Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.     

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2,7-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.     

A hierarchy of lipid constructs for the sperm plasma membrane

We have presented a series of lipid constructs as models of the sperm plasma membrane. We also isolated the plasma membrane from rabbit sperm cells and characterized the lipid composition. The behavior of these various membrane systems was evaluated using a vesicle leakage assay, in which surfactant (nonoxynol-9, N-9; or benzalkonium chloride, BZK) exposure induced membrane permeabilization. These studies shed light on the relative importance and significance of particular components present in the lipid constructs. In particular, a highly unsaturated phospholipid component characterized by an ether-linkage to position 1 of the glycerol backbone (as opposed to the more conventional ester linkage) as well as the presence of sulfogalactosyl ceramide were found to have an effect on the surfactant-induced leakage response. The presence of cholesterol had the greatest influence on membrane behavior. The construct series also demonstrated the ability of the surfactants studied to discriminate between different membrane systems. We found that N-9 displayed little sensitivity to membrane composition while BZK showed specific behavior with the various membrane systems.     

Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells

Summary.  The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia (Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscuta spp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress. Received June 12, 2002; accepted August 26, 2002; published online March 11, 2003     

Exotic aqueous behavior of synthetic lipids: formation of vesicular nanotubes

The work reported herein deals with the synthesis and the aggregation behavior studies of synthetic lipids bearing a non-ionic polar head made up of a tris(hydroxymethyl) aminomethane (tris) moiety linked with an aminoglycerol interface. The hydrophobic chains with variable lengths were grafted onto the hydroxyl functions of the aminoglycerol residue through ester or carbamate bonds. Tiny chemical modifications within this family of non-ionic surfactants brought about major variations in their aggregation behavior. They formed vesicles, tubules, and also small stable end-capped tubules - called vesicular nanotubes -, when the polar head bore two heptadecyl chains linked through a carbamate bond. Various techniques (nanosizer measurements, freeze fracture electron microscopy (FFEM), transmission electron microscopy (TEM), carboxyfluorescein (CF)) encapsulation were used to specify the structure of these assemblies. Notably, the vesicular nanotubes exhibited a small size, a fair polydispersity, great stability in an aqueous solution (up to 1 year) and a good efficiency to entrap and slowly release a probe such as carboxyfluoresceine: all these properties are perfectly suitable for their use as potential drug carriers.     

Osmotically induced fluid-phase uptake of fluorescent markers by protoplasts ofChenopodium album

Summary Protoplasts fromChenopodium album suspension culture show large, up to 5-fold, changes in surface area upon hypertonic or hypotonie treatment. These surface area variations cannot be explained by elastic stretching of the plasmalemma. An exchange of membrane material between the plasmalemma and an internal membrane source takes place. Fluid-phase uptake experiments with the fluorescence dyes 5, 6-carboxyfluorescein and Lucifer yellow CH demonstrated that osmotic shrinkage of protoplasts is accompanied by vesicular uptake of the external medium into protoplast cytoplasm. Confocal laser scanning microscopy, as well as conventional fluorescence microscopy, revealed the number, diameter and distribution of the osmocytotic vesicles at different osmotic levels. The rate of osmocytotic vesicle uptake was higher in the presence of calcium chloride than in the presence of EDTA in the external medium. At 6.9 mM calcium chloride we observed a loss of vesicular fluorescence upon returning protoplasts to 0.4 M from 0.8 M sorbitol.     

Separating the isomers—Efficient synthesis of the N-hydroxysuccinimide esters of 5 and 6-carboxyfluorescein diacetate and 5 and 6-carboxyrhodamine B

Diacetate protection of 5 and 6-carboxyfluorescein followed by synthesis of the N-hydroxysuccinimide esters allowed ready separation of the two isomers on a multi-gram scale. The 5 and 6-carboxyrhodamine B N-hydroxysuccinimide esters were also readily synthesised and separated.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

Peculiar behaviour of rabbit thymocytes in interaction with liposomes of different compositions shown by fluorescence polarization studies,lipid analysis,and uptake of vesicle-entrapped carboxyfluorescein

In order to obtain more information on membrane phenomena occurring at the cell surface of rabbit thymocytes we have performed experiments aimed at altering the lipid composition of the plasma membrane. Thymocytes were incubated at 37°C with phospholipid vesicles of different compositions. Vesicle-cell interaction was followed by measuring the degree of fluorescence polarization and the uptake of vesicle-entrapped carboxyfluorescein. Neutral and negatively charged liposomes prepared from egg phosphatidylcholine are currently used in investigations of vesicle-cell interaction. In this report we show that these liposomes do not interact with rabbit thymocytes as is evident from unaltered lipid fluidity measured in whole cells and in isolated plasma membranes. This was confirmed by experiments with vesicle-entrapped carboxyfluorescein showing hardly any uptake of the fluorophor from neutral and negatively charged egg phosphatidylcholine liposomes. Using both techniques substantial interaction was found with positively charged egg phosphatidylcholine liposomes and with liposomes prepared from soybean lecithin which is composed of a variety of phospholipids. The results of these experiments were supported by lipid analysis of cells treated with soybean lecithin liposomes. Increase in phosphatidylcholine contents of mixed phospholipid vesicles was further shown to result in decreased vesicle-cell interaction. From measurements of the quantity of carboxyfluorescein inside cells and the total amount of cell-associated carboxyfluorescein it is concluded that adsorption plays a prominent role in interaction between liposomes and rabbit lymphocytes. The grade of maturation of lymphocytes was also found to affect vesicle-cell interaction. The more mature thymocytes took up more vesicle-entrapped carboxyfluorescein from soybean liposomes than immature thymocytes. Mesenteric lymph node cells exhibited a still stronger interaction. The role of vesicle and cell surface charge and membrane fluidity of both vesicles and cells in interaction between liposomes and rabbit thymocytes is discussed.