Embryotropic effect of glycosaminoglycans and receptors in development of porcine pre-implantation embryos

The present study investigated the expression of receptors for glycosaminoglycans (GAGs) and the effect of GAGs supplementation on development of porcine IVF embryos. Total RNA was prepared from oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts. The expression of hyaluronic acid receptor (CD44) and heparin (HP) interacting protein (HIP) was determined using RT-PCR and Southern blot analysis. The CD44 and HIP mRNA were detected from in vitro matured oocytes and all stages of pre-implantation embryos. The IVF embryos were cultured in modified NCSU-23 medium supplemented with various concentrations (0, 0.1, 0.5 or 1.0 mg/mL) of hyaluronic acid (HA) or heparin. Supplementing with 0.5 mg/mL HA significantly increased total cell number compared to other experimental groups, due to increase in trophectoderm cells. Supplementing with 1.0 mg/mL, HP significantly increased blastocyst formation rate compared to the control group. Supplementing media, in which IVF embryos were cultured with 0.5 mg/mL HA + 1.0 mg/mL HP, significantly increased blastocyst formation rate compared to the control group. In conclusion, the present study demonstrated the expression of HA and HP receptors and the embryotrophic effect of HA or HP on porcine IVF embryos.     

Thermodynamics of partitioning of chitinase and laminarinase in a soya lecithin liposome system and their antifungal effect against Fusarium oxysporum

Abstract

The goal of the present work was to compare the partitioning behavior of chitinase and laminarinase (from Trichoderma spp.) in soya lecithin liposomes at different temperatures and examine the activity of the resulting microencapsulated enzymes against Fusarium oxysporum. In both cases the partition coefficients (K_o/w) were greater than 1, indicating affinity of the enzymes for microencapsulation in liposomes. Enthalpy calculations indicated that the process was endothermic in the case of laminarinase and exothermic in the case of chitinase. Soya lecithin liposomes were stable for more than 20 days. The stability of the immobilized enzyme was increased in the case of chitinase, but was not changed in the case of laminarinase. Although the antifungal effects of individual immobilized preparations decreased after microencapsulation compared with non-immobilized enzymes, they were increased by the synergistic effect of both encapsulated enzymes. The application of free or immobilized enzymes was also shown to enhance the inhibitory effect of the chemical fungicide, thiabendazole, on F. oxysporum.     

Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival

Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post-thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition.     

Chelating and antiradical effect of rutin during peroxidation of lipids from microsomes and liposomes

The antioxidative effect of rutin (vitamin P) on Fe2+-induced lipid peroxidation (LPO) in bovine heart microsomes and lecithin liposomes was studied. It was shown that the LPO-induced inhibition of microsomes and liposomes in the presence of rutin occurs via two mechanisms, i.e., association of Fe2+ ions to form an inactive complex and a direct interaction between rutin and free radicals. The contribution of these mechanisms depends on the composition of the reaction mixture. In bovine heart microsomes and liposomes, ascorbic acid has a dual activity towards LPO. At high concentrations of Fe2+ necessary for LPO induction (approximately 1 x 10(-3) M), ascorbic acid blocks LPO, whereas at low Fe2+ concentrations (less than 1 x 10(-4) M) it has a prooxidative effect. A combined use of ascorbic acid and rutin results in an additive antioxidative effect at high Fe2+ concentrations (approximately 1.10(-3) M). However, at low Fe2+ concentrations rutin acts as an antagonist of the prooxidative effect of ascorbic acid.     

Microviscosity in lecithin liposomes: effect of nicotinic acid

We have studied the effect of nicotinic acid, a drug commonly used as a vasodilatory agent and also for the treatment of hypercholesterolemia, on the fluidity profile of liposomes of egg lecithin and dipalmitoyl lecithin, using a fluorescent polarization probe. In both cases the drug decreases the membrane fluidity and for cholesterol-probed liposomes, it disrupts the "intermediate fluid condition" induced by cholesterol. The drug also affects the activation energy for diffusion in the hydrophobic region of the liposomes.     

Various properties of urease encapsulated into liposomes

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.     

Cholesterol crystal nucleation from enzymatically modified low-density lipoproteins: combined effect of sphingomyelinase and cholesterol esterase

An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. F?rster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.     

Circular dichroism study of membrane dynamics. Effects of carboxymethyl-chitin

The circular dichroism (CD) active phospholipid bis(4'-n-octanoxyazobenzene-4-carboxyl)-L-alpha-phosphatidylcholin e (CDPC) was used to study the effects of carboxymethyl-chitin (CM-chitin) on the membrane dynamics. For this purpose, CD and electronic spectra were observed for a mixture of CM-chitin and liposomes composed of CDPC and egg lecithin (EPC) (50% CDPC-EPC-liposomes), and for a mixture of CM-chitin, 50% CDPC-EPC-liposomes, and EPC-liposomes. CM-chitin at concentrations higher than ca. 10(-5) M induced the increase of absorbance at 600 nm synchronized with the dilution of CDPC in 50% CDPC-EPC-liposomes by EPC matrix, indicating that CM-chitin induces the fusion among a few liposomes and/or the lipid transfer through the transient liposome fusion. Temperature dependent CD spectra of a mixture of the 50% CDCP-EPC-liposomes and CM-chitin suggested that even high concentration of CM-chitin has no significant perturbation of the lipid organization in the membranes. The effects of CM-chitin on the leakage of [3H]sucrose from the EPC-liposomes were also studied. By the presence of 5 X 10(-6) M CM-chitin, the half-life for leakage of [3H]sucrose in plasma was increased by 4 times. This effect of CM-chitin was reduced by chitinase, while no effect was observed with lysozyme, suggesting that CM-chitin molecules are adsorbed on the liposome surface and stabilize the liposomes against the plasma proteins.     

Study of the mechanism of permeabilization of lecithin liposomes and rat liver mitochondria by the antimicrobial drug triclosan

The effect of the antimicrobial compound triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol) on the permeability of lecithin liposomes and rat liver mitochondria was studied. It was found that triclosan was able to increase nonspecific permeability of liposomes in a dose-dependent manner, which was detected by the release of the fluorescent probe sulforhodamine B (SRB) from vesicles. A partial release of SRB occurs instantly at the moment of triclosan addition, which is followed by a slow leakage of the dye. The triclosan-induced release of SRB from liposomes grew as pH of the medium was decreased from 9.5 to 7.5. As revealed by the laurdan generalized polarization (GP) technique, triclosan increased laurdan GP in lecithin liposomes, indicating a decrease in membrane fluidity. Measurements of GP as a function of fluorescence excitation wavelength gave an ascending line for triclosan-containing liposomes, which can be interpreted as phase heterogeneity of the lipid/triclosan system. Dynamic light scattering experiments also showed that at a high triclosan-to-lipid molar ratio (~ 0.5), a population of smaller light-scattering particles (~ 0.4 of the size of liposomes) appear in the system. Experiments with rat liver mitochondria demonstrated that triclosan (10–70 μM) induced a high-amplitude cyclosporin А-insensitive swelling of the organelles accompanied the release of cytochrome c. On the basis of the results obtained, possible mechanisms of the toxic effect of triclosan in eukaryotic cells are discussed.     

Correlations for the partition behavior of proteins in aqueous two-phase systems: effect of overall protein concentration

The effect of protein concentration in partitioning in PEG/salt aqueous two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000/phosphate system with 3% w/w NaCl was used for alpha-amylase. The concentration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on their physicochemical properties. In the top phase, maximum protein concentration was determined mainly by a steric exclusion effect of PEG, and hydrophobic interaction between PEG and proteins. In the bottom phase, maximum concentration was determined mainly by a salting-out effect of the salts present. As the ionic strength was increased in the systems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting-out effect. Amyloglucosidase had a very low maximum concentration in the PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the salt-rich bottom phase due to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and, hence, a stronger salting-out effect. The protein concentration in each of the two phases was correlated with a "saturation"-type equation. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between the "saturation" equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. (c) 1996 John Wiley & Sons, Inc.     

Permeability and integrity properties of lecithin-sphingomyelin liposomes.

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acid's composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C24: 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1:1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1:1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1:1 molar ratio. (8) When the temperature is abruptly lowered to about 2 degrees C, lipsomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.     

Effect of alpha-tocopherol incorporation of glucose permeability and phase transition of lecithin liposomes.

Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.     

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon^® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.     

The biphasic effect of calcium on lipid peroxidation

Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and ufasomes (from linolenic acid and methyl linolenate) with the aid of an O2-(.) -generating system (Fe2+ + ascorbate) were studied. It was shown that stimulation of LPO by low Ca2+ concentrations (10(-6)-10(-5)M) was due to its ability to release Fe2+ ions bound to negatively charged (phosphate or carboxylic) lipid groups (of lecithin or linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion radicals and was not observed in LPO systems independent of O2- generation (e.g., Fe2+ + cumol hydroperoxide).     

Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.     

A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution

The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.     

Synergistic effect of phospholipid-based liposomes and propylthiouracil on U-937 cell growth

Scientific evidence indicates that exogenous phospholipids in the form of liposomes can affect cell growth. Effects of liposomes on cell growth depend on several factors including composition of liposomes, lipid concentration, and type of cells studied. Because phagocytic cells such as monocytes and macrophages are natural targets of liposomes, intracellular delivery of drugs to modulate cellular activity of these cells is of interest. We explored the effects of phospholipid-based liposomes composed of soy bean phosphatidylcholine (PC) as the main lipid component on U-937 cell growth. Effects of charge-imposing lipids and cholesterol were also studied. In addition, we investigated whether phospholipid-based liposomes would exert any interaction on cell growth with propylthiouracil, a drug with known antiproliferative activity. We found that PC in the form of extruded liposomes had intrinsic antiproliferative activity on U-937 cells at concentrations of 200 microM and up without any appreciable cytotoxicity. Phosphatidylserine and phosphatidylglycerol, but not dicetlylphosphate, at 10 mol% increased growth retardation activity of PC liposomes. Cholesterol at 30 mol% did not have any effect on cell growth, except for liposomes composed of PC and phosphatidylserine, where growth retardation was negated in the presence of cholesterol. Synergistic effect on cell growth was seen with certain liposome compositions when 5.5 microg/mL of propylthiouracil was coincubated. The results of this study suggest that the effects of exogenous lipids on cell growth should be taken into consideration when PC-based liposomes are to be used as drug delivery systems, especially when the targets are cells with phagocytic activity.     

Effect of valinomycin on the structure of water-lecithin liposomes]

It has been established that water--lecithin liposomes in the heptane phase are formed in the course of equilibration of lecithin solution in heptane with water phase containing Na, K-picrate. The salts penetrate both the water nucleus of liposomes and their lecithin shell. The addition of valinomycin to this system does not change the water content in liposomes, but considerably increases the salt absorbed by the lipid shell of liposomes. A characteristic S-shape curve showing the relation between picrate extraction and the valinomycin concentration might be explained as an indication of a cooperative change of the structure of lecithin shell. This phase transition is induced by one valinomycin molecule per 10(3)--10(4) lecithin molecules.     

Binding of spin-labeled local anesthetics to phosphatidylcholine and phosphatidylserine liposomes

A homologous series of spin-labeled local anesthetics, 2-[N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-p-alkoxybenzoates were shown to bind to phosphatidylcholine and phosphatidylserine liposomes. Under similar conditions, 70% of the ethoxy homolog (R2C) of these spin-labeled local anesthetics bound to synthetic dipalmitoyl lecithin while 98% bound to phosphatidylserine liposomes. Five percent of R2C's bound signal could be released by 4 mm calcium from phosphatidylserine liposomes, but calcium had no effect on R2C bound to synthetic lecithin. The butoxy (R4C) and hexyloxy (R6C) homologs bound to phosphatidylcholine in the order R6C > R4C. All of R6C and all of R4C were bound to phosphatidylserine liposomes, while only 90% of R6C bound to synthetic dipalmitoyl lecithin. Calcium was incapable of displacing bound R4C or R6C from either phosphatidylcholine or phosphatidylserine liposomes. The results are discussed in light of anesthetic binding by electrostatic and Van der Waal's forces to phospholipids.