Yosef Graziani

Water permeability of bilayer lipid membranes: Sterol-lipid interaction.

SummaryBilayer lipid membranes were generated in an aqueous medium from synthetic, egg or plant phosphatidyl choline (PC) or from plant monogalactosyl diglyceride (MG). The water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. The osmotic permeability coefficient,Pos, was markedly affected by the number of double bonds in the fatty acid conjugates of the lipids: the greater the degree of unsaturation, the higher the value ofPos. The temperature dependence ofPos of the lipid membranes was studied over a range of 29 to 40°C. The experimental activation energy,Ea, estimated from the linear plots of log (Pos)versus 1/T, was significantly higher for MG membranes (17 kcal/mole) than for the various PC membranes (11 to 13 kcal/mole), probably owing to hydrogen bonding between MG and water molecules. In comparison with PC membranes, the membranes generated from PC and cholesterol (1∶1 molar ratio) had lowerPos but similarEa values. Likewise, either stigmasterol or β-sitosterol decreasedPos of MG membranes, whileEa was not affected by the sterols. MG-cholesterol membranes were specifically characterized by a unique value ofEa (−36 kcal/mole) thus indicating temperature dependent structural changes.

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinase activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.

Regulation of cyclic amp level and synthesis of dna, rna and protein by quercetin in ehrlich ascites tumor cells.

Abstract Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10 −4 M as well as 10 −2 M theophylline and 10 −3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [ 3 H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [ 3 H]uridine and [ 3 H]- L -leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10 −3 M), the specific inhibitor of Na + -K + pump system, did not alter the incorporation of [ 3 H]thymidine and [ 3 ]uridine, but decreased the incorporation of [ 3 H]- L -leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [ 3 H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [ 3 H]uridine and abolished completely their effect on incorporation of [ 3 H]-L-leucine.

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.

Elevation of cyclic AMP level in Ehrlich ascites tumor cells by quercetin

Abstract Quercetin, a flavonoid known to cause inhibition of ATPase activity and cell growth in vitro , increases cyclic AMP levels in Ehrlich ascites tumor cells. The possibility of interrelationship between cyclic AMP metabolism and (Na + + K + )ATPase activity is discussed.

cAMP-independent protein kinase activity is correlated with growth of rat mammary tumors

An important portion of the protein kinase activity in the 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary tumour is inhibited by the bioflavonoid quercetin (10(-4) M). By partial purification on a DEAE Cellulose column it was shown that the quercetin-inhibitable enzyme activity can be eluted in a separate peak which contains markedly reduced cAMP-dependent protein kinase activity. Since quercetin does not affect the cAMP-dependent protein kinase activity, this drug becomes a potent tool for the quantitation of this special activity in the tumor. By hormonal manipulation, namely ovariectomy and estrogen treatment, it was shown that changes in the growth rate of the tumour were closely correlated with the magnitude of these special protein kinase activities. These results suggest a possible cause-and-effect relationship between cyclic AMP-independent protein kinase activity and tumour malignancy in this chemically induced tumor. These data are similar to recent findings in viral-induced malignant transformation.

Inhibition of glucose efflux from human erythrocytes by hashish components

Abstract Glucose efflux from human erythrocytes under zero- trans conditions is inhibited by the hashish components Δ′-tetrahydrocannabinol and cannabidiol at concentrations above 5 × 10 −7 M. The inhibitory effect is rapid and apparently readily reversible. Increasing concentrations of alcohols up to 20 mM do not change the rate of glucose efflux but they do amplify the extent of efflux inhibition by a given dose of hashish component. Methanol, ethanol and propanol show this amplifying effect in a decreasing order.

Bilayer lipid membrane as a model for vasopressin, prostaglandin and ca2+ effects on water permeability

Abstract 1. 1. Bilayer lipid membranes were generated in an aqueous medium from egg phosphatidylcholine and the water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. 2. 2. In agreement with previous reports, vasopressin increased the water permeability coefficient of the lipid membrane. Cyclic AMP, over a wide concentration range, had little or no effect on the water permeability, substantiating a direct effect of the antidiuretic hormone on the membrane. 3. 3. Similar to observations in biological systems, Ca 2+ and prostaglandin E 1 inhibited, apparently competitively, the action of vasopressin on the water flow of the model membrane. The inhibitory effects were observed if Ca 2+ and prostaglandin E 1 were added prior to, but not after, vasopressin. 4. 4. The formation of a vasopressin-lipid membrane complex stabilized by electrostatic and hydrophobic interactions is proposed. 5. 5. Unlike valinomycin, neither vasopressin nor cyclic AMP changed significantly the electrical conductance of the bilayer membranes in a medium containing NaCl and KCl. 6. 6. It is concluded that bilayer lipid membranes apparently possess the characteristics of the proposed site of action of vasopressin in biological membranes related to water transport, while the second site, related to Na + transport, is lacking.