Spectroscopic and thermodynamic characterization of the interaction between sugar-stabilised silver nanoparticles and wheat germ agglutinin (WGA), a chitin binding lectin.

Nanomaterials have lately been investigated in agriculture as eco-friendly and effective antifungal agents. Many nanomaterials, notably metal nanoparticles, have strong antifungal properties. Among metal nanoparticles, Ag nanoparticles have received the most attention as antifungal agents. Many plant lectins have been identified as antifungal agents. Conjugating AgNPs with antifungal lectins is thus expected to improve Ag nanoparticle antifungal efficacy. Understanding the molecular interactions and physical features of lectin-sugar-stabilised nanoparticle conjugates is critical for future applications. WGA has traditionally been used as an anti-tumor and antifungal agent. To investigate the prospect of developing an effective biocompatible antifungal system with applications in medicine and agriculture, fluorescence spectroscopy was used to investigate the interaction between sugar-stabilised silver nanoparticles and WGA. During the association, protein intrinsic fluorescence emission is suppressed by about ∼15 % at saturation, with no significant shift in fluorescence emission maxima. Binding tests reveal a strong bond. Stern-Volmer analysis of the quenching data indicates that the interaction happens via a static quenching process that induces complex formation. The study of hemagglutination activity and interaction experiments in the presence of particular sugar shows that the lectin's sugar-binding site is separate from the nanoparticle-binding site, and cell recognition is conserved in the lectin-nanoparticle complex. The Van't Hoff plot thermodynamic parameters suggest that the contact is hydrophobic. The fact that ΔGo is negative shows that the binding is a spontaneous process. CD spectroscopy experiments reveal that the lectin's secondary structure is not affected while binding to the nanoparticle. Our findings suggest that a stable WGA-silver nanoparticle combination may emerge for a variety of applications.

Interaction of sugar stabilised silver nanoparticles with Momordica charantia seed lectin, a type II ribosome inactivating protein.

Sugar-stabilised nanomaterials have received a lot of attention in cancer therapy in recent years due to their pronounced application as specific targeting agents and maximizing their therapeutic potential while bypassing off-target effects. Lectins, the carbohydrate-binding proteins, are capable of binding to receptors present on the target cell/tissue and interact with transformed glycans better than normal cells. Besides some of the lectins exhibit anticancer activity. Conjugating sugar-stabilised NPs with lectins there for is expected to multiply the potential for the early diagnosis of cancer cells and the specific release of drugs into the tumor site. Because of the prospective applications of lectin-sugar-stabilised nanoparticle conjugates, it is important to understand their molecular interaction and physicochemical properties. Momordica charantia Seed Lectin (MCL) is a type II RIP and has been known as an anti-tumor agent. Investigation of the interaction between sugar-stabilised silver nanoparticles and MCL has been performed by fluorescence spectroscopy to explore the possibility of creating an effective biocompatible drug delivery system against cancer cells. In this regard interaction between lectin and NPs should be well-preserved, while recognizing the specific cell surface sugar. Therefore experiments were carried out in the presence and absence of specific sugar galactose. Protein intrinsic fluorescence emission is quenched at ~ 20% at saturation during the interaction without any significant shift in fluorescence emission maximum. Binding experiments reveal a good affinity. Tetrameric MCL binds to a single nanoparticle. Stern-Volmer analysis of the quenching data suggests that the interaction is via static quenching leading to complex formation. Hemagglutination experiments together with interaction studies in the presence of specific sugar show that the sugar-binding site of the lectin is distinct from the nanoparticle-binding site and cell recognition is very much intact even after binding to AgNPs. Our results propose the possibility of developing MCL-silver nanoparticle conjugate with high stability and multiple properties in the diagnosis and treatment of cancer.

Interaction of N-acyltaurines with phosphatidylcholines.

N-acyltaurines (NATs) are biologically active amphiphilic lipids. They come under the group of compounds known as N-acyl amino acids. NATs were first detected in the brain and other tissues in mice lacking the enzyme fatty acid amide hydrolase FAAH (-/-). N-arachidonoyltaurine (20:4 NAT) acts as an excellent ligand for the subset of transient receptor potential (TRP) channels, especially vanilloid type channels TRPV1 and TRPV4. Also, hydrophobic and hydrophilic regions of NATs enable them to interact with membrane lipids. Here, we have investigated the interaction of NATs, N-myristoyltaurine (NMT), and N-palmitoyltaurine (NPT) with their corresponding diacyl phosphatidylcholines (PCs), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylchoine (DPPC). The miscibility and phase behavior of the hydrated binary mixtures have been investigated by differential scanning calorimetry (DSC). Studies on the interaction of NMT/NPT with DMPC/DPPC revealed that the two amphiphiles mix well up to 50 mol% of NAT and phase separation is observed at higher contents of the NAT. The phase transition of the equimolar mixtures of NAT:PC (50:50) studied by fluorescence, also supported the DSC results. PXRD and FTIR analysis show that the NAT:PC equimolar mixture (50:50) forms different supramolecular structures when compared to that of individual NATs and PCs. From transmission electron microscopic studies it is observed that the equimolar mixtures of NMT and NPT with their corresponding diacylphosphatidylcholines (50:50, mol/mol) forms unilamellar vesicles whose diameter range between 30 and 50 nm.

Function and therapeutic potential of N-acyl amino acids.

N-acyl amino acids (NAAs) are amphiphilic molecules, with different potential fatty acid and head group moieties. NAAs are the largest family of anandamide congener lipids discovered to date. In recent years, several NAAs have been identified as potential ligands, engaging novel binding sites and mechanisms for modulation of membrane proteins such as G-protein coupled receptors (GPRs), nuclear receptors, ion channels, and transporters. NAAs play a key role in a variety of physiological functions as lipid signaling molecules. Understanding the structure, function roles, and pharmacological potential of these NAAs is still in its infancy, and the biochemical roles are also mostly unknown. This review will provide a summary of the literature on NAAs and emphasize their therapeutic potential.

Highly selective rapid colorimetric sensing of Pb2+ ion in water samples and paint based on metal induced aggregation of N-decanoyltromethamine capped gold nanoparticles.

Lead is highly toxic. The detection of lead in the environmental bodies is difficult, because it is colourless and odourless. Herein, we report the synthesis of gold nanoparticles (AuNPs) using the interdigitized vesicles formed by N-decanoyltromethamine (NDTM). AuNPs stabilized by NDTM was pink in colour with spherical shape and the size is 29 ± 7 nm. The optical property of the NDTM-AuNPs was explored for the first time to detect toxic chemical, Pb2+. The addition of toxic metal ion Pb2+ to NDTM-AuNPs rapidly (< 1 min) alters the colour from pink to violet due to aggregation, which was confirmed by particle size analyser and TEM. The aggregation induced colour changes were realized via broad spectra in UV-Vis spectroscopy. NDTM-AuNPs showed a selective and sensitive spectrophotometric signal with Pb2+ when compared with other metal ions. The colorimetric change as a function of Pb2+ concentration gave a linear response in the range of 0-30 µM (R2 = 0.9942). The detection limit was found at 10 µM by naked eye and 0.35 µM by spectrophotometry. The proposed method was successfully applied for the determination of Pb2+ ions in tap water and sewage water. Moreover, as a proof of concept, the NDTM-AuNPs sensor system was applied for the detection of lead in commercial paints. The results of the quantitative estimation of lead in paints by NDTM-AuNPs colorimetric sensor were as good as the standard method, atomic absorption spectroscopy.

Characterization of the molecular packing, thermotropic phase behaviour and critical micellar concentration of a homologous series of N-acyltaurines (n = 9-18). PXRD, DSC and fluorescence spectroscopic studies.

N-acyltaurines (NATs) are amides of fatty acids that can be structurally related to endocannabinoids. They show interesting physiological and pharmacological properties. We have synthesized a homologous series of NATs with saturated acyl chains (n = 9-18) and investigated their supramolecular structure and thermotropic phase transitions by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). The d-spacings obtained from PXRD increase linearly with chain length with an increment of ∼0.847 Å per additional CH2 moiety suggesting that NATs adopt a tilted bilayer structure with similar packing in crystal lattice. Results obtained from DSC studies indicate that the endothermic transition temperature (Tt) of NATs showed a gradually increasing trend with increasing acyl chain length. The enthalpy (ΔHt) and entropy (ΔSt) of transition show odd-even alternations with odd-chain compounds having higher values than the even-chain compounds. The critical micellar concentration (CMC) of NATs was determined in water at room temperature by fluorescence spectroscopy by monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonic acid (ANS). The CMCs of NATs were found to decrease with increase in acyl chain length. The present results provide a thermodynamic and structural basis for investigating the interaction of NATs with other membrane lipids and proteins, which in turn can shed light in understanding how they function in vivo (in biological membranes).

Remarkable Effect of Jacalin in Diminishing the Protein Corona Interference in the Antibacterial Activity of Pectin-Capped Copper Sulfide Nanoparticles.

Herein, we report a new strategy based on jacalin functionalization to diminish the impact of biological fluids in the antibacterial applications of nanoparticles (NPs). Precoating pectin-capped copper sulfide NPs (pCuS) with bovine serum albumin produced a protein corona, which affects the antibacterial activity of pCuS. It was found that the minimum inhibitory concentration (MIC) increases fourfold because of the formation of the protein corona. Interestingly, the pCuS functionalized with jacalin enhance the targeting capabilities through bacterial cell surface glycan recognition with no interference from the protein corona. The MIC of pCuS decreases 16-fold on functionalization with jacalin. Mechanistic studies indicated that the pCuS functionalized with jacalin impede the protein corona interference and induce bacterial cell death by impairing the GSH/reactive oxygen species balance and disrupting the bacteria cell membrane. As a proof of concept, we used a bacteria-infected zebrafish animal model to demonstrate the interference of biological fluids in the antibacterial activity of NPs. Infected zebrafish treated with 1× MIC of pCuS failed to recover from the infection, but 4× MIC rescues the fish. The requirement of a high dose of NPs to treat the infection confirms the interference of biological fluids in nanotherapeutic applications. At the same time, the jacalin-pCuS complex rescues the infected fish at 16-fold lesser MIC. The results obtained from this study suggest that jacalin-mediated NP targeting may have broad implications in the development of future nanomedicine.

In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.

Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein.

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained >90% activity and 80-90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85-90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30-40%) and λmax blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

Structure and thermotropic phase behavior of a homologous series of bioactive N-acyldopamines.

N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting biological and pharmacological properties. In the present study, a homologous series of NADAs with varying acyl chains (n = 12-20) have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry state the transition temperatures, enthalpies, and entropies of NADAs exhibit odd-even alternation with the values corresponding to the even chain length series being slightly higher. Both even and odd chain length NADAs display a linear dependence of the transition enthalpies and entropies on the chain length. However, odd-even alternation was not observed in the calorimetric properties upon hydration, although the transition enthalpies and entropies exhibit linear dependence. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between the hydroxyl groups of the dopamine moieties of opposing layers and N-H···O hydrogen bonds between the amide groups of adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they function in vivo, e.g., as signaling molecules in the modulation of pain.

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~41°C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λ(max)~352nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) 'signature' GLTP-fold Trp fluorescence response, i.e., intensity decrease (~30%) accompanied by strongly blue-shifted λ(max) (~14nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold.

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp96-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Structure, phase behaviour and membrane interactions of N-acylethanolamines and N-acylphosphatidylethanolamines.

N-Acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are naturally occurring membrane lipids, whose content increases dramatically in a variety of organisms when subjected to stress, suggesting that they may play a role in the stress-combating mechanisms of organisms. In the light of this, it is of great interest to characterize the structure, physical properties, phase transitions and membrane interactions of these two classes of lipids. This review will present the current status of our understanding of the structure and phase behaviour of NAEs and NAPEs and their interaction with major membrane lipids, namely phosphatidylcholine, phosphatidylethanolamine and cholesterol. The relevance of such interactions to the putative stress-combating and membrane stabilizing properties of these lipids will also be discussed.

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains.

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10-20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc(21) with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.

Interaction of N-myristoylethanolamine with cholesterol investigated in a Langmuir film at the air-water interface.

The dramatic increase in the content of N-acylethanolamines (NAEs) having different acyl chains in various tissues when subjected to stress has resulted in significant interest in investigations on these molecules. Previous studies suggested that N-myristoylethanolamine (NMEA) and cholesterol interact to form a 1:1 (mol/mol) complex. In studies reported here, pressure-area isotherms of Langmuir films at the air-water interface have shown that at low fractions of cholesterol, the average area per molecule is lower than that predicted for ideal mixing, whereas at high cholesterol content the observed molecular area is higher, with a cross-over point at the equimolar composition. A plausible model that can explain these observations is the following: addition of small amounts of cholesterol to NMEA results in a reorientation of the NMEA molecules from the tilted disposition in the crystalline state to the vertical and stabilization of the intermolecular interactions, leading to the formation of a compact monolayer film, whereas at the other end of the composition diagram, addition of small amounts of NMEA to cholesterol leads to a tilting of the cholesterol molecules resulting in an increase in the average area per molecule. In Brewster angle microscopy experiments, a stable and bright homogeneous condensed phase was observed at a relatively low applied pressure of 2 mN.m(-1) for the NMEA:Chol. (1:1, mol/mol) mixture, whereas all other samples required significantly higher pressures (>10 mN.m(-1)) to form a homogeneous condensed phase. These observations are consistent with the formation of a 1:1 stoichiometric complex between NMEA and cholesterol and suggest that increase in the content of NAEs under stress may modulate the composition and dynamics of lipid rafts in biological membranes, resulting in alterations in signaling events involving them, which may be relevant to the putative cytoprotective and stress-combating ability of NAEs.

Miscibility and phase behavior of N-acylethanolamine/diacylphosphatidylethanolamine binary mixtures of matched acyl chainlengths (N=14, 16).

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and (31)P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by (31)P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with (31)P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65 degrees C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.

Studies on the critical micellar concentration and phase transitions of stearoylcarnitine.

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 microM at pH 3.0 to ca. 25 microM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 microM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5 degrees C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (Tt) shifts to ca. 50.5 degrees C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1 degrees C at pH 3.0, which increased with increasing pH up to a value of 55.3 degrees C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and Tt suggest that the pKa of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes).