stimulated through surface IgG, using Western blot and immunoprecipitation. Stimulation through surface IgG induced the tyrosine phosphorylation of GTPase-activating protein (GAP) and two associated proteins, a 190-Kd protein and a 62-Kd protein, within 1 minute and in a dose-dependent manner. This tyrosine phosphorylation was blocked by Genistein (Extrasynthese, Genay, France). These data suggest that GTPase- activating protein is involved in a signal transduction pathway initiated from surface IgG in human B cells.

scholarly journals Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

1995 ◽  
Vol 15 (12) ◽  
pp. 6777-6784 ◽  
Author(s):  
C A Pickett ◽  
A Gutierrez-Hartmann
Keyword(s):  
Signal Transduction ◽  
Transcription Factors ◽  
Growth Factor ◽  
Map Kinase ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Epidermal Growth ◽  
Dose Dependent

We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types.

scholarly journals Chlorotetracycline as a fluorescent Ca2+ probe in pancreatic islet cells.

1978 ◽  
Vol 76 (3) ◽  
pp. 652-674 ◽  
Author(s):  
I B Täljedal
Keyword(s):  
Plasma Membranes ◽  
Islet Cells ◽  
Dependent Manner ◽  
Cell Surfaces ◽  
Noticeable Effect ◽  
Cell Plasma ◽  
Glass Capillaries ◽  
Asymmetrically Distributed ◽  
Dose Dependent ◽  
Dispersed Cells

Pancreatic islets, or suspensions of islet cells, from noninbred ob/ob-mice were incubated with chlorotetracycline and analyzed for Ca2+-dependent fluorescence in a microscope. Unless logarithmically transformed, signals from islets were asymmetrically distributed with unstable variance. Signals from cells pelleted in glass capillaries were more homogeneous and depended linearly on the thickness of the sample. The effect of sample thickness and a significant enhancement of fluorescence by alloxan suggest that beta-cells were involved in producing the signal from whole islets. The signal from dispersed cells was probably diagnostic of Ca2+ in beta-cell plasma membranes because it was suppressed by La3+ and had a spectrum indicative of an apolar micromilieu; fluorescent staining of cell surfaces was directly seen at high magnification. Fluorescence from cells was enhanced by 0.5-10 mM Ca2+ in a dose-dependent manner, whereas less than 0.5 mM Ca2+ saturated the probe alone in methanol. The signal from islets or dispersed cells was suppressed by 5 mM theophylline; that from cells was also suppressed by 0.5 mM 3-isobutyl-1-methylxanthine, 1.2 or 15 mM Mg2+, 3-20 mM D-glucose, and, to a lesser extent, 20 mM 3-O-methyl-D-glucose. D-glucose was more inhibitory in the absence than in the presence of Mg2+, as if Mg2+ and D-glucose influenced the same Ca2+ pool. L-glucose, D-mannopheptulose, or diazoxide had no noticeable effect and 20 mM bicarbonate was stimulatory. The results suggest that microscopy of chlorotetracycline-stained cells can aid in characterizing calcium pools of importance for secretion. Initiation of insulin release may be associated with an increas

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1129-1137 ◽  
Author(s):  
P.E. Visconti ◽  
J.L. Bailey ◽  
G.D. Moore ◽  
D. Pan ◽  
P. Olds-Clarke ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Bovine Serum ◽  
Female Reproductive Tract ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Mode of Action of the Hypertrehalosaemic Peptides from the American Cockroach

1985 ◽  
Vol 40 (9-10) ◽  
pp. 670-676 ◽  
Author(s):  
Gerd Gäde
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mode Of Action ◽  
Glycogen Phosphorylase ◽  
Fat Body ◽  
American Cockroach ◽  
Dependent Manner ◽  
Low Concentrations ◽  
First Time ◽  
Dose Dependent

Abstract Although crude extracts of cockroach (Periplaneta amencana) corpora cardiaca have been shown previously to affect the activity of adenylate cyclase and phosphorylase, we demonstrate in the present study for the first time that low concentrations (0.5 to 5 pmol) of the synthetic myoactive peptides. M I and M II, also affect these systems; these myoactive peptides are identical to the hypertrehalosaemic hormones I and II, and cause an increase in the concentration of the second messenger cyclic AMP in the fat body.In addition, both octapeptides activate fat body glycogen phosphorylase and promote breakdown of fat body glycogen. Both peptides increase the levels to haemolymph carbohydrate in a dose-dependent manner.

scholarly journals The Influence of Bee Venom Melittin on the Functioning of the Immune System and the Contractile Activity of the Insect Heart—A Preliminary Study

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 494 ◽  
Author(s):  
Jan Lubawy ◽  
Arkadiusz Urbański ◽  
Lucyna Mrówczyńska ◽  
Eliza Matuszewska ◽  
Agata Światły-Błaszkiewicz ◽  
...  
Keyword(s):  
Immune System ◽  
Contractile Activity ◽  
Model Organism ◽  
Dependent Manner ◽  
Physiological Studies ◽  
Almost All ◽  
First Time ◽  
Dose Dependent ◽  
Positive Effect

Melittin (MEL) is a basic polypeptide originally purified from honeybee venom. MEL exhibits a broad spectrum of biological activity. However, almost all studies on MEL activity have been carried out on vertebrate models or cell lines. Recently, due to cheap breeding and the possibility of extrapolating the results of the research to vertebrates, insects have been used for various bioassays and comparative physiological studies. For these reasons, it is valuable to examine the influence of melittin on insect physiology. Here, for the first time, we report the immunotropic and cardiotropic effects of melittin on the beetle Tenebrio molitor as a model insect. After melittin injection at 10−7 M and 10−3 M, the number of apoptotic cells in the haemolymph increased in a dose-dependent manner. The pro-apoptotic action of MEL was likely compensated by increasing the total number of haemocytes. However, the injection of MEL did not cause any changes in the percent of phagocytic haemocytes or in the phenoloxidase activity. In an in vitro bioassay with a semi-isolated Tenebrio heart, MEL induced a slight chronotropic-positive effect only at a higher concentration (10−4 M). Preliminary results indicated that melittin exerts pleiotropic effects on the functioning of the immune system and the endogenous contractile activity of the heart. Some of the induced responses in T. molitor resemble the reactions observed in vertebrate models. Therefore, the T. molitor beetle may be a convenient invertebrate model organism for comparative physiological studies and for the identification of new properties and mechanisms of action of melittin and related compounds.

Cutaneous Adverse Effects With HER1/EGFR-Targeted Agents: Is There a Silver Lining?

2005 ◽  
Vol 23 (22) ◽  
pp. 5235-5246 ◽  
Author(s):  
Román Peréz-Soler ◽  
Leonard Saltz
Keyword(s):  
Surrogate Marker ◽  
Growth Factor Receptor ◽  
Dependent Manner ◽  
Activity Data ◽  
Egfr Inhibition ◽  
The Face ◽  
Epidermal Growth ◽  
Management Recommendations ◽  
The Relationship ◽  
Dose Dependent

The human epidermal growth factor receptor (HER1/EGFR) is dysregulated in many solid tumors, making it an attractive target for anticancer therapy. A number of agents that target this receptor are in use or in development. A specific adverse effect common to this class of agent is a papulopustular rash, usually on the face and upper torso, which generally occurs in a dose-dependent manner. Little is known about the etiology of this rash, and there are no clear evidence-based management recommendations. Histologic data indicate that rash may be caused by HER1/EGFR inhibition in skin, although this has not been confirmed. Findings suggest that there is a relationship between the development of rash and response and/or survival, making rash a potential surrogate marker of activity. Data from multiple studies with cetuximab and erlotinib show a consistent relationship between rash and response, as well as between rash and survival. The relationship between rash and clinical outcome is currently less consistent for gefitinib. Some studies report a correlation, whereas others do not. The cause of the possible relationship between rash and clinical benefit remains unclear at this time, and additional studies are needed to determine the clinical utility of this observation.

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