AY9944 inhibits early activation of phosphatidylinositol metabolism in concanavalin A-stimulated lymphocytes.
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Biomedical subjects
Publications and source records attributed to C Rampini.
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Amphiphilic molecules AY 9944 and chlorpromazine (CPZ) inhibited DNA synthesis in Concanavalin A-stimulated lymphocytes in a dose-dependent manner. While AY 9944 strongly decreased 7-dehydrocholesterol conversion to cholesterol, CPZ did not significantly affect this reaction. Moreover, the inhibitory effect of AY 9944 and CPZ on DNA synthesis took place in the presence of cholesterol in the culture medium. These findings suggest that the mechanism of inhibition of DNA synthesis by AY 9944 or CPZ is not related to endogenous cholesterol synthesis or exogenous cholesterol supply. Results are discussed in relation to the amphiphilic properties of AY 9944 and CPZ and to the interaction of these drugs with membranes or other intracellular targets such as calmodulin.
Concanavalin A-mediated stimulation of 32P-phosphate incorporation into phospholipids of human peripheral lymphocytes is comparatively studied in normal and phosphate-depleted media. In the phosphate-depleted medium, 2 hours after the start of cell activation, the stimulation sharply decreases for phosphatidylinositol (6.5-fold) and for phosphatidylcholine (in the latter case, the stimulation is even replaced by a slight inhibition of the incorporation). These results must be related to the rate-limiting effect of inorganic phosphate on ATP formation and thus on phospholipid synthesis, an effect which may be particularly pronounced when there is both phosphate depletion and cell activation.
The metabolism of gangliosides was studied during the in vitro differentiation of both normal quail myoblasts and myoblasts which have been transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV). These transformed cells can be maintained undifferentiated if incubated at 35 degrees C, but they will differentiate when shifted to 41 degrees C. (D. Montarras and M. Y. Fiszman (1983) J. Biol. Chem. 258, 3882-3888). The analysis of [14C]Glucosamine-labeled gangliosides by two-dimensional thin-layer chromatography reveals variations in the metabolism of the gangliosides during the process of differentiation. During the formation of myotubes, it was observed that the accumulation of GD1a is reduced, while the accumulation of GD3 is increased. Therefore, this results in the variation of the ratio GD3/GD1a which increases from 1.8 to 25 in the case of clones of transformed myoblasts, and from 0.5 to 1.7 in the case of uninfected myoblasts. These variations which have been observed seem to be specific of the myogenic differentiation since they cannot be reproduced when differentiation is inhibited by BUdR treatment or when fibroblasts reach confluency and are blocked in the G1 phase of cell cycle. Furthermore, the transformed myoblasts in vitro are shown to be a good model system since their gangliosides composition is very similar to that of muscle cells in vivo.
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The rate of 32P incorporation into phospholipids of EHSVi transformed fibroblasts was studied by pulse experiments (2 hours) in cells which are detached from the support by trypsin and either maintained in suspension or replated and in the process of adhering. This is performed comparatively to cells in monolayers. Cells are taken during or after the log phase. Both global incorporation into phospholipids and, in the case of cells in log phase, percentage of incorporation into phosphatidylinositol are lowered, after detachment from the support, in suspended cells. They are stimulated by a new contact with the support. In the same conditions, percentages of incorporation into phosphatidylethanolamine and phosphatidylcholine remain at values they have in cells in monolayers. These values are characteristic of the physiological state of cells in monolayers, in relation with medium factors exhaustion.
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Phospholipid metabolism of 3T3 mouse fibroblasts has been studied after serum stimulation of arrested cells. The study of [32Pi] incorporation shows : a) in the case of PE and PC an early peak of incorporation in the G1 phase of the cell cycle, 6 hours after serum addition; b) in the case of PI an intense initial increasing of the incorporation which continues up to a S phase peak. The study of the disappearance of [32P] Phosphate from the different phospholipids points out: a) at the beginning of serum stimulation, an intense breakdown of PI, that continues through the G1 and S phases. Except at the onset, the breakdown of PI, is at any time exactly compensated for by synthesis : the two phenomena are closely linked; b) a synthesis of PE, from PC probably, at the G1 phase, 4 hours after the serum addition and the beginning of the chase experiment.
Partial inhibition, by serum starvation, of multiplication of SV3T3 transformed fibroblasts was studied by cultivating cells in a medium containing 1% of serum. Two different and successive states are described: a first step, with a rate of multiplication for 24 hrs, which is lowered (2 instead of 3 in a normal medium containing 10% of serum) but which remains constant (i.e. exponential multiplication), the observed inhibition being reversed by normal medium; a second step, where the rate of multiplication for 24 hrs, decreases very fast, and which is not reversible.
The sensitivity of transformed EHSVi fibroblasts to the antibody-complement lytic system was evaluated, at various antibody concentrations (Ac), in conditions in which a variation of the lytic activity proportional to Ac3, 1 may be observed. This points out, not the expected cooperation between IgG molecules in activating C1 within a definite lytic unit, but a cooperation between the lytic units themselves, and therefore the existence of a cell defence.
E coli cells are harvested at early stationary phase and, first, incubated in phosphate buffer without energetic metabolites, conditions which invole the formation of an excess of diphosphatidylglycerol. If, in a second stage, they are cultivated again in normal medium, the diphosphatidylglycerol is metabolized very fast. It is mainly hydrolyzed, by activation of a specific phospholipase D, with formation of phosphatidylglycerol and phosphatidic acid. The latter is immediatly metabolized in bacteria in phosphatidylglycerol and phosphatidylethanolamine, both about equal in amount.
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The phospholipid composition and turnover in normal and in SV 40 transformed hamster fibroblasts were studied. The amount of phospholipid phosphorus relative to protein is lower in transformed hamster fibroblasts than in normal fibroblasts. This amount decreases with increasing cell density until stationary growth is reached. The decrease is largest for the normal fibroblasts. In transformed cells, less sphingomyelin and more diphosphatidyl glycerol are found than in normal cells. The turnover of 32P in sphingomyelin is slower in transformed cells than in normal cells ; the contrary is observed with diphosphatidyl glycerol. On the other hand, in transformed cells, phosphatidyl ethanolamine has a faster turnover than phosphatidyl choline, whereas the contrary is observed in normal cells. Finally, the change to stationary growth slows down the turnover of 32P of all phospholipids, this decrease being more important in transformed cells.
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