Preferential interactions of fluorescent probe Prodan with cholesterol.

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal Lalpha, 435 nm for the gel L'beta, and 507 nm for the interdigitated gel LbetaI phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 x 10(4) in the interdigitated gel, 1.8 x 10(4) in the noninterdigitated gel, and 7. 6 x 10(4) in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 x 10(4) for the liquid crystal and the gel phase, and 5.1 x 10(4) in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.

Role of cholesterol in the modulation of interdigitation in phosphatidylethanols.

Phosphatidylethanol (Peth) is formed in biological membranes when ethanol replaces water in the transphosphatidylation reaction catalyzed by phospholipase D. This charged lipid accumulates in the presence of ethanol, and it has unusual properties that can influence membrane structure and function. We have previously shown that dimyristoylphosphatidylethanol (DMPeth) and dipalmitoylphosphatidylethanol (DPPeth) form the interdigitated gel phase in the presence of Tris-HCl [O.P. Bondar, E.S. Rowe, Biophys. J., 71 (1996) 1440-1449]. In the present investigation, differential scanning calorimetry (DSC) and fluorescence have been used to investigate the effect of cholesterol on the phase behavior of DPPeth and DMPeth. Our results show that cholesterol prevents the formation of the interdigitated phase in the presence of Tris-HCl, and that ethanol counters this influence and restores the ability of these lipids to interdigitate. Pyrene-PC fluorescence probe was used in this investigation and gave results that were in agreement with the conclusions based on the DSC study.

Flavonols--new fluorescent membrane probes for studying the interdigitation of lipid bilayers.

Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy-4'-(15-azacrown-5) flavone (FRC) have been investigated as new fluorescence probes for studying the formation of the interdigitated gel phase in lipid bilayers. The formation of the interdigitated gel phase in the saturated symmetrical phosphatidylcholines (PCs) and phosphatidylethanol (Peth) in the presence of ethanol has been well studied. The present study examines the behavior of these new probes in PC-ethanol and Peth-ethanol systems, as well as in PC-cholesterol and Peth-cholesterol vesicles. The present results demonstrate that both flavonols give distinctively different spectra in interdigitated lipid compared to non-interdigitated lipids, when examined in lipids in which the interdigitation behavior is known. This makes them useful for determinations of the structural state of unknown lipids, and for following the transitions between interdigitated and non-interdigitated phases. However, in the presence of cholesterol, only FCR gave appropriate indications of interdigitation. The results with FME in the presence of cholesterol were not consistent with the known behavior of the lipids examined; instead, FME appears to be located preferentially in the cholesterol-rich non-interdigitated regions of the bilayer.

Effect of vitamin D3 and 1,25(OH)2D3 on growth of neoplastically derived cell lines and their alkaline phosphatase activity.

Inhibitory effects of 1,25(OH)2D3 and D3 on growth of four neoplastically derived cells were observed in human acute leukemia cell culture CEM-C-1 and CEM-C-7, human cervical carcinoma cell lines C-4-1 and human epithelioid carcinoma cells of cervix HeLa S3K. Concurrently, in dexamethasone-responsive cells C-4-1 and HeLa S3K there was a 1,25(OH)2D3 and D3 induced elevation of alkaline phosphatase with 1,25(OH)2D3 showing the greater effects. It is supposed that vitamins D3-induced alkaline phosphatase activity in malignant cells, which is proposed to be a possible marker of cell differentiation, can be associated with the membrane effects of these vitamins.

Thermotropic properties of phosphatidylethanols.

Phosphatidylethanol is formed when ethanol substitutes in the transphosphatidylation reaction catalyzed by phospholipase D. The structural and thermotropic properties of dipalmitoylphosphatidylethanol and dimyristoylphosphatidylethanol have been studied using differential scanning calorimetry, fluorescence spectroscopy, and 31P nuclear magnetic resonance. These lipids exist in a bilayer phase with no indication of nonbilayer phase formation, as shown by 31P nuclear magnetic resonance. It was found that the phase behavior of these phospholipids before and during the main chain melting transition is different in 50 mM Tris buffer compared to salt solutions. The phase transition behavior and the 6-propionyl-2-(dimethylamino)naphthalene (Prodan) fluorescence spectra for both lipids are consistent with the formation of the interdigitated gel phase under certain conditions. Both lipids become interdigitated in Tris-HCl, and ethanol enhances the formation of this phase. Comparative studies of the 6-propionyl-2-(dimethylamino)naphthalene spectra in dipalmitoylphosphatidylglycerol, dielaidoylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine further elucidate the value and limitations of this probe as a diagnostic tool for lipid structure.

Differential scanning calorimetric study of the effect of vitamin D3 on the thermotropic phase behavior of lipids model systems.

Differential scanning calorimetry has been used to investigate the effects of vitamin D3 on the physical properties of model membranes including pure phosphatidylcholines (PC's) of chain length from 14 to 18, pure dipalmitoylphosphatidylethanolamine (DPPE), and various mixtures of these lipids. The results demonstrate that the interactions of vitamin D3 with PC's are dependent on acyl chain lengths. It was found that vitamin D3 reduces the transition temperatures of PC's and PE's, broadening the transition and reducing the enthalpy, eventually abolishing the transition. The interaction of vitamin D3 with PC varies with chain length; the transition of DMPC is abolished by only 15 mol% vitamin D3 whereas 45 mol% vitamin D3 is required to abolish the transition of DSPC. These variations in vitamin D3 lipid interactions are further explored in various mixtures. In the mixture studies it is shown that vitamin D3 affects the mixing properties of the lipids in the mixtures. The results suggest that the presence of vitamin D3 in lipids can affect the lateral phase distributions of lipids, and thereby have important effects on membrane function.

Effects of farnesol on the thermotropic behavior of dimyristoylphosphatidylcholine.

Differential scanning calorimetry (DSC) and DPH fluorescence anisotropy have been used to investigate the effects of trans-trans farnesol on the physical properties of model membranes and extracted cell lipids. Farnesol was shown to have a significant effect on the gel to liquid-crystal phase transition temperature, the enthalpy of the transition and the transition co-operativity for extruded vesicles of dimyristoylphosphatidylcholine (DMPC). The phase transition of DMPC vesicles was eliminated at 25 mol% farnesol. Farnesol decreased the fluorescence anisotropy of the lipids extracted from human leukemia line CEM-C1 cells.

[The effect of cholesterol and dicetylphosphate on the physical parameters of liposomes]. / Vliianie kholesterina i ditsetilfosfata na fizicheskie parametry liposom.

The physical parameters of liposomes from phosphatidylcholine with cholesterol or dicetylphosphate were investigated. The sizes, electrophoretic mobility, surface density charges of these liposomes were estimated. It has been shown that dicetylphosphate, as well as cholesterol increases the thickness of bilayer of the lecithin liposomes and at the same time the area occupied by one lipid molecule has not been changed by dicetylphosphate in contrast to cholesterol.

[Comparative characteristics of color reactions to ecdysteroids]. / Sravnitel'naia kharakteristika tsvetnykh reaktsii na ékdisteroidy.

The colour reactions used to determine sterols have been compared to find most sensitive method of quantitative determination of ecdysteroids. It is shown that the Liberman-Burchard reaction is most sensitive to most ecdysteroids, while the Chugaev reaction is most specific.

[The enhancement under the action of a limited diet of the antimetastatic effect due to macrophage activation in mice with Lewis carcinoma]. / Usilenie pod vozdeistviem ogranichennogo ratsiona antimetastaticheskogo éffekta, obuslovlennogo aktivatsiei makrofagov, u myshei s kartsinomoi L'iuis.

An antimetastatic effect associated with macrophage activation by liposome-encapsulated glucosaminylmuramyldipeptide was found to enhance in malnourished mice with the Lewis lung carcinoma. These changes were not matched by further increase in the functional activity of macrophages. It has been suggested that enhancement of the antimetastatic effect in malnourished animals is due to the inhibition of neovascularization necessary for the beginning of metastatic exponential growth. The induction of neovascularization may be caused by the tumor necrosis factor, main product of activated macrophages.

[The effect of muramyl dipeptide analog, GMPD, incorporated into liposomes of various composition on the functional activity of macrophages]. / Vliianie analoga muramildipeptida--GMDP,--zakliuchennogo v liposomy razlichnogo sostava, na funktsional'nuiu aktivnost' makrofagov.

Liposomes of different composition and N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMPD) encapsulated in them are studied for their effect on the functional activity of macrophages by means of determining the 5'-nucleotidase and adenosine deaminase activity in the in vivo experiments. It is shown that both liposomes and GMDP encapsulated in them increase the activity of adenoside deaminase and decrease that of 5'-nucleotidase. This evidences for a change in the adenosine metabolism in the alveolar and peritoneal macrophages and an increase in the functional activity of cells which resulted from that rise. The manifestation of the process depends both on the lipid composition of liposomes and their charge. The observed increase in the functional activity of the alveolar macrophages under the effect of liposomes and GMDP encapsulated in them correlates with inhibition of the lung metastases development in mice.

[Surface charge of erythrocyte membrane during disorders of lipid metabolism from the data of microelectrophoresis, H+-titration and fluorescence studies]. / Poverkhnostnyi zariad membran éritrotsitov pri narusheniiakh lipidnogo obmena po dannym mikroélektroforeza, H+-titrovaniia i fluorestsentnykh issledovanii.

The surface charges and the isoelectric points (pI) as well as changes in these values depending on the lipid composition are determined for erythrocyte membranes of rat, rabbit, bull and man under conditions of hypercholesterinemia, atherosclerosis, D-hypovitaminosis and in experiments in vitro using the techniques of microelectrophoresis and positive charged ligands (proton, probe astraphloxin) binding. The ionogenic groups forming charge and essential differences in their nature (pK) and quantity depending on the erythrocyte type are found. Inverse correlation between the content of cholesterol and cholesterol ethers and the value of erythrocyte membrane surface charges was established for each sample examined. A comparative evaluation of the methods used was conducted.

[Antimetastatic effect of a liposome-enclosed analog of muramyl dipeptide]. / Antimetastaticheskii éffekt analoga muramildipeptida, zakliuchennogov liposomy.

The influence of a new, liposome-encapsulated muramyldipeptide analog--GMDP--on Lewis lung carcinoma spreading was studied in mice in which primary tumor had been removed. The drug was found to significantly decrease the extent and number of lung metastases, as compared to mice which had not received GMDP. This was matched by recovery of alveolar and splenic macrophages functional activity, as assessed by adenosine deaminase and 5'-nucleotidase levels in these cells.

[Effect of GMDP encapsulated into liposomes on metastatic spread of Lewis lung carcinoma]. / Vliianie GMDP, zakliuchennogo v liposomy, na metastazirovanie kartsinomy legkogo L'iuis.

N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP), a new liposome-encapsulated muramyl dipeptide analog, was studied for its effect on the Lewis lung carcinoma metastatic spreading as well as on the adenosine deaminase (ADA) and 5'-nucleotidase (5-N) activity in the alveolar and peritoneal mice macrophages. The drug administration was found to cause a sharp dose-dependent decrease in the lung metastases volume and number as compared to those in mice not treated with GMDP. The antimetastatic effect of GMDP is accompanied by an increase in the functional macrophage activity determined by ADA and 5-N level in these cells.

[Physico-chemical properties of IgG characteristic for normal growth]. / Fiziko-khimicheskie svoistva IgG, kharakternogo dlia normal'nogo rosta.

It is shown that IgG typical of the normal growth appears in blood of rats with the regenerating liver and in pregnant ones and its capacity to form complexes with alpha-globulins insoluble in acid medium, isoelectric point and molecular mass are similar to these features in IgG typical of the malignant growth. At the same time hydrophobic properties and energy stability of this protein are close to those of IgG in control animals. The accessibility of ionogenic groups for titration in the studied protein varies like that observed in IgG typical of the malignant growth but is less pronounced. The data obtained evidence for the fact that subpopulations of IgG molecules with similar but not identical properties appear in blood of animals with the normal and malignant growth.

[Effect of monochromatic light on oxygen transport across the multibilayer lecithin film pigmented wih carotene]. / Vliianie monokhromaticheskogo osveshcheniia na perenos kisloroda cherez mul'tibisloinuiu plenku letsitina, pigmentirovannuiu karotinom.

The permeability of beta-carotene-pigmented lecithin multibilayer was studied under the effect of monochromatic illumination for O2 in media with various ionic composition. It is shown that illumination of film within the 400 to 500 nm wavelengths causes a decrease in the permeability of the multibilayer for O2. The effect of light depends on the cation type. Hydration of the multilbilayer within the NaCl solution recovers the permeability of the system after switching of the light. In CaC12 solutions the light reaction is more pronounced but after the light switching off there is no the reversible recovery of the permeability for O2.

[Hypocholesterolemic effect of phytoecdysones during experimental hypercholesterolemia in rats]. / Gipokholesterinemicheskoe deistvie fitoékzidonov pri éksperimental'noi giperkholesterinemii u krys.

The most active hypocholesterolemic preparation, affecting the absorption of 3H1-cholesterol in small intestine of rats with experimental hypercholesterolemia, proved to be I-hydroxyecdisterone; the activity was gradually decreased in the following series of preparations: ecdisterone, steroid sapogenine allyogenone, ciasterone and ecdisterone 25 acetate. Thus, the hypocholesterolemic activity of the preparations was increased with an increase in the content of hydroxy groups in the molecules. After daily administration of ecdisterone at a dose of 2.5 mg/kg into the animals with hypercholesterolemia within 3, 6 and 8 weeks, content of cholesterol in blood plasma was decreased by 7.0%, 16.9% and 29%, respectively; this phenomenon was accompanied by a decrease of the cholesterol content in erythrocyte membranes as well as in microfilaments of erythrocyte border by 26% and 34%, respectively. Amount of phospholipids and the ratio cholesterol/phospholipids were also normalized in the membranes studied. The data obtained suggest that competitive relationship occurs between cholesterol and the hypocholesterolemic drugs during binding with the membrane sites depending on their concentrations in blood plasma, intestine lumen and on the content in the membranes.

[Changes in charge density of cholesterol-enriched erythrocyte membranes]. / Izmenenie plotnosti zariadov membran éritrotsitov pri obogashchenii ikh kholesterinom.

Application of potentiometric titration and fluorescent probes showed that density of negative charges of erythrocyte membranes decreases with their cholesterol enrichment by different methods (keeping animals on a diet causing hypercholesterinemia, application of cholesterol-lecithin liposomes in experiments in vitro). These effects are largely due to cholesterol-induced in accessibility for titration of a definite amount of carboxylic, imidazole and secondary phosphate groups of the membrane components and to an increase in the number of titrated epsilon-NH2-groups resulted from the weakening of electrostatic interaction of the membrane charges. The found shifts of pK groups dissociating in the region of pH neutral values and shifts in the fluorescence peaks of a positively charged probe of astraphloxin towards the spectral short-wave region with cholesterol enrichment of the membranes evidence for changes in microenvironment of the studied polar groups. A separate quantitative expression of the cholesterol-induced effects of viscosity increase and a density decrease of negative charges of membranes affecting the processes of astraphloxin binding is shown to be possible.

[Method for estimating data of potentiometric titration of membrane fragments by computers]. / Metod otsenki dannykh potentsiometricheskogo titrovaniia fragmentov membran s pomoshchiu EVM.

The flow chart of the computer programme is described for estimation of the quantity and pK values of separate ionogenic sites of the membrane based on the data of potentiometric titration with application of graphic method for calculation of parametres of titration differential curves. Its application makes it possible to reduce the time for calculating the potentiometric curves, to chose minima in the differential curve more objectively for characterization the pK values and to calculate areas of its different plots, which characterize binding capacity for ionogenic groups. The programme was written in the Analytic language for the MIR-2 computer.