Solid-phase Lipid Research Articles

Lipid-Based Nanoformulations for Drug Delivery: An Ongoing Perspective

Oils and lipids help make water-insoluble drugs soluble by dispersing them in an aqueous medium with the help of a surfactant and enabling their absorption across the gut barrier. The emergence of microemulsions (thermodynamically stable), nanoemulsions (kinetically stable), and self-emulsifying drug delivery systems added unique characteristics that make them suitable for prolonged storage and controlled release. In the 1990s, solid-phase lipids were introduced to reduce drug leakage from nanoparticles and prolong drug release. Manipulating the structure of emulsions and solid lipid nanoparticles has enabled multifunctional nanoparticles and the loading of therapeutic macromolecules such as proteins, nucleic acid, vaccines, etc. Phospholipids and surfactants with a well-defined polar head and carbon chain have been used to prepare bilayer vesicles known as liposomes and niosomes, respectively. The increasing knowledge of targeting ligands and external factors to gain control over pharmacokinetics and the ever-increasing number of synthetic lipids are expected to make lipid nanoparticles and vesicular systems a preferred choice for the encapsulation and targeted delivery of therapeutic agents. This review discusses different lipids and oil-based nanoparticulate systems for the delivery of water-insoluble drugs. The salient features of each system are highlighted, and special emphasis is given to studies that compare them.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4nM and 66.4nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. Invitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

Improvements in extraction yield by solid phase lipid extraction from liquid infant formula and human milk, and the fatty acid distribution in milk TAG analyzed by joint JOCS/AOCS official method Ch 3a-19.

The Röse-Gottlieb method is one of the most widely used methods for extracting lipids from milk samples. However, we found that lipid recovery from liquid infant formula and human breast milk was lower than expected. Better lipid recovery from these liquid matrices was obtained by solid phase extraction using silica gel; ~10% more could be recovered from liquid infant formula and ruminant milk, and 25% more from human breast milk. However, the method is not recommended for lipid extraction from dried whole milk powders.

Solid lipid nanoparticles as a drug delivery system to across the blood-brain barrier

Solid lipid nanoparticles (SLNs) were prepared by biocompatible and biodegradable solid-phase lipids. β-elemene is a safe natural essential oil with broad-spectrum anti-tumor activity. However, its clinical application has been adversely affected by its poor water solubility and limited bioavailability. SLN incorporation is a potential strategy to bypass the blood-brain barrier, the most important factor limiting the bioactivity of neurotherapeutics. The SLNs-β has the same efficacy as commercially available elemene in vitro and an enhanced brain drug accumulation in vivo. The survival rate data was promising and acute toxicity experiment proved its safety. All these data suggested that SLN-β is a safe and effective drug delivery system, especially for brain tumor therapy, and warrants further development.

Relationships Between Bilayer Phase and Equilibration Rates of Patman and Laurdan

Patman is a fluorescent membrane probe related to laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in laurdan and fifteen in patman) and the presence of a trimethylammonium forming a positively-charged head on patman. Studies exploring patman as a probe to detect membrane properties during apoptosis revealed that the two edges of the emission spectrum (435 and 500 nm) stabilize at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, patman equilibrated more rapidly with bilayers in the liquid disordered phase than in the solid ordered phase. With solid phase membranes, the fluorescence stabilized faster at 435 nm than at 500 nm. Similar yet more subtle results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 500 nm than at 435 nm. To determine whether these results reflected specific properties of patman, the experiments were repeated with laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with patman for solid phase membranes but greater than with patman for melted bilayers. Third, at the phase transition temperature, equilibration rates favored 435 nm over 500 nm, the opposite of the result obtained with patman. Computer simulations were used to assist with interpretation of these results.

Light-Induced Permeability Changes in Liposomes Containing Photo-Polymerizable Phospholipids

We have designed a novel class of light-triggerable liposomes prepared from a photo-polymerizable phospholipid DC8,9PC (1,2- bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine). Exposure to UV radiation (254 nm) for 0-45 minutes (25 0C) resulted in photo-polymerization of DC8,9PC in these liposomes and the release of an encapsulated fluorescent dye (calcein). Photopolymerization and permeability changes did not occur from UV-triggered Egg PC/DC8,9PC liposomes. We propose that phase separation and packing of polymerizable lipids in the liposome bilayer are major determinants of photo-polymerization resulting in the formation of local defects and/or lipidic pores in the liposome membrane. Differential Scanning Calorimetry show phase transition peaks at 36.8 0C and 41.6 0C, respectively, in liposomes composed of DPPC:DC8,9PC (9:1 mole ratio) indicating that the reactions occurred while these lipids are in the gel phase (25 0C). Our results indicate that DC8,9PC and DPPC molecules undergo de-mixing in the gel phase. This hypothesis is supported by Molecular Dynamics simulations that indicate separation of DC8,9PC and DPPC in the solid phase lipid bilayer. Cryo-electron microscopic images of the liposomes show major changes in liposome morphology after UV irradiation. When an appropriate tunable photo-sensitizer dye is included in the aqueous compartment of these liposomes, release of contents is triggered by excitation with a laser at the wavelength of the encapsulated dye. Inhibition of release in the presence of oxygen radical scavengers indicate that the mechanism of release involves chemical changes in DC8,9PC unrelated to photo-polymerization. The laser-mediated chemical modifications in DC8,9PC are being analyzed by MS, LC, GC and NMR. We are further developing these liposomes for undergo triggered release of chemotherapeutic agents (e.g. doxorubicin) and are testing their efficacy in vitro and in vivo.

Anomalous Subdiffusion in Heterogeneous Lipid Bilayers

Fluorescence photobleaching recovery (FPR) is commonly used to measure lipid and protein diffusion in cellular membranes. Typically, a model wherein diffusion is constant with time and the mean-squared displacement is directly proportional to time is used to analyze the results; however, in nonhomogeneous systems such as cellular membranes, anomalous subdiffusion may occur. In anomalous subdiffusion, the diffusion coefficient, D, decreases with time and thus the mean-squared displacement is proportional to some power of time less than 1. Although theory predicts that diffusion can be anomalous through protein interactions or obstruction, the complex composition of cellular membranes has made the actual origin and consequences of anomalous diffusion in phospholipid bilayers unclear. In this study, we use atomic force microscopy to detect and measure the amount of the solid phase in supported bilayers that contain coexisting fluid- and solid-phase lipids. Solid-phase domains in bilayers have been shown to a...

Solid-phase lipid synthesis (SPLS)-2: incidental discovery of organogelators based on artificial glycolipids

Here we report a new solid-phase (glyco)lipid synthesis (SPLS), which gives traceless glycolipids using an acetal linker. During the synthesis, we incidentally found some of the artificial glycolipids act as good organogelators, the property of which is greatly dependent on the modular saccharide structure.

Pulmonary Surfactant Protein A Interacts with Gel-Like Regions in Monolayers of Pulmonary Surfactant Lipid Extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 μg/ml SP-A and 0, 1.64, or 5 mM CaCl 2. In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca 2+. Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures ( π ∼5 mN/m) and the domains grew in size with increasing π. Above 25 mN/m, the domain size decreased with increasing π. The amount of observable dark phase was maximal at 18% of the total film area at π ∼25 mN/m, then decreased to ∼3% at π ∼40 mN/m. The addition of 0.16 μg/ml SP-A with 0 or 1.64 mM Ca 2+ in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to ∼25% between π of 10–28 mN/m. Monolayer features in the presence of 5 mM Ca 2+ and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca 2+. PSLE films were spread on a subphase containing 0.16 μg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca 2+, TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca 2+ resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.

Solid phase lipid synthesis (SPLS) for construction of an artificial glycolipid library

A simple solid phase lipid synthesis (SPLS) is proposed for construction of an artificial glycolipid library; this method is so convenient and flexible that we have efficiently constructed a glycolipid library; some of these glycolipids formed into stable bilayer aggregates in aqueous solution, suggesting that the glycolipids obtained by SPLS are useful as a suitable model of naturally occurring glycolipids.

Lactoferrin-lipid A-lipopolysaccharide interaction: inhibition by anti-human lactoferrin monoclonal antibody AGM 10.14.

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.

Models of stratum corneum intercellular membranes: 2H NMR of macroscopically oriented multilayers

Deuterium NMR was used to characterize model membrane systems approximating the composition of the intercellular lipid lamellae of mammalian stratum corneum (SC). The SC models, equimolar mixtures of ceramide:cholesterol:palmitic acid (CER:CHOL:PA) at pH 5.2, were contrasted with the sphingomyelin:CHOL:PA (SPM:CHOL:PA) system, where the SPM differs from the CER only in the presence of a phosphocholine headgroup. The lipids were prepared both as oriented samples and as multilamellar dispersions, and contained either perdeuterated palmitic acid (PA-d31) or [2,2,3,4,6-2H5]CHOL (CHOL-d5). SPM:CHOL:PA-d31 formed liquid-ordered membranes over a wide range of temperatures, with a maximum order parameter of approximately 0.4 at 50 degrees C for positions C3-C10 (the plateau region). The quadrupolar splitting at C2 was significantly smaller, suggesting an orientational change at this position, possibly because of hydrogen bonding with water and/or other surface components. A comparison of the longitudinal relaxation times obtained at theta = 0 degrees and 90 degrees (where theta is the angle between the normal to the glass plates and the magnetic field) revealed a significant T1Z anisotropy for all positions. In contrast to the behavior observed with the SPM system, lipid mixtures containing CER exhibited a complex polymorphism. Between 20 and 50 degrees C, a significant portion of the entire membrane (as monitored by both PA-d31 and CHOL-d5) was found to exist as a solid phase, with the remainder either a gel or liquid-ordered phase. The proportion of solid decreased as the temperature was increased and disappeared entirely above 50 degrees C. Between 50 and 70 degrees C, the membrane underwent a liquid-ordered to isotropic phase transition. These transitions were reversible but displayed considerable hysteresis, especially the conversion from a fluid phase to solid. The order profiles, relaxation behavior, and angular dependence of these parameters suggest strongly that both the liquid-ordered CER- and SPM-membranes are bilayers. The unusual phase behavior observed for the CER-system, particularly the observation of solid-phase lipid at physiological temperatures, may provide insight into the functioning of the permeability barrier of stratum corneum.

Gas chromatographic measurement of 3- and 4-thia fatty acids incorporated into various classes of rat liver lipids during feeding experiments.

A practical procedure is described for the quantitative measurement of the amount of acyl units derived from tetradecylthioacetic acid (effecting hypolipemia in rats) and tetradecylthiopropionic acid (effecting hyperlipidemia). The procedure involves three main successive steps: (1) extraction; (2) solid-phase lipid class separation yielding free fatty acids, phospholipids, triacylglycerides, cholesterol esters, and diacylglycerides without crosscontamination; and (3) gas chromatography of hydrolyzed lipids derivatized to picolinyl esters, combined with unambiguous identification by gas chromatography-mass spectrometry. The overall recoveries of heptadecanoyl lipids added as internal standards during extraction were 94-96%, except for cholesteryl heptadecanoate where the recovery was 60% owing to incomplete hydrolysis. Recoveries of thia fatty acids from samples spiked with these compounds were 95%. Flame-ionization response factors were found to be 0.92 and 0.81 for the tetradecylthioacetic acid and tetradecylthiopropionic acid picolinyl esters, respectively, compared to that of heptadecanoic acid. The lower limit of quantitation was 25 pmol as injected. Measurement of the amount of thia fatty acyl units in rat plasma and in liver lipids 4 h after administration of single doses by gastric intubation indicated efficient absorbtion and rapid incorporation into liver lipids, particularly in the phospholipid fraction. Both plasma clearance and channelling into lipids was slower for the 4-thia fatty acid.

Lipopolysaccharide epitope specificity and binding cross reactivity of the human IgM anti-lipid a monoclonal antibody SdJ5-1.17.15

The binding properties and specificity of the SdJ5-1.17.15 (SdJ5) human IgM monoclonal antibody (mAb), prepared against S. minnesota R595 heat killed whole organisms, were assessed by dot blot assay in vitro. In that assay, lipopolysaccharide (LPS) related antigens, immobilized on nitrocellulose membrane, were reacted with the test and control human IgM antibodies. Results indicated the SdJ5 mAb reacted specifically with lipid A, a component of LPS, from a variety of bacterial species, but not with the whole LPS molecule. The inability of the mAb to react with whole LPS in dot blot assay was attributed to the possible effect of the solid phase on epitope exposure or structure. This hypothesis was tested by inhibition studies which indicated that liquid phase adsorption with LPS abolished or greatly reduced the specific recognition of solid phase lipid A by the mAb. These results indicated that LPS-associated antigens were recognized by the SdJ5 anti-lipid A mAb in liquid, but not solid, phase. In an attempt to identify the SdJ5-specific epitope on lipid A, the pattern of reactivities of different lipid A analogues with the mAb were examined by dot blot assay. Results indicated that a combination of the fatty acid side chains and the phosphate groups of lipid A (both groups being implicated as important for sepsis mediation) were either directly involved in the epitope structure or affected the exposure of epitope in solid phase. Because SdJ5 recognizes an LPS epitope on lipid A closely associated with endotoxin activity, this mAb could potentially be a useful therapeutic agent against septic shock.

Partition of parinaroylphosphatidylethanolamines and parinaroylphosphatidylglycerols in immiscible phospholipid mixtures

Partitioning of two parinaroyl phosphatidylethanolamines and two parinaroyl phosphatidylglycerols between solid and fluid phase phospholipids was examined. Fluorescence quantum yields and fluorescence polarization measurements were used to calculate K p s/t, the solid to fluid partition coefficient of each probe (Sklar, L.A., Miljanich, G.P. and Dratz, E.A. (1979) Biochemistry 18, 1707–1716). In the immiscible mixture dipalmitoylphosphatidylcholine and dilinoleylphosphatidylcholine, both 1-palmitoyl-2- trans-parinaroylphosphatidylethanolamine and 1-palmitoyl-2- trans-parinaroylphosphatidylglycerol partitioned preferentially into solid phase lipid with mean K p s/t values (calculated from quantum yields) of 3.4 ± 1.5 and 2.1 ± 0.7, respectively. In contrast, 1-oleoyl-2- cis-parinaroylphosphatidylethanolamine and 1-oleoyl-2- cis-parinaroylphosphatidylglycerol partitioned preferentially into fluid phase lipid in the same model system with mean K p s/t values (calculated from quantum yields) of 0.44 ± 0.26 and 0.16 ± 0.07, respectively. Fluorescence polarization data on the same four parinaroyl phospholipids in mixtures of solid-phase dimyristoylphosphatidyl ethanolamine and fluid-phase dilinoleoylphosphatidylglycerol were similar to those obtained in the immiscible phosphatidylcholine system, demonstrating that the partitioning of these probes is not strongly dependent on head group. Knowledge of the partition properties of these fluorescent probes is relevant to use of these probes in investigation of the phase behavior of Escherichia coli inner membrane lipids, since phosphatidylethanolamine and phosphatidylglycerol species account for approximately 95% of these lipids.

Hydrolytic action of phospholipase A 2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A 2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A 2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A 2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

An enzyme caught in action: Direct imaging of hydrolytic function and domain formation of phospholipase A 2 in phosphatidylcholine monolayers

Phospholipase A 2, a ubiquitous lipolytic enzyme that actively catalyses hydrolysis of phospholipids, has been studied as a model for enzyme-substrate reactions, as a membrane structural probe, and as a model for lipid-protein interactions. Its mechanism of action remains largely controversial. We report here for the first time direct microscopic observation of the lipolytic action of fluorescently marked phospholipase A 2 ( Naja naja naja) against phosphatidylcholine monolayers in the lipid phase transition region. Under these conditions, phospholipase A 2 is shown to target and hydrolyse solid-phase lipid domains of L-α-dipalmitoylphosphatidylcholine. In addition, after a critical extent of monolayer hydrolysis, the enzyme itself aggregates into regular, visible proteinaceous domains within the lipid monolayer. Solid-phase lipid hydrolysis indicates a preferential hydrolytic environment for phospholipase A 2 while enzyme domain formation points to a possible allosteric inhibition mechanism by hydrolysis products.

Fluorescence depolarization of cis- and trans-parinaric acids in artificial and red cell membranes resolved by a double hindered rotational model.

Although steady-state anisotropy measurements of phase-sensitive probes provide a qualitative description of the phase behavior of biomembranes, there is little information about the physical state of lipid domains. We have developed a ground-state double hindered rotator model (DHR) for fluorescence anisotropy decay, in which probes possess separate rotational correlation times and r infinity in each phase. To validate the model, multifrequency differential phase angles (delta) and modulation amplitudes (lambda) were measured in a two-compartment cuvette with combinations of POPOP, TMA-DPH, and DPH in isotropic solvents and in DPPC liposomes. Rotational parameters obtained by fitting the DHR model were similar to those of a single hindered rotator model fitted to data obtained separately for each probe. As predicted by the model, negative delta and decreasing lambda with increasing modulation frequency were obtained when fluorophores in isotropic solvents were paired with fluorophores in DPPC liposomes. The rotational parameters of the phase-sensitive fluorophores cis-parinaric (cPnA) and trans-parinaric (tPnA) acid in DPPC/DMPC (1:0, 0:1, and 1:1) liposomes were determined at 15-40 degrees C. Two lifetimes (1 and 3 ns) were obtained above the phase transition temperature (Tc); greater than 95% of the fluorescence intensity was described by two lifetimes (3-9 and 12-32 ns) below Tc. Negative delta values were obtained when solid-phase lipid was present. r infinity varied from 0.26-0.32 below to 0.11-0.14 above Tc; at intermediate T, where two phases coexists, r infinity values were approximately 0.23 and approximately 0.31. These data indicate very hindered PnA rotation in solid-phase lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a possible immunogen: requirements for tumor-associated antigen and immunogen.

A mouse monoclonal antibody M2590, previously established after immunization of mice (C57BL/6) with syngeneic melanoma B16 cells and showing preferential reactivity with various types of melanoma over other tumor and normal cells or tissues, was shown to be directed to GM3 ganglioside. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concept of a tumor-associated antigen and immunogen. Studies on the reactivity of M2590 antibody with various cells having different GM3 density at their cell surface, including cells treated with sialidase, liposomes, and solid-phase lipid layer containing different GM3 concentrations, have indicated that 1) reactivity of the antibody M2590 depends greatly on the density of GM3 exposed at the cell surface, on liposomes, or on solid phase; and 2) there is a threshold density that is recognized by the antibody in all-or-none fashion. In addition, the antibody M2590 reacts not only with GM3 but also with GM3 lactone, and the binding affinity of the antibody to GM3 lactone is strikingly higher than to GM3; however, the antibody does not react with GM3 ethyl ester. GM3 lactone was detected in melanoma as 3H-labeled GM3 gangliosidol after melanoma cells were directly treated with NaB[3H]4. A comparative immunization of BALB/c mice with GM3 and GM3 lactone showed that GM3 lactone is a much stronger immunogen than GM3, although the antibody elicited reacts with both GM3 and its lactone. Thus, the real immunogen could be GM3 lactone, although it is a minor membrane component.

31P NMR spectra of rod outer segment and sarcoplasmic reticulum membranes show no evidence of immobilized components due to lipid-protein interactions.

31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22 degrees C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (L alpha) phase at 22 degrees C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P.L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to approximately 7 phospholipids/rhodopsin molecule and approximately 11 phospholipids/Ca2+-ATPase molecule, respectively.